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The global viralization of policies to contain the spreading of the COVID-19 pandemic: Analyses of school closures and first reported cases
BackgroundOn January 30 th 2020, the World Health Organization (WHO) declared a international health emergency due to the unprecedented phenomenon of COVID-19. After this declaration countries swiftly implemented a variety of health policies. In this work we examine how rapid countries responded to this pandemic using two events: the day in which the first case of COVID-19 was reported, and first day in which countries used school closure as one of the measures to avoid outbreaks. We also assessed how countries' health systems, globalization, economic development, political systems, and economic integration to China, Republic of Korea and Italy increased the speed of adoption.MethodsWe compiled information from multiple sources, from December 31 st 2019 to June 1 st 2020, to trace when 172 countries reported their first COVID-19 case and implemented school closure to contain outbreaks. We applied cross-national Weibull survival analysis to evaluate the global speed of detection of first COVID-19 reported cases and school closure.ResultsTen days after WHO declared COVID-19 to be an international emergency, relative to seven days from this declaration, countries were 28 (95% CI: 12-77) times more likely to report first COVID-19 cases and 42 (95% CI: 22-90) times more likely to close schools. One standard deviation increase in the epidemic security index rises the rate of report first cases by 37% (Hazard Ratio (HR) 1.37 (95% CI: 1.09-1.72) and delays the adoption for school closures by 36% (HR 0.64 (95% CI:0.50-0.82). One standard deviation increase in the PLOS ONE PLOS ONE | https://doi.
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# Introduction
In the past thirty years a strong tendency towards isomorphism has been identified across nation-states. That is countries are likely to have similar public policy responses when faced with challenges that occur at the national or international levels. In short, we have observed in this period a trend of global convergence of multiple policies such as democracy, terrorist laws, privatization, human rights among others. With the current exposure to COVID-19 pandemic, countries rapidly realigned the allocation of public resources after China informed the World Health Organization's (WHO) authorities (December 31st 2019), and Italy issued a state emergency decree the same date when WHO declared a global health emergency (January 30th 2020). As such this phenomenon provides a unique opportunity to revisit the thesis of global convergence with a rather exceptionally short time span of five months.
Simultaneously, the debate over what measures should have been implemented to prevent deaths associated with vulnerable populations or at least decrease the lethality of this virus has been fierce. While some measures seemed to be more effective than others in Singaporeand Republic of Korea-including aggressive technological tracing, massive testing, and isolation of cases and extensive quarantining of contacts-many concerns were raised since some public health recommendations directly affected liberties and economies, and thus the overall functioning of countries, regions and the world. Even though countries' decisions to tackle the pandemic is suggesting a strong case of global convergence yet little is known of what makes countries to more rapidly adopt such policies. Indeed, the presence of uneven public health resources across countries, different levels of integration with the world, China, Republic of Korea and Italy, respect for liberties, may have all constrained differently countries' swiftness to rapid response. In short, the COVID-19 pandemic offers an exclusive opportunity to explore how countries transit this public health emergency by rapidly introducing different policies.
In this work, we examine the speed of isomorphism using two events associated with the COVID-19 pandemic: 1) the day in which the first case of COVID-19 was reported in each country, and 2) the first day in which countries nationally used school closure as one of the measures to reduce the spread of this virus. We chose school closing since its implementation informs a sense of urgency under uncertain conditions while little was known about transmission in children.
Since countries are not similarly prepared, we hypothesise that countries with greater global political and economic integration, and neither equally distant to China, South Korea nor Italy, will respond at different speeds to these two events. We advance two groups of hypotheses, one for the detection of the first case of COVID-19, and another for school closures. Inwe formalize each hypothesis for both events.
# Methods and data
To test the speed of the global convergence thesis in the context of the COVID-19 pandemic and what makes countries more rapidly adopt policies designed to detect and stop its contagion, we compiled and analyzed data on 172 countries between December 31 st 2019 and June 1 st 2020 from multiple sources and applied survival analysis. Each source to carry out the analyses is described below and open repositories.
## I) outcomes
We analyzed two events: i) date in which the first case of COVID-19 was reported; and ii) date in which schools were closed at the national level. (In France the first date of school closure occurred at the provincial level in March 7 th 2020, however the national decision to implement this measure took place in March 16 th; this date was used to code when the adoption occurred in this country). Countries in which a national decision was not taken before June 1 st 2020 were regarded as not having implemented this measure (i.e. censored). To check for the robustness of this design, we also carried out analyses with countries in which national decision was not taken before this date and used the last date in which a state or province had reported closure and results were consistent. Information to detect dates in which first cases of COVID-19 were reported, and school closure was carried out was gathered from UNESCOas well as from governments' websites and national and international newspapers (S1 File contains all sources per country). To verify information on when first cases of COVID-19 were reported per country, we also used the open depository worldometer. 1.1 After the WHO declared a global health emergency, countries will be more likely to report first cases of COVID-19. 1.2 Countries with health systems designed to respond and mitigate more rapidly the spread of an epidemic, higher gross development product (GDP) per capita, more populated, more globally integrated and with tighter economic ties to China, Republic of Korea or Italy will be more rapid in reporting to the first detected case.
2. School closures 2.1 Countries more globally integrated will be more exposed to the influence of the WHO recommendations to tackle the pandemic and therefore more rapidly to adopt school closure. 2.2 Countries with health systems designed to respond and mitigate more rapidly the spread of an epidemic will delay the implementation of school closures since they have better knowledge to prevent a stringent measure such as school closures 2.3 Countries with higher GDP will delay the implementation of school closures since this measure has a direct impact in the economy. 2.4 Less democratic countries will be swifter in implementing this measure since a vertical response of this nature implies a direct limitation of freedom of assembly, which in these countries may not be regarded as a fundamental right. 2.5 Countries more economically integrated with China, Republic of Korea and Italy will be more rapidly closing schools since closeness to these countries will raise higher public health concerns to stop the spreading of the COVID-19.
https://doi.org/10.1371/journal.pone.0248828.t001
## Ii) determinants of early response
We use the following variables to explore what makes countries adopt more rapidly or more slowly the two events. Epidemic security index. We expect this variable to increase the hazard rate of reporting the first case since these countries would have more capacity to detect the presence of the virus in the population. We expect this variable to be associated with a less rapid adoption of school closure since other measures are likely to be assessed before setting in place a very restrictive measure in the population. These data have been obtained from the Global Health Security Index. We used data corresponding to the dimension "rapidly responding to and mitigating the spread of an epidemic" which gathers national information on: Emergency preparedness and response planning; exercising response plans; emergency response operation; linking public health and security authorities; risk communication; access to communications infrastructure; and trade and travel restrictions. To facilitate interpretation of this index we transformed values to z-scores.
Globalization index. This a measure of globalization and global influence, considering social, political and economic dimensions. We expect regimes with higher levels of social, political, and economic integration in the global system to be more exposed to WHO recommendations and thus more rapidly to report a first case of COVID-19 and implement the closure of schools. These data have been obtained from Gygli. To facilitate interpretation of this index we transformed values to z-scores.
Gross domestic product per capita. We employ a measure of gross domestic product (GDP) per capita (purchasing power parity for 2000 US$). We log-transformed this variable to avoid influence of outliers because of the skewed distribution. We expect this variable to increase the speed of adoption of reporting the first case since early detection will help them to determine more rapidly what course of actions to follow. We also expect this variable to delay the implementation of school closure since this measure can affect the functioning of the economy by reducing mobilization of their citizens and thus affecting productivity. On the other hand, a poorer nation-state, which lacks financial means, advanced technological measures to accelerate testing, or efficient health systems, may turn to wide school closure simply because this is its most available policy. These data have been obtained from the World Bank.
Population size. Countries with larger populations may be more concerned on how to avoid an outbreak crisis in severely impacting the health system, and thus more rapidly testing and finding the presence of the virus in the population. On the other hand, larger populations may require higher levels of internal coordination to effectively close schools and therefore may delay the implementation of this measure. These data have been obtained from the WB.
Democracy index. We adopt a measure of democracy, which identifies nations along a scale ranging from 0 ('strongly autocratic') to 100 ('strongly democratic'). The Democracy Index is based on five categories: electoral process and pluralism; civil liberties; the functioning of government; political participation; and political culture. Regimes that enjoy higher levels of democracy may delay the implementation of school closing since this measure contradicts core values and beliefs associated with respecting personal liberties. These data have been obtained from the Economic Intelligence Unit database for the year 2019. To facilitate interpretation of this index we transformed values to z-scores.
Economic integration to China. This is a measure of how much integrated a country is to China's exports. We measured the total of all products exported value to each country from China divided by GDP. The larger the proportion of this value, the more integrated to China's commerce a country is. This is a proxy to measure economic integration. We chose this country since the first world case of COVID-19 was thereby reported. Countries more integrated with China are expected to both increase the speed of reporting the first case of COVID-19 as well as introducing school closure to avoid the spreading of the virus. These data have been obtained from United Nations Comtrade database.
Economic integration to Republic of Korea. This is a measure of how much integrated a country is to Republic of Korea's exports. We measured the total of all products exported value to each country from Republic of Korea divided by GDP. The larger the proportion of this value, the more integrated to Republic of Korea's commerce a country is. We chose this country since it was the second one in reporting a severe outbreak of COVID-19. Countries more integrated with Republic of Korea are expected to both increase the speed of reporting the first case of COVID-19 as well as introducing school closure to avoid the spreading of the virus. These data have been obtained from United Nations Comtrade database.
Economic integration to Italy. This is a measure of how much integrated a country is to Italy's exports. We measured the total of all products exported value to each country from Italy divided by GDP. The larger the proportion of this value, the more integrated to Italy's commerce a country is. We chose Italy since this was the most shocked country in reporting high levels of lethality at the time WHO declared a global emergency. Countries more integrated with Italy are expected to both increase the speed of reporting the first case of COVID-19 as well as introducing school closure to avoid the spreading of the virus. These data have been obtained from United Nations Comtrade database.
For each determinant we used the last year in which countries reported the respective information.
# Iii) methods
To obtain valid estimates to examine policy adoption, we employ survival analysis. This method allows explaining events occurring to countries over a specified period. Survival analysis has been used for various types of events ranging from decolonizationto policy adoption. We particularly use the Weibull hazard function since its ρ value can be used to interpret whether policy adoption significantly increases during the observed period. The Weibull function (h 0 (t)) is specified as h 0 (t) = ρ � t ρ −1 . If ρ is less than 1, the speed of policy adoption (i.e. hazard of failure) decreases with time, while if it is greater than 1, the speed of the policy adoption increases with time. We hypothesize that, if the thesis of convergence is supported, the ρ value will be greater than 1, because it would run counter to the heterogeneity bias. In reporting the results we call this shape parameter "speed,"as its sign and magnitude provide information on whether baseline adoption increases or slows during the observed period. For the thesis of convergence to be supported by the results, the parameter ρ should increase significantly, because it would run counter to the heterogeneity bias. However, a lower parameter ρ can be the product of high-hazard countries, which leave behind the group of low-hazard cases leading to the suggestion that the overall parameter has declined with time. If the convergence process was a response to a national stimulus, with those countries most predisposed to reporting or adopting first, then the parameter would not increase as the first adopters were censored. If instead an ongoing global diffusion process is boosting the adoption of the two events, a significant increase in the parameter of the models should be observed.
It is important to notice that since outcomes could be a result of modeling countries as if they had been equally or not exposed to the same time risk, we defined three different onsets of risk: i) December 31 st , 2019, when China alerted WHO's authorities to a cluster of pneumonia in Wuhan; ii) January 31 st , 2020, when WHO declared COVID-19 to be a global health emergency; and iii) the first case detected in each country. The first two were used to assess when the first case of COVID-19 was reported per country, and each onset of risk were used to predict school closure. Information to determine the two first onsets were derived from WHO's press conferences.
Since unobserved heterogeneity could also arise from information that countries share due to their regional closeness, implying that unobserved processes could bias the results of the parameters, we adjusted the precision of the estimates for their adoption rates in reference to 22 regional clusters based on the United Nations geoscheme(S2 File has the regional cluster list with the countries). In other words, each regional cluster was assigned a random effect-whose distribution does not depend on the observed variables-to model the potential impact of information exchange among countries within each cluster.
When needed, differences in the association of parameters were tested by comparing the value of d/SEd to the standard normal distribution, where d is the difference between the two estimates, and 〖SE〗 _d = p (〖SE〗 _1^2+〖SE〗 _2^2) is the standard error of the difference.
We carried out several sensitivity analyses to (1) indirectly assess whether the results were robust to model specification and (2) using alternative distributions (exponential, and Gompertz models) (Tables S3.1 and S3.2 in S3 File). We also carried out sensitivity analysis with countries in which national decisions were not taken but had begun a process by closing school in states or provinces. In this case we used the date in which the last state or province had close schools.1 in S4 File). We also use linear regression and negative binomial models assuming that countries were independent of each other at the time of closing schools (Tables S5.1 and S5.2 in S5 File). We used Stata/SE 14.0 for all the analyses (codes available in S6 File).
# Results
## Global viralization of the covid-19 and school closures
Since December 31 st 2019, up until June 1 st 2020, we tracked 194 member states of United Nations, and successfully complied information for both events for 186 countries. This sample corresponds to 99,65% of the world population. In this period, 172 countries had reported the presence of COVID-19 in their territories, and 165 closed their schools at the national level.the cumulative distribution of both events. In that period Australia, Russia, Seychelles, and United States closed schools on a regional or state basis rather than nationally, and Botswana El Salvador, Guinea-Bissau, Kyrgyzstan, Lao People's Democratic Republic, Libya, Malawi, Montenegro, Myanmar, San Marino, Saint Kitts and Nevis, Sao Tome and Principe, Syrian Arab Republic and Yemen close schools before the first case was reported. Intime. For the first onset, countries are 6 times more likely to report first cases after ten days, than after 7 days ((10/7) 5.85-1 ). Whereas after the WHO declared the global emergency, countries were more than 28 times more likely to report cases (10/7) 10.34-1 ). In other words, the speed of reporting first cases is faster after the WHO declared a global emergency. In (S8.1 we observe the distribution of both structural parameters from day 1 to day 14, with 7 days as the base to compare. In terms of whether national characteristics explain a more rapid response to report, we observe that health systems designed to respond and mitigate the spread of an epidemic, GDP per capita and population significantly increased the hazard ratio of this event in both models. After the WHO declared a global emergency, we observe that one standard deviation increase in the epidemic security index, and a 1-log increase in GDP per capita and population were more likely to increase the rate of report first cases by 37% (Hazard Ratio (HR) 1.37 (95% CI: 1.00,1.02)) 74% (HR 1.74 (95% CI:1.29, 2.35)) and 34% (HR 1.34 (95% CI:1.19, 1.52)). Regarding the other three variables only economic integration to Republic of Korea suggests a more rapid response to detect the first case before the WHO declared a global emergency. More specifically, a country is 1% (HR: 1.00 (95%CI: 1.00, 1.00)) more likely to identify a first case the more commercially integrated to Republic of Korea is.
## Global spreading of first reported cases and national characteristics
## Global spreading of school closures and national characteristics
In, we report results regarding school closures as policies adopted to curb down the pandemic at the national level. The structural parameter speed of adoption (ρ) captures significant increases of 11.48 (95% CI: 9.67, 13.62), and 8.52 (95%CI: 6.64, 10.94), indicating that regardless of the onset chosen the speed of closing schools has progressively augmented (as. However, opting for different onset of risk changes the global speed of adoption of this policy. Ten days after the WHO declares a global health emergency, countries are 42 times more likely to close schools than after 7 days ((10/7) 11.48-1 ), whereas after each country reported its first case, countries were 15 more times likely close schools ((10/7) 8.52-1 ). In (S8.2. Weibull models predicting first reported case of COVID-19.
## Outcome
First reported case of COVID-19 we observe the distribution of both structural parameters from day 1 to day 14, with 7 days as the base to compare. In terms of national characteristics, and regardless of the model, we observe a significant decreased in hazard ratios of relations to the epidemic security index, and concomitant increases in the globalization index. After each country reported a first case, for a one standard deviation increase in the epidemic security index, delays of school closures by 36% (HR 0.64 95% CI: 0.51, 0.80) in standard deviation units are observed. Whereas, for a one standard deviation increase in the globalization index, accelerations of school closures by 74% (HR 1.74 95% CI: 1.34, 2.24) in standard deviation units are noticed. We also observe that, after the WHO declared the international emergency, countries more commercially integrated to Italy were 1% (HR 1.00 (95%CI: 1.00, 1.00) more rapid to adopt this measure. GDP per capita, population, democracy and economic integration to Republic of Korea were not associated with time to the adoption of school closure. Sensitivity analyses showed that results were robust to model specification, and alternative distributions (including countries in which a national decision was not yet taken using the date in which the last state or province implemented school closure (S3.1, S3.
## Onset december 31st, 2019-china reports to who's authorities the epidemic in wuhan
# Discussion
The viralization of COVID-19 and policies to contain it across the world has been swift. At least 172 countries reported the presence of a case after China informed WHO's authorities of the Wuhan's cluster. From the date in which WHO declared a global emergency, January 31 st 2020, up until June 1 st 2020, the average of detecting the first case was slightly more than one month. Once countries detected the first case, it took them in average two weeks to close schools at the national level. In this paper, we advanced two groups of hypotheses to understand the speed of this global phenomenon, and our preliminary findings partially confirmed them. After the global declaration of emergency of the WHO, countries were more rapidly to detect a first COVID-19 case. Further, if countries had well designed health systems to respond to an epidemic, were rich and highly populated were more likely to report a first COVID-19 case in their territories. Countries were also faster in adopting school closure, if they were more globally integrated and had stronger ties to Italy, but slower if their health system was better prepared to contain an epidemic. To sum up an interaction of international with national forces is likely to explain the speed of different layers associated with this phenomenon.
We are witnessing indeed an unprecedent case of global convergence. Previous studies of policy adoption which focused on periods of more than 30 years, using country-year as unit of analysis, have detected increases in the shape speed of adoption parameter ρ above 2 when measuring decision to implement privatization programsor road safety polices. In this study, using country-day as unit of analysis, the most conservative parameter ρ is higher than 5, suggesting a unique case of rapid global convergence. An important finding in this regard is that the speed of detection the first case of COVID-19 was much higher after the global emergency declaration than when China notified to WHO's authorities. After the global emergency declaration countries were significantly more rapid to detect the first case of COVID-19. This suggests that after the WHO scaled its global response, countries were more likely to report the presence of positive cases, and in better condition to initiate the implementation of health and related policies to contain the outbreak. Hence the importance of international organizations with high credibility in times of high uncertainty.
Our results indicate that those with better prepared health systems, richer and more populated were associated with higher hazard rates to inform the presence of first cases. These findings could be signaling more capacity to test and increases in the probability that citizens from these countries were returning from COVID-19 risk zones. To detect whether more populated countries for instance responded more rapidly due to concerns of observing outbreaks, more information is needed to determine how countries were targeting the identification of these cases. Nevertheless, to assess the robustness of the variable Epidemic security index, we also used a variable from the same global index 'early detection and reporting epidemics of potential international concern,' (which systematizes information regarding the quality and presence of laboratory systems, real time surveillance and reporting systems, epidemiology workforce, and data integration between human/animal/environmental health sectors), and results were robust in direction and magnitude when compared to how well prepared the health systems were when targeting an epidemic (results available in S7.1 Table in S7 File).
Our second group of hypotheses of why countries may adopt more rapidly or delay the policy of school closure identified again the importance of global and national factors. First, the presence of well-prepared health systems designed to tackle potential outbreaks was associated with delays in implementing school closures. This could be associated with a better health management and understanding of when the introduction of more stringent measures is necessary. This is particularly salient at the time that knowledge regarding population transmission characteristics was still developing. Second, how well connected the country is to the global system, measured with the globalization index, increases the speed of implementing this policy. A possible explanation of this finding is that more open countries are likely to receive faster information of what measures should be set in place in a global emergency. Further, countries with higher level of integration to the global system are more exposed to influences of international organizations, in which the role of WHO, in a global health crisis can be indeed very relevant. It is important to note however that this policy was not a top priority in the set of recommendations diffused by the WHO to contain the spread of COVID-19 . Indeed the WHO's first report regarding China, only recommended the possibility of considering closing schools for countries in which imported cases of COVID-19 had been reported, but only after some simulations were carried out. This recommendation could have been taken by countries which reported the first case, as a positive signal to proceed, even if countries did not have the capacity to develop the recommended simulations. In reference to our finding of integration to Italy, the rapid adoption of school closure of these countries could be explained by the constant information received of the progression of the outbreak in which school closure was one of the measures taken. This resembles a process of policy diffusion whereby policies are triggered by the events happening in other closely related country. As it has been pointed out, this particular process of diffusion emerges when high levels of uncertainty are present. In short, countries with closer economic ties to Italy were more rapid to adopt this measure since this could help them anticipating more successfully how to tackle a likely outbreak. Lastly in terms of economic and political capacities, we observed that none of the variables representing these dimensions at the national level were adequate to predict how fast or slow countries implemented school closures. This ultimately reinforces the importance of global forces and considering adequate health national variables when assessing which responses were applied.
While this study has limitations it also opened new questions to better understand this phenomenon. First, since we only conceptualize countries which nationally determined the closures of schools as adoption cases, other analyses are needed to understand patterns of countries where closure of schools was decided at a subnational level, like United States or the Russian Federation. However, to check for the robustness of our results, we also carry out analyses in which we used the last date in which a state or province closed schools in countries where a national decision was not reached, and results were consistent (see analysis in S4 File). Second, while our preliminary analyses showed specific patterns of globalization to examine some aspects of the implication of COVID-19, we should emphasize the need for greater precision and granularity when examining more critically the processes of policy diffusion of school closures and first case reporting at the global level. Future analyses should attempt to better capture how actors, who transit international and nation social networks, debated, accepted and in some cases rejected the implementation of school closure within the context of other recommended policies but also more controversial ones such as full lockdowns. While findings of the current study highlight the great importance of time in the diffusion of policies, particularly in the context of a pandemic, these results call for a larger expansion of the way we understand the actions of political and economic actors and scientists, in national and international arenas. While the rapid detection of first cases may have triggered national efforts to avoid outbreaks, it is very much unclear the extent under which the rapid or slower adoption of school closure, and other policies, will have an impact in reducing both the spread and lethality of COVID-19. A more sophisticated analysis at the end of this pandemic, with inclusion of data on the effects of school closures and other implemented interventions will inform future policies about timing of implementation of such policies and their efficacy.
## Supporting information
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Penile cancer: about ten cases at the University Hospital of Rabat, review of the literature
The aim of our study was to report the status of penile cancer sites in the urology department at the University Hospital of Rabat and evaluate long-term results of surgical treatment of this cancer. Patients and Methods: Between 1989 and 2015, 10 patients were treated for penile cancer.10 cases were retrospectively reviewed and the following data were recorded: mode of revelation, seat, staging, TNM stage, treatment, evolution and survival. The mean age of patients was 58,1 years (48-81 years). All patients had squamous cell carcinoma of the penis. Six patients had a partial amputation of the penis, and three patients underwent total amputation. The median size of the lesion was 4.25 cm (1.5-8 cm). All tumors had a distal seat (gland-Furrow balanopreputial), 8 were localized and non-invasive (PT1 -PT2) and 2 had infiltrated the urethra (PT3). Four patients had lymph node localization. A single bilateral lymphadenectomy was performed and was positive only on one side, with a node <3 cm and no extracapsular extension. Two patients were referred for chemotherapy, a neoadjuvant referred to basic (Bleomycin -Methotrexate, Cisplatin) the other in a palliative goal. Median follow-up was 42 months (6 -72mois). Four patients died, one of which was presented immediately with metastatic mode. Six patients were alive at last node or local recurrence negative. Cancer of the penis seems rare in Morocco. His oncologic and functional outcomes (sexual and urinary) depend on the precocity of the treatment. The surgery of lymph node resection with lymphadenectomy remains the reference treatment.
# Introduction
Tumors of the penis are the rarest tumors of the genitourinary system, it represents 0.5% of malignant tumors of man [bib_ref] Penile tumors (Tumeurs du penis), Mottet [/bib_ref]. Penile carcinoma is mostly a squamous cell carcinoma (SCC) but other types of carcinoma exist as well [bib_ref] Basaloid squamous cell carcinoma of the penis with papillary features: a clinicopathologic..., Cubilla [/bib_ref]. The incidence of penile cancer increases with age [bib_ref] Epidemiologic profile, sexual history, pathologic features, and human papillomavirus status of 103..., Chaux [/bib_ref] , with an age peak during the sixth decade of life. However, the disease does occur in younger men.
Several risk factors for penile cancer have been identified by a review of the literature published since 1966.One of the most important risk factor for developing penile canceris the Human papilloma virus infection. It seems that neonatal circumcision reduces the incidence of penile cancer in countries and cultures where this is routinely practiced. Squamous cell carcinoma can have several clinical aspects: ulcerating or budding, localized or invading the whole structure of the penis.
To appreciate the evolution and expansion of these tumors, several diagnostic tests have been proposed: Ultrasound, CT or MRI and more recently FDG-positron emission tomography. There are a variety of treatment modalities for the penile cancer and are always adapted to TNM stage, tumor grade. The prognosis is pejorative: 80% 5-year survival for N0 and N + 50%.
# Methods
This is a retrospective study interesting ten patients. The following data were recorded: history, diagnosis, mode of revelation, the seat height, balance sheet preparation extension stage (TNM 2009), rank, the margin of resection and therapeutic attitude tick. The patients were seen every 3 months during the first year and then every 6 mounth in the absence of oncological recurrence.
# Results
Patient characteristics were detailed in [fig_ref] Table 1: main characteristics of patients [/fig_ref]. The mean age at diagnosis was 58,1 years (46-81 years). The mode of revelation in all patients was the presence of a macroscopic lesion , the median tumor size was 5,1cm (1.5-8 cm). All tumors had a distal seat (gland-Furrow balano-preputial), 8 were located and non-invasive (pT1 -pT2) and 2 infiltrated the urethra (pT3) . Three patients had lymph node localization. One of the patients had T3 emblem of inguinal lymph node metastases and liver metastases. Tumor grade was always ? 2. Nine patients underwent surgical treatment for early amputation of the penis or partial penectomy , [fig_ref] Figure 4: Resected specimen [/fig_ref] , [fig_ref] Figure 5: Perineal urethrostomy [/fig_ref]. Two patients were referred for chemotherapy, a neo referred based adjuvants (Bleomycin -Methotrexate, Cisplatin) the other in a palliative goal. Median follow-up was 46 months (6 -72mois) 4 patients died [fig_ref] Table 1: main characteristics of patients [/fig_ref].
# Discussion
The average age of cancer diagnosis in our study (58,1 years) is consistent with the literature: incidence maximum occurs after 50 years [bib_ref] Epidemiologic profile, sexual history, pathologic features, and human papillomavirus status of 103..., Chaux [/bib_ref]. The location was most often distal (prepuce and glans) according to what is described. One of the most important risk factor for developing penile cancer is the Human papilloma virus infection. It has been identified in 70-100% of intraepithelial neoplasia and in 30-40% of invasive penile cancer tissue samples [bib_ref] HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of..., Stankiewicz [/bib_ref] HPV subtypes most commonly found in penile cancer are types 16 and 18 [bib_ref] HPV in the etiology of human cancer, Muñoz [/bib_ref].
Penile carcinoma is most frequent in uncircumcised males than in those circumcised in infancy, for example: Non-Jewish versus Jewish population in the West and non-Muslim versus Muslim population in India [bib_ref] Discriptiveepiderniologhy of the cancers of male genital organs in greater, Yeok [/bib_ref]. Circumcision at birth however, does not always protect an individual from developing penile cancer.Also circumcision beyond infancy is also not protective against the development of penile cancer [bib_ref] Epidennological and clinical aspects of carcinoma of penis at kenyatta national hospital, Mangoha [/bib_ref]. An other risk factor of the development of invasive penile cancer is the Phimosis [bib_ref] Epidemiological aspects of penile cancer in Rio de Janeiro: evaluation of 230..., Koifman [/bib_ref] , probably due to associated chronic infection. A further risk factor suggested by epidemiological studies is cigarette smoking, which is associated with a 4.5-fold increased risk (95%) [bib_ref] Penile cancer: importance of circumcision, human papillomavirus and smoking in in situ..., Daling [/bib_ref].
Low socio-economic status and a low level of education are other epidemiological factors associated with penile cancer. In our study, only two cases of HPV have been diagnosed on biopsy by the presence of malpigiennes cells infected with the virus who take an aspect quite pathognomonic called koilocyte (large cells with vacuolated cytoplasm with enlarged nuclei and hyperplastic sometimes multinucleated).
Squamous cell carcinoma accounts for more than 95% of cases of malignant diseases of the penis. Other malignant lesions of the penis unrelated to penile SCC are melanocytic lesions, mesenchymaltumours, lymphomas and secondary tumours, i.e. metastases. These are all much less common than penile SCC. Aggressive sarcoma of different types occurring in the penis have been reported.
In our study assessing the local expansion of the penis was based on the penis examination and ultrasound. Ultrasound can give information about infiltration of the corpora [bib_ref] Primary and secondary malignancies of the penis: ultrasound features, Bertolotto [/bib_ref]. Magnetic resonance imaging (MRI) in combination with an artificial erection with prostaglandin E 1 can also be used for excluding tumour invasion of the corpora cavernosa if organ-preservation is planned and preoperative decisions are needed [bib_ref] The role of magnetic resonance imaging in the local staging of penile..., Kayes [/bib_ref].
All patients had a deep biopsy for histological confirmation and appreciation of the depth of infiltration. The areas of palpation for lymph node assessmentshould be systematic and bilateral. If lymphadenopathy palpated, we should do a needle aspiration cytology of (the) node (s), associated with ultrasound guidance. She increased the micrometastases detection rates in the sentinel lymph node biopsy but few expert center used in practice. When the clinical examination is difficult (obese patients) or if clinical inguinal lymph node involvement (evaluation of pelvic areas) Inguinal and pelvic CT scan isrecommended. It is important to notice that there is no pelvic lymph node involvement that has been demonstrated without inguinal lymph node involvement [bib_ref] Lymph node metastasis in intermediate-risk penile squa-mous cell cancer: a two-centre experience, Leijte [/bib_ref].
The imagery is not essential for normal inguinal lymphadenopathy area without palpated. All patients had at least one abdominopelvic CT scan to define the nodal status in the most comprehensive way possible, but one for which an MRI was performed. MRI has proven superior to CT in the detection of inguinal lymph nodes. The sentinel node was most effective in patients CN0 that cN +. It is not recommended in patients with a palpable lymphadenopathy. The nodal risk is material and requires care in case of penile tumor ? pT1bG2 or when palpated adenopathy .
An assessment of distant metastases should be performed in patients with positive inguinal nodes. There is no established tumour marker for penile cancer. Regarding surgical treatment, six patients with pT1 N0 M0 tumor site in the glans had a partial penectomy, the other three pT2 N2 M0, pT2 N1 M0, pT3 N0 M0 have experienced a complete amputation with negative margins. The last one had a palliative chemo. So following the recommendations of CCAFU, patients with infiltrating lesions ? PT2 had surgery by partial or total penectomy. In case of partial amputation, the length of the remaining penis must be at least 3cm.
In clinically lymph-node positive patients, surgical staging by inguinal lymphadenectomy is indicated. Radical inguinal lymphadenectomy carries a significant morbidity related to problems of lymph drainage from the legs and wound healing. While morbidity can be as high as 50% [bib_ref] Radical open inguinal lymphadenectomy for penile carcinoma: surgical technique, early complications and..., Koifman [/bib_ref]. Wound infections (1.2-1.4%), skin necrosis (0.6-4.7%) lymphedema (5-13.9%) and lymphocele formation (2.1-4%) were the most commonly reported complications in recent series. Therapeutic radical inguinal lymphadenectomy can be life-saving but it may be underused for fear of associated morbidity [bib_ref] PubMed | Google Scholar 17. Catalona WJ. Modified inguinal lymphadenectomy for carcinoma..., Lughezzani [/bib_ref]. The superficial inguinal lymphadenectomy modified involves ablation of superficial inguinal nodes located medial to the great saphenous vein . Morbidity is low at about 6.8% of early complications and 3.4% of late complications [bib_ref] Morbidity of inguinal lymphadenectomy for invasive penile carcinoma, Bouchot [/bib_ref]. If a modified inguinal lymphadenectomy is performed and confirmed lymph node involvement (or extemporaneous final review), it should always be taken for totalization (lymphadenectomy).
In our study, we performed a single bilateral inguinal lymph node dissection, the dissection was negative right and left we found a single node metastases <3 cm without capsular which did not require a pelvic lymphadenectomy ipsilateral metastasis is recommended if there are more than two nodes or metastasized to a lymph node capsular.
Cancer of the penis is a little chemo-sensitive tumor chemotherapy and no product has the marketing authorization (MA) for use in cancer of the penis. Overall, the results of neoadjuvant or adjuvant chemotherapy does not suggest the interest on survival and local control, given the small number of cases in the series and the absence of randomization.
Two of our patients were referred for chemotherapy, one (pT2N2M0) to neoadjuvant referred to basic (Bleomycin -Methotrexate, Cisplatin) in the general condition does not allow to make a inguinal lymphadenectomy, the other (pT3N3M + ) in a palliative goal.There is no consensus in the manner and frequency of monitoring. The follow-up interval and strategies for patients with penile cancer are directed by the initial treatment of the primary lesion and regional lymph nodes. In a multicentre study, during the first two years 74.3% of all recurrences, 66.4% of local recurrences, 86.1% of regional recurrences and 100% of distant recurrences were detected. In the same study, 92.2% of all recurrences occurred within the first 5 years and all recurrences seen after 5 years were local recurrences or new primary lesions. Therefore, an intensive follow-up regimen during the first 2 years is rational, with less intensive follow-up needed thereafter for a minimum of 5 years. Generally, follow-up should continue thereafter but may be omitted in well-educated and motivated patients who reliably continue to carry out regular self-examination [bib_ref] Recurrence patterns of squamous cell carcinoma of the penis: recommendations for follow-up..., Leijte [/bib_ref].
Our patients had clinical monitoring penile and lymph nodes, quarterly in the first year and then every six months in the absence of oncological recurrence.
# Conclusion
The low incidence of penile tumors in the general population remains a real obstacle to the publication of consistent series of patients likely to generate well codified therapeutic management. Human papilloma virus infection is an important risk factor for developing penile cancer. Neonatal circumcision reduces the incidence of penile cancer. The treatment of cancers of the penis is usually surgical roughly in combination with chemotherapy in the case of lymph node. The main prognostic factor is lymph node involvement justifying appropriate care from diagnosis. [fig_ref] Table 1: main characteristics of patients [/fig_ref] : Main characteristics of patients : Cauliflower appearance of the tumor : Infiltration of the urethra (HEx100) : Appearance after total penectomy
# Table and figures
[fig] Figure 4, Figure 7: Resected specimenFigure 5: Perineal urethrostomy Figure 6: Dyskeratotic cells with tumor proliferation (HEx400) Cutaneous coatings adjacent to the tumor showing parakeratosis. Koilocytespresence (HEx200) [/fig]
[fig] Figure 1, Figure 2, Figure 3: Cauliflower appearance of the tumor Infiltration of the urethra (HEx100) Appearance after total penectomy [/fig]
[fig] Figure 4: Resected specimen [/fig]
[fig] Figure 5: Perineal urethrostomy [/fig]
[fig] Figure 6, Figure 7: Dyskeratotic cells with tumor proliferation (HEx400) Cutaneous coatings adjacent to the tumor showing parakeratosis. Koilocytespresence (HEx200) [/fig]
[table] Table 1: main characteristics of patients [/table]
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A Randomized Trial of Low-Dose Aspirin in the Prevention of Clinical Type 2 Diabetes in Women
OBJECTIVE -Subclinical inflammation is linked with the development of type 2 diabetes, and epidemiologic data suggest that this association may be stronger in women. Although small clinical studies have shown a prominent hypoglycemic effect of short-term high-dose aspirin, no randomized trials have directly evaluated the efficacy of aspirin in diabetes prevention at doses acceptable for use in routine clinical practice. We evaluated whether chronic low-dose aspirin prevents the development of clinical diabetes among initially healthy American women.RESEARCH DESIGN AND METHODS -Subjects were enrolled in the Women'sHealth Study, a 10-year randomized double-blind, placebo-controlled trial of aspirin and vitamin E for primary prevention of cardiovascular disease and cancer. Between 1992 and 1995, 38,716 women aged Ն45 years and free of clinical diabetes were randomly assigned to either low-dose aspirin or placebo (median follow-up 10.2 years). Documented clinical type 2 diabetes was prospectively evaluated throughout the trial.RESULTS -Among women randomly assigned to receive aspirin (n ϭ 19,326) or placebo (n ϭ 19,390), there was no statistically significant difference in the incidence of type 2 diabetes. There were 849 cases of diabetes in the aspirin group and 847 in the placebo group (rate ratio 1.01 [95% CI 0.91-1.11]). Stratification by diabetes risk factors including age, BMI, family history of diabetes, physical activity, A1C, and high-sensitivity C-reactive protein did not support a modulating effect of these variables. Analyses accounting for treatment duration and adherence similarly found no beneficial effects.CONCLUSIONS -These data suggest that long-term low-dose aspirin does not prevent the development of clinical type 2 diabetes in initially healthy women. Diabetes Care 32:3
T he ability of salicylates, such as aspirin, to reduce glucose levels was described Ͼ125 years ago. This effect was largely forgotten until the emergence of recent data linking inflammation with the development of type 2 diabetes. A wealth of experimental and epidemiological evidence now indicates that insulin resistance and type 2 diabetes are, in part, obesity-linked inflammatory disorders [bib_ref] Inflammation and insulin resistance, Shoelson [/bib_ref] , and the presence of subclinical inflammation is now known to be a potent indicator of risk for this disease. This relationship may be of particular importance in the pathogenesis of diabetes in women among whom obesity-triggered inflammation may be heightened compared with that in men [bib_ref] Sex differences in the prediction of type 2 diabetes by inflammatory markers:..., Thorand [/bib_ref]. These findings have spurred interest in the use of antiinflammatory drugs in diabetes prevention and treatment [bib_ref] Salsalate improves glycemia and inflammatory parameters in obese young adults, Fleischman [/bib_ref]. However, data pertaining to this novel approach are sparse with no large-scale randomized studies available to date.
Aspirin is an anti-inflammatory agent with pleiotropic actions, many of which remain poorly understood. The cellular and molecular mechanisms of the hypoglycemic response to aspirin are an area of active investigation but likely involve anti-inflammatory pathways distinct from effects on prostaglandin synthesis [bib_ref] Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis..., Gao [/bib_ref]. Several small clinical studies [bib_ref] Aspirin stimulates insulin and glucagon secretion and increases glucose tolerance in normal..., Micossi [/bib_ref] [bib_ref] The effect of acetylsalicylic acid on plasma glucose and the response of..., Prince [/bib_ref] [bib_ref] Mechanism by which highdose aspirin improves glucose metabolism in type 2 diabetes, Hundal [/bib_ref] have reported that short-term high-dose aspirin (3-10 g/day for 3 days-3 weeks) improves glucose handling and may ameliorate insulin resistance in diabetic patients, albeit with a high rate of side effects. Although data are not available on the hypoglycemic action of low-dose aspirin, several short-term clinical trials demonstrated that aspirin triggers the production of anti-inflammatory mediators [bib_ref] Aspirin triggers antiinflammatory 15-epi-lipoxin A4 and inhibits thromboxane in a randomized human..., Chiang [/bib_ref] and lowers systemic levels of inflammatory biomarkers at doses as low as 30 mg/day [bib_ref] Thromboxane-dependent CD40 ligand release in type 2 diabetes mellitus, Santilli [/bib_ref]. Whether chronic lowdose aspirin therapy has favorable clinical effects is unknown.
We assessed whether long-term lowdose aspirin therapy reduces the incidence of clinical type 2 diabetes in the randomized treatment arms of the Women's Health Study (WHS). The WHS tested the efficacy of low-dose aspirin in the primary prevention of cardiovascular disease and cancer over a 10-year period in a large group of initially healthy women. The occurrence of clinical diabetes was ascertained prospectively throughout the trial.
RESEARCH DESIGN AND METHODS -The WHS was a 2 ϫ 2 factorial trial evaluating the balance of risks and benefits of low-dose aspirin (100 mg every other day; Bayer Healthcare) and vitamin E (600 IU ␣-tocopherol every other day; Natural Source Vitamin E Association) in the primary prevention of cardiovascular disease and cancer [bib_ref] A randomized trial of low-dose aspirin in the primary prevention of cardiovascular..., Ridker [/bib_ref] [bib_ref] Low-dose aspirin in the primary prevention of cancer: the Women's Health Study:..., Cook [/bib_ref] [bib_ref] Vitamin E in the primary prevention of cardiovascular disease and cancer: the..., Lee [/bib_ref]. The dose of 100 mg every other day was chosen to be the lowest dose that would have a cardioprotective effect while minimizing gastrointestinal side effects. Although not a prespecified primary end point of the WHS, clinical diabetes was a key outcome of interest and prospectively ascertained throughout the duration of the trial.
Written informed consent was obtained from all women. The study was approved by the institutional review board of the Brigham and Women's Hospital and monitored by an external data and safety monitoring board.
## Setting and participants
Details of the study design have previously been described [bib_ref] A randomized trial of low-dose aspirin in the primary prevention of cardiovascular..., Ridker [/bib_ref] [bib_ref] Baseline characteristics of participants in the Women's Health Study, Rexrode [/bib_ref]. Women were eligible if they were at least 45 years of age without a previous history of cancer (except nonmelanoma skin cancer), cardiovascular disease, or other major chronic illness; had no history of adverse effects from aspirin; were not taking aspirin or nonsteroidal anti-inflammatory drugs (or were willing to forgo their use during the trial); and were not taking anticoagulants or individual supplements of vitamin A, E, or -carotene more than once a week. A total of 39,876 women were willing, eligible, and compliant during a 3-month placebo run-in period and underwent random assignment: 19,934 were assigned to receive aspirin and 19,942 to receive placebo. In the present analyses, we excluded women with reported physician-diagnosed diabetes at baseline (n ϭ 1,160), leaving a total of 38,716 women free of clinical diabetes at entry into the trial; 19,326 were assigned to receive aspirin and 19,390 to receive placebo. Additional details including a Consolidated Standards of Reporting Trials (CONSORT) flow diagram are provided in supplemental data and supplemental [fig_ref] Figure 1 -: Cumulative incidence of type 2 diabetes in the aspirin and placebo groups [/fig_ref] (available in an online appendix at http://dx.doi.org/10. 2337/dc08-1206).
## Randomization and interventions
With use of a computer-generated table of random numbers, treatment assignments were made within seven age-groups (45-49, 50 -54, 55-59, 60 -64, 65-69, 70 -74, and Ն75 years) using block sizes of 16. Random assignment took place from 30 April 1993 through 24 January 1996. Each year, women received calendar packs that contained amber capsules (vitamin E or placebo) and white pills (aspirin or placebo) on alternate days. Every 6 months for the first year and annually thereafter, they also received follow-up questionnaires inquiring about compliance with pill-taking, potential adverse effects, occurrence of end points, and risk factors. Study medications were continued in blinded fashion through the scheduled end of the trial (31 March 2004).
## Compliance
On the basis of self-reported adherence, compliance, defined as taking at least two-thirds of their aspirin or matching placebo tablets, was 76.1% at 5 years and 67.0% at 10 years. Averaged throughout the trial, compliance was slightly higher in the placebo (73.7%) group than in the active (72.5%) group (P ϭ 0.004). Nontrial use of aspirin or aspirin-containing products on Ն4 days/month ("drop-ins") was 11.6% at 5 years and 19.2% at 10 years. Averaged throughout the trial, it was somewhat higher in the placebo (13.0%) group than in the active (12.7%) group (P ϭ 0.10).
## Outcomes and follow-up
Details regarding the ascertainment of incident diabetes in the WHS have been reported previously [bib_ref] Vitamin E and risk of type 2 diabetes in the Women's Health..., Liu [/bib_ref]. Information on newly diagnosed diabetes was collected on every follow-up questionnaire from baseline through the end of the trial. All participants were asked annually "In the past year, were you newly diagnosed with diabetes mellitus?" Subjects also provided the month and year of diagnosis. Confirmation of diabetes was conducted in a blinded fashion using American Diabetes Association diagnostic criteria (18). Selfreported cases were investigated by either telephone interview conducted by a physician or a previously validated selfadministered supplemental questionnaire that inquired about symptoms, diagnostic testing, and use of diabetes medications. The response rate was high with Ͼ90% of women who reported incident diabetes responding to either the telephone interview or supplemental questionnaire. On the basis of responses to the supplemental questionnaire, 77.2% of those with confirmed cases reported use of antidiabetic agents. Only women with confirmed cases were analyzed in this report. Because the vast majority of diabetes diagnosed at age Յ45 years is of the type 2 variant, incident diabetes in the WHS is considered to be type 2 diabetes.
Glucose screening rates were assessed during follow-up. When asked about screening for diabetes on the 9-year questionnaire, 71.8 and 68.2% of nondiabetic women reported having a fasting glucose test performed within the preceding 5 and 3 years, respectively. Screening rates were equivalent between the two treatment arms: 68.3 versus 68.1% in the preceding 3 years in the aspirin and placebo groups, respectively (P ϭ 0.78). These values are similar to contemporaneous diabetes screening rates; among patients in a U.S. managed care population, the occurrence of any glucose testing (random or fasting) over a 3-year period was 71.5% for women Ն45 years [bib_ref] Opportunistic screening for diabetes in routine clinical practice, Ealovega [/bib_ref].
# Statistical analysis
All primary analyses were performed on an intention-to-treat basis. We used Cox proportional hazards models to estimate the rate ratio (RR) and 95% CIs, comparing event rates in the aspirin and placebo groups after adjustment for age and other randomized treatment assignments (vitamin E and -carotene, which was a component of the trial for a median of 2.1 years) [bib_ref] Hennekens CH: -Carotene supplementation and incidence of cancer and cardiovascular disease: the..., Lee [/bib_ref]. The proportionality assumption of constant hazards over time was tested using an interaction term of aspirin with the logarithm of time. The divergence of diabetes incidence over time between groups was estimated using Kaplan-Meier survival curves and the logrank test was computed to compare curves.
Exploratory subgroup analyses were conducted to examine the effect of aspirin according to the baseline presence of major risk factors for type 2 diabetes including age-group, BMI group, family history of diabetes in a first-degree relative, physical activity, menopausal status, and hormone therapy. Categories are specified in [fig_ref] Table 1 -: Baseline characteristics of the study population [/fig_ref]. Among women providing baseline blood specimens (n ϭ 27,167), subgroup analyses were performed after stratification by levels of total cholesterol, LDL cholesterol, HDL cholesterol, the total cholesterol-to-HDL cholesterol ratio, non-HDL cholesterol, A1C, and highsensitivity C-reactive protein (hsCRP).
To estimate the effect of treatment duration, we fit two separate proportional hazards models to the experience of the first 5 years and after 5 years of follow-up. To assess the impact of potentially undiagnosed diabetes at baseline, we conducted sensitivity analyses in which women with diabetes diagnosed during the first 2 and 5 years of follow-up were excluded. To examine the effect of actual as opposed to assigned aspirin use, we performed additional analyses in which subjects were censored if and when they stopped tak-
## Aspirin does not lower diabetes incidence
ing at least two-thirds of their study pills. To assess the effect of nontrial aspirin use, we also performed analyses in which censoring occurred when women either stopped taking at least two-thirds of their study pills or reported outside use of aspirin or aspirin-containing products for Ն4 days/month.
All analyses were performed with SAS (version 9.1; SAS Institute, Cary, NC). A two-sided significance level of 0.05 was used.
RESULTS -The aspirin and placebo groups were similar with respect to baseline characteristics [fig_ref] Table 1 -: Baseline characteristics of the study population [/fig_ref]. Among women providing blood specimens, there were also no significant differences in lipid levels, A1C, or hsCRP. The median duration of follow-up was 10.2 years (mean 9.8 years). At completion of the trial, 1,696 cases of confirmed incident clinical type 2 diabetes had occurred. There were 849 cases in the aspirin group and 847 cases in the placebo group, with no significant risk reduction (RR for aspirin versus placebo 1.01 [95% CI 0.91-1.11]).
The cumulative incidence curves according to treatment assignment were similar throughout follow-up [fig_ref] Figure 1 -: Cumulative incidence of type 2 diabetes in the aspirin and placebo groups [/fig_ref] (log-rank P ϭ 0.92). A test of the proportionality hazards assumption showed no deviation from proportionality (P ϭ 0.27). In separate analyses that considered newly diagnosed diabetes in the first 5 years of follow-up versus thereafter, aspirin therapy was associated with an RR of 0.99 (95% CI 0.86 -1.15) during the first 5 years and 1.01 (0.90 -1.15) after 5 years. We found no difference when patients with potentially undiagnosed diabetes documented during the first 2 or 5 years of follow-up were excluded. In these analyses the RRs were 0.99 (0.89 -1.09) and 1.01 (0.90 -1.15), respectively.
There was no evidence that any diabetes risk factors considered modified the effect of aspirin on diabetes incidence ( , P ϭ 0.053), these findings must be interpreted with caution given both the lack of a consistent risk increase across biomarker categories and the large number of subgroups examined. There was no statistically significant interaction for any of the biomarkers assessed (P interaction ϭ 0.39 for total cholesterol, 0.26 for LDL cholesterol, 0.13 for the total cholesterol-to-HDL cholesterol ratio, and Ն0.2 for all others).
Because compliance diminished over time, sensitivity analyses were performed that censored noncompliant women at the time they stopped taking at least twothirds of their study pills during the preceding year. In this analysis, there was also no significant benefit of aspirin (RR 1.02 [95% CI 0.91-1.15]). When women were censored at the time they either stopped taking at least two-thirds of their study pills or started outside aspirin or aspirin-containing medications on Ն4 days/month, findings were similarly nonsignificant. In addition, there was no evidence of effect modification by other randomized treatments. With regard to safety, as expected, there were slightly higher rates of clinically significant bleeding episodes and other side effects in the aspirin arm compared with placebo: 4.5 vs. [bib_ref] Sex differences in the prediction of type 2 diabetes by inflammatory markers:..., Thorand [/bib_ref] CONCLUSIONS -In this largescale, long-term trial of initially healthy women, there was no association of 100 Data are % or median (interquartile range). *Family history of diabetes in a first-degree relative (mother, father, sister, or brother). †Physical activity defined by number of episodes of vigorous physical activity per week.
mg aspirin on alternate days with the overall incidence of clinical type 2 diabetes. Treatment duration, Յ5 years or Ͼ5 years, did not have an impact on our results, and the treatment effect did not vary significantly across subgroups of women at high risk for diabetes due to the presence of clinical risk factors, dyslipidemia, elevated A1C, or hsCRP. There was no difference when women with early cases, presumably undiagnosed at baseline, were excluded or in analyses accounting for adherence to randomized assignment. Aspirin therapy was associated with a higher incidence of clinically important bleeding events. The increasing incidence of diabetes, high treatment costs, and disproportionate impact on cardiovascular disease in women highlight the need to identify prevention strategies applicable on a broad population basis. Preventive measures with potential dual effects on cardiovascular and diabetes risk reduction are particularly appealing because "cardiometabolic" risk factors often coincide in the same individual. The main findings of the Women's Health Study were published previously and demonstrated that longterm treatment with low-dose aspirin, although resulting in no significant benefit or harm on the end point of any first major cardiovascular event, did significantly reduce the risk of stroke overall (RR 0.83 [95% CI 0.69 -0.99]) and myocardial infarction among women aged Ͼ65 years (0.66 [0.44 -0.97]) [bib_ref] A randomized trial of low-dose aspirin in the primary prevention of cardiovascular..., Ridker [/bib_ref]. The current analysis addresses the important question of whether low-dose aspirin has added benefits for diabetes prevention in this large population of otherwise healthy women. This report provides the only randomized data available in this regard.
One of the least appreciated pharmacological actions of aspirin is its ability to lower glucose levels. Attempts to delineate this effect have been made in several small clinical studies. Among both nondiabetic and diabetic patients, short-term high-dose aspirin (3-10 g/day for up to 3 weeks) is consistently associated with higher basal and stimulated insulin concentrations and reduced glucose excursion during glucose tolerance testing . However, whether aspirin has a net beneficial contribution to glucose homeostasis has been controversial, with several studies suggesting that any favorable hypoglycemic action is offset by deterioration in insulin sensitivity [bib_ref] Aspirin causes tissue insensitivity to insulin in normal man, Newman [/bib_ref] [bib_ref] Acetyl-salicylic acid impairs insulin-mediated glucose utilization and reduces insulin clearance in healthy..., Bratusch-Marrain [/bib_ref]. Higher doses of aspirin given over a longer period were more recently found to have broad therapeutic benefits in a detailed study of nine patients with overt type 2 diabetes (10). At the end of 2 weeks, aspirin at a dose of 7 g/day lowered fasting glucose (ϳ25%) and C-reactive protein (ϳ15%) while improving glucose tolerance (ϳ20%), reducing basal hepatic glucose production (20%), and increasing insulin-stimulated peripheral glucose uptake (20%). Importantly, however, the high dose of aspirin used in all of these studies is known to cause serious side effects.
The current analysis directly evaluated whether a low dose of aspirin acceptable for long-term use in routine clinical practice is effective in reducing clinical type 2 diabetes. Although we found no evidence to support this approach, there are three main possibilities that could ac-count for our null results. First, the dose of aspirin used in this study may be insufficient to impart a clinical benefit. As noted, prior studies evaluating the glucose metabolic effects of aspirin have used far higher doses. Data pertaining to potential hypoglycemic actions of low-dose aspirin have not been available. However, short-term treatment with doses as low as 30 -81 mg/day have been shown to improve several systemic inflammatory parameters, including lowering soluble CD40 ligand and promoting counterregulatory anti-inflammatory mediators such as 15-epi-lipoxin A 4 [bib_ref] Aspirin triggers antiinflammatory 15-epi-lipoxin A4 and inhibits thromboxane in a randomized human..., Chiang [/bib_ref] [bib_ref] Thromboxane-dependent CD40 ligand release in type 2 diabetes mellitus, Santilli [/bib_ref]. Despite these latter findings, our data demonstrate that chronic treatment with 100 mg aspirin on alternate days does not prevent clinical diabetes. However, intermediate doses as high as 1.3 g/day have been used in long-term clinical trials of cardiovascular disease prevention, and these settings may offer additional opportunities to evaluate this issue if diabetes incidence was also ascertained.
Second, women enrolled in the WHS were generally at low risk for diabetes as evidenced by the low prevalence of obesity, predominantly non-Hispanic white ethnicity, and lower rates of clinical diabetes compared with similarly aged women in the overall U.S. population. It is possible that the use of aspirin in a higher risk group may have led to detection of a beneficial effect. However, in subgroup analyses of women with highrisk features we did not find strong support for differential effects. Furthermore, there was no benefit among women having evidence of inflammation as reflected by elevated hsCRP.
Third, underdiagnosis of diabetes may have influenced our results. Women did not undergo systematic screening for diabetes or glucose intolerance as a part of the study; thus, we detected clinical diabetes as ascertained during routine clinical practice rather than all women with biochemical evidence of disease. However, any misclassification due to unrecognized diabetes would be nondifferential between treatment arms and unlikely to lead to important alterations in the estimation of relative effects. In addition, we are reassured that reported diabetes screening rates among participants in our study were similar to contemporaneous screening rates in the general population and equivalent in the two treatment groups.
Major strengths of this analysis include the randomized setting, large study Data are % unless otherwise indicated. Analyses involving biomarkers are restricted to women providing baseline blood specimens (n ϭ 27,167). *RR of clinical diabetes in the aspirin versus placebo group adjusted for age and randomized treatment assignment to vitamin E and -carotene. †Family history of diabetes in a first-degree relative (mother, father, sister, or brother). ‡Physical activity defined by number of episodes of vigorous physical activity per week. HT, hormone therapy. population, long treatment duration, large number of events, and ability to examine several large high-risk subgroups of women. In addition, baseline blood specimens were provided by roughly 70% of women in whom hsCRP and A1C levels were available. Importantly, systematic data were also collected on the occurrence of significant bleeding events.
## Aspirin does not lower diabetes incidence
Limitations of this study have been alluded to previously. An important limitation is the lack of systematic screening for more sensitive measures of glucose intolerance and insulin resistance during follow-up. While the costs associated with such diagnostic testing are prohibitive in this large-scale setting, we cannot with the current data determine the potential impact of low-dose aspirin on these subclinical markers of incipient disease. In summary, aspirin at a dose of 100 mg on alternate days is not effective for the prevention of clinical type 2 diabetes among otherwise healthy women at generally low risk for this disease. Our data do not pertain to other salicylate agents currently being evaluated for diabetes treatment or to intermediate or high doses of long-term aspirin in primary prevention. However, even at the low dose evaluated in this trial, the use of aspirin was associated with a significant increase in clinically important bleeding events and any potential benefit at higher doses, if found, must take into account this potential for excess risk.
[fig] Figure 1 -: Cumulative incidence of type 2 diabetes in the aspirin and placebo groups. [/fig]
[table] Table 1 -: Baseline characteristics of the study population [/table]
[table] Table 2 -: Incidence rates and rate ratio of type 2 diabetes in clinically important subgroups [/table]
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Lumbar chronic subdural hematoma mimicking an intradural extramedullary tumor: A case report
Background: Chronic spinal subdural hematomas are extremely rare with only 28 cases reported in the literature. Nevertheless, they should be considered among the differential diagnoses for spinal intradural/extramedullary lesions. Case Report: A 65-year-old male presented with progressive back pain and right S1 radiculopathy. Magnetic resonance imaging scan revealed a right-sided posterolateral intradural/extramedullary lesion at the L5-S1 level. It was hyperintense on T1 and hypointense on T2-weighted images; on the short TI inversion recovery sequence it was hyperintense. The lesion was excised through a right L5 hemilaminectomy, and the patient was neurologically intact postoperatively. Histopathology revealed a chronic subdural hematoma. Conclusion: Chronic spinal subdural hematoma can mimic intradural extramedullary spinal tumors even in the absence of trauma and/or coagulopathies.
# Introduction
Spinal subdural hematomas (SDHs) are rare, accounting for only 4.1% of all spinal hemorrhages. [bib_ref] Acute cervical spinal subdural hematoma not related to head injury, Kim [/bib_ref] There are only 28 cases of spinal subdural hematomas reported in the literature. [bib_ref] Spinal chronic subdural hematoma mimicking intradural tumor in a patient with history..., Abuzayed [/bib_ref] Most occur in the thoracic and/or thoracolumbar regions. [bib_ref] Chronic spinal subdural hematomas, Khosla [/bib_ref] Here, we report a chronic SDH occurring in a 65-year-old male at the L5-S1 level mimicking an intradural extramedullary tumor.
## Case report
## Clinical and radiographic presentation
In the absence of trauma or a history of coagulopathy, a 65-year-old male presented with a progressive right lower extremity L5/S1 radiculopathy.
On physical examination, straight leg raising was positive on the right side at 70 degrees and the right Achilles response was absent; there was no sensory or motor deficit.
Standing lateral dynamic X-rays showed a grade 1 listhesis at the L4-L5 level [ resonance imaging (MRI) documented a right-sided, posterolateral intradural/extramedullary lesion at the L5-S1 level [ ]. It was hyperintense on T1 and hypointense on the T2-weighted images; the short TI inversion recovery sequence showed it was hyperintense.
## Surgery
The patient underwent a right L5 microscope-assisted hemilaminectomy. When the dura was opened longitudinally, it revealed a dark blood clot within a semi-transparent neomembrane [ ]. It was easily removed with blunt dissection; the covering membrane was easily separated from the surrounding arachnoid mater. Due to the grade I spondylolisthesis at the L4-L5 level, an L4-L5 transforaminal interbody fusion (TLIF) was performed. Postoperatively, the patient had no complaints, including no neurological deficit. Radiological MR follow up [ ] revealed no residual pathology and adequate decompression. Postoperative X-rays documented the TLIF at L4-L5 [ and d].
## Histopathology
Histopathology showed membranes with loose connective tissue containing scattered lymphocytes, siderophages, many eosinophils within areas of hemorrhage, and spindly, fibroblasts. These findings confirmed the diagnosis of a chronic SDH [ [fig_ref] Figure 5: Histopathological section of chronic subdural hematoma [/fig_ref] ].
# Discussion
Spontaneous spinal SDHs without coagulopathy are rare conditions. There are two major theories regarding the etiology of these lesions. First, they may be attributed to cranial subarachnoid hemorrhages (SAH) that have extended into the spinal subarachnoid space and spinal subdural space by exceeding or lacerating the arachnoid membrane. Second, minor trauma increases both the intrathoracic pressure and intraluminal pressure of the vessels in the subarachnoid space. When cerebrospinal fluid pressure momentarily lags behind the intravascular pressure, vessels may ruptures resulting in SAH. [bib_ref] Spontaneous spinal subdural hematoma with spontaneous resolution, Kang [/bib_ref] MRI is the best imaging modality for diagnosing spinal SDH. The radiological differential diagnoses include schwannoma, meningioma, intradural lipoma, and spinal SDH. There are no definite guidelines for the management of these lesions. Those without neurological deficits may be treated with nonoperative management and serial MRI monitoring.
The operative treatment for spinal SDH includes wide laminectomy for evacuation of the hematoma. [bib_ref] Spontaneous Spinal Subdural Hematoma with Simultaneous Cranial Subarachnoid Hemorrhage, Jung [/bib_ref] Here, a right L5 hemilaminectomy preserved normal anatomical structures. Early diagnosis using MRI imaging with surgical confirmation of the pathology may prevent disastrous neurological deterioration and is the key to prevent potential severe neurological deficits.
# Conclusion
The differentiate diagnosis for spinal intradural/ extramedullary lesions should include chronic spinal SDH even without the history of trauma or coagulopathies. The early diagnosis and treatment of these lesions optimizes outcomes.
## Financial support and sponsorship
Nil.
## Conflicts of interest
There are no conflicts of interest.
[fig] Figure 3, Figure 4, Figure 1, Figure 2: Intraoperative photograph of chronic SDH: blood clots within semi-transparent neo-membrane Radiological follow up with T2WI showed no residual pathology and decompression of spinal cord The standing lateral dynamic x-rays showed grade 1 listhesis at L4-L5 level(a and b). TLIF was done at an L4-L5 level with percutaneous posterior screw fixation (c and d) Preoperative MRI revealed an intradural extramedullary mass lesion at the level of L5-S1 on right postero-lateral aspect. The lesion was hyperintense in T1WI (a and d), and hypointense on T2WI (b and e) with hyperintense to the cord on STIR sequence (c and f) [/fig]
[fig] Figure 5: Histopathological section of chronic subdural hematoma [/fig]
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Effect of hand washing and personal hygiene on hand food mouth disease
There are no specific treatment drugs and vaccine for Hand Foot and Mouth Disease (HFMD). Taking effective preventive measures is particularly important for control of HFMD infection. The objective of this study is to evaluate the effect of intervention of intensive education on hand hygiene on HFMD.We randomized 64 villages into intervention and control groups in Handan, Hebei province, China. Parents and caregivers of children 6 to 40 months age group in intervention villages received intensive education on hand hygiene. Control group received general education. The intervention period was from April 1 to July 31, 2011 and April 1 to July 31, 2012. We measured and compare the knowledge and incidences of HFMD between 2 groups.We collected 6484 questionnaires, including 3583 in the intervention group [response rate: 96% (3583/3726)] and 2901 in the control group [response rate: 90% (2901/3224)]. We observed that hand washing habit of children and parent, knowledge of HFMD of parents, children's daily cleaning habits scores improved in the intervention group and higher than that in the control group at both the end of year 1 (April 1-July 31, 2011)and year 2 (April 1-July 31, 2012). The incidence of HFMD (2.1%) in intervention group was significantly lower than that in control group (4.2%) at year 2 (x 2 = 22.138, P <.001). The positive percent of coli-form on the hand swabs in intervention group (2.00%) were significantly lower than that in control group (9.45%) at the end of year 2.The intervention of intensive education on hand hygiene effectively improved the personal hygiene both of children and parents, as well as reduced the incidence of HFMD. We suggested expanding the intervention measures in community to prevent HFMD.Abbreviations: EV71 = Enterovirus 71, HFMD = hand, foot, and mouth disease.
# Introduction
Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by various Enteroviruses (such as Coxsackievirus A16, other Coxsackieviruses, and Enterovirus 71 [EV71]), which primarily affects infants and children under 5 years of age. [bib_ref] Epidemiological characteristics and control strategy of HFMD, Xu [/bib_ref] It is usually a mild and self-limiting disease; however, recently many severe HFMD outbreaks caused by EV71 have occurred worldwide. These outbreaks are characterized by high numbers of neurological complications, which often led to death or permanent paralysis, causing significant concerns in the public health community. [bib_ref] Enterovirus 71 infection of the nervous system, Guo [/bib_ref] Up to now, no specific treatment or vaccine is currently available for HFMD.
Studies conducted in China and Korea have identified risk factors for HFMD, including low socioeconomic status, existence of open-air defecation, poor sewage treatment, poor handwashing and other poor personal hygienic habits (such as biting fingers). [bib_ref] Hand foot and mouth disease epidemiology and preventive measures, Lai [/bib_ref] [bib_ref] Hand foot and mouth disease popular trend, Cai [/bib_ref] [bib_ref] Transmission of seasonal outbreak of childhood enteroviral aseptic meningitis and Hand-foot-mouth disease, Park [/bib_ref] [bib_ref] Study the epidemiological characteristics of HFMD and risk factors in Guangdong Province, Guo [/bib_ref] [bib_ref] Control study of the risk factors in HFMD children in Tianjin, Xu [/bib_ref] [bib_ref] The evaluation of health education on controlling intestinal infectious diseases, Xiao [/bib_ref] Among these factors, open-air defecation and poor sewage treatment usually require expensive social engineering solutions, whereas risk factors such as hand-washing and other personal hygienic habits may be achieved by low-cost measures such as health education.
Observational studies have strongly shown the association between poor hand-hygiene and HFMD. [bib_ref] Transmission of seasonal outbreak of childhood enteroviral aseptic meningitis and Hand-foot-mouth disease, Park [/bib_ref] [bib_ref] Study the epidemiological characteristics of HFMD and risk factors in Guangdong Province, Guo [/bib_ref] [bib_ref] Control study of the risk factors in HFMD children in Tianjin, Xu [/bib_ref] However, no interventional studies have been conducted to verify this association. In 2011, we conducted a community-based trial to evaluate the effect of intervention of intensive education on hand hygiene on HFMD, and to provide scientific evidence for developing strategies for preventing and controlling HFMD.
# Methods
This study was approved by the Institutional Review Board of Hebei Center for Disease Control and Prevention, China. The institutional review board stated that written consents from patients were not required for this study because the identification numbers and personal information about participants were not included in the secondary files. All participants provided their verbal, informed consent.
We conducted an intervention trial in rural areas in Handan Prefecture, Hebei Province in Northeastern China. In 2010, the Prefecture had an estimated population of 8900000 persons. The attack rate among children 6 to 40 months age group was1652/100000 which was higher than that in other age groups in 2010. So we targeted the intervention at children in the 6 to 40 months age group because of their susceptibility to HFMD. For feasibility reasons, and to minimize cross-contamination of intervention effect, the intervention was administered at the village level rather than at sub-village levels.
We selected 4 counties with the highest incidences of HFMD during 2009 to 2010 in the prefecture as the study sites. Before intervention in 2010, the incidence of HFMD among target children in these 4 countries was about 5%, based on the data from the national notifiable disease surveillance system, which had made HFMD a notifiable disease in 2008. Expecting that the intervention would reduce the incidence by 30%, with a = 0.05 and a power of 90%, we calculated that each group would need 2900 children to participate. Assuming a 10% dropout rate, the sample size for each group would be about 3190 children.
Census and childhood immunization data showed that in Handan Prefecture, each administrative village had an average of about 500 residents, and each household had an average of 0.2 children in the targeted age group. Therefore, each group would need 30 (=3190/0.2/500) administrative villages. We random chose 64 administrative villages from all the 483 villages whose incidence rates were in the mid-pack in the 4 countries, that is, 3% to 10%, to participate in this study. Those 64 villages were randomized into intervention and control groups; each group had 32 villages.
Parents and caregivers in the 32 intervention villages received intensive education on hand hygiene. Posters on hand-washing were posted on every street corner and popular gathering places; messages describing the importance and correct procedures of handwashing were written on the villages' information blackboards and broadcasted over the radio station of the villages. Brochures and leaflets on preventing HFMD were distributed to all parents in the intervention villages. Also, the Prefecture's Center for Disease Control and Prevention conducted train-the-trainer sessions for all village doctors on knowledge of HFMD and correct hand-washing methods. The village doctors were then asked to train the parents in their villages. The specific training received by the village doctors, who then trained the parents, included the following: 1) Six steps of hand washing; 2) When hand-washing is needed; 3) Preparation of separate towels for each child and boiling them at least once a week; 4) Preparation of separate eating utensils for each child; 5) Cleaning of toys at least once a week, when being taken back home or after sharing with other children; 6) Minimizing outside activities for sick children with respiratory disease, gastrointestinal disease, or HFMD, and keeping the sick children at home as much as possible. All of the intervention messages we used were taken from the official website of China center for disease control and prevention.
Parents in the 32 control villages received general health education that was implemented in the whole prefecture, which included: general knowledge of hand-washing, drinking boiled water, and avoiding activities in the crowded places.
The intervention was implemented in 2 consecutive HFMD epidemic seasons (i.e., April 1-July 31) in 2011 and 2012, respectively with the same method of intervention.
## Questionnaire and data collection
We designed a standardized questionnaire to collect the following information from target children parents by face to face interviewing: demographic information, health conditions and family water conditions, the behavior of washing their hand among parents and children, the habits of cleaning children's toys and daily necessities, the knowledge of HFMD among parents, the history of HFMD among children.
The scores given for each question were shown in . We computed a hand-washing score and a cleaning habit score for each child; and a hand-washing and an HFMD knowledge score for each caregiver of the child. For hand-washing habits of children, cleaning habits of daily necessities and hand-washing habits of parents, the higher score they got the better health habit they had. For the HFMD knowledge of parents, all the options for each question were correct. The subject will get 1 point once he choice 1 option. He will get more points if he could choice more options. The maximum points were 4 or 5.
Before the intervention, a baseline survey was conducted in both intervention and control villages on children's handwashing habits and parents' knowledge for HFMD. The village doctors collected the information with a standard questionnaire by interviewing children's parents in person. At the end of each intervention period, the same survey was repeated.
During intervention periods, the village doctors collected data on respiratory symptoms, gastroenteritis symptoms and symptoms of HFMD among children and their family members weekly, either by telephone or in person. Respiratory symptoms include a high fever, usually with a temperature higher than 38°C, and 1 or 2 symptoms such as headache, nasal discharge, cough, and sore throat. Gastrointestinal symptoms were defined as persistent or intermittent abdominal pain for more than 24 hours, diarrhea for more than 3 times in 24 hours, and vomiting more than for 3 times in 24 hours, 1 of which was monitored. A HFMD case was defined as onset with fever and rash on hand, food or mouth.
In addition to the questionnaire survey, the village doctors randomly selected 100 households before, during, and at the end of each of the 2 intervention periods, and swabbed the hands of the children and their parents (or caregivers). The hand swabs were cultured for coliform bacteria at the laboratory of the Prefecture's Center for Disease Control and Prevention.
# Data analysis
We analyzed the effect of the intervention by using multivariable linear regression. The stepwise method is used to analyze the multivariate linear regression. The dependent variable was the score of hand-washing habit of the child, the score of daily cleaning habit of the child, the score of the hand-washing habit of the parents or caregivers, the knowledge score of HFMD of the parents. We selected the following independent variables based on the literatures which showed the influence factors of HFMD: variables which are relevant to immune (age and gender), health condition (family income, water source, outdoor toilets, and sewage ditch around the house), health habit (degree of education, intervention) and history of HFMD. We compared the percentages of gastrointestinal symptoms, respiratory symptoms and HFMD between intervention and control group by x 2 test. We compared the coliform-positive rates of hand swabs between the subjects in intervention and control groups by x 2 test before, during and after the intervention.
# Results
In the baseline survey, 32 intervention villages and 31 control villages participated. We collected 6484 questionnaires, including Based on these questionnaires, the average age of the child in control group was 22.50 ± 8.4 months, compared to 22.46 ± 8.5 months in intervention group (t = 0.190, P = .850). 87% of the parents in the intervention group had a secondary school or higher education; compared to 88% in the control group (x 2 = 0.2, P = .658). There was no significant statistical difference between 2 groups for hygienic risk factors such as having a private well as water source, having outdoor toilets, presence of sewage ditch around the house . Before intervention, the 2 group had nearly identical average hand-washing scores of children (11.2 for intervention group vs 11.3 for control group; t = À0.762, P = .446), hand-washing scores of parents/caregivers (17.0 for intervention group vs 17.2 for control group; t = À1.385, P = 0.166), knowledge scores on HFMD of parents/caregivers (8.0 for intervention group vs 8.1 for control group; t = À1.594, P = .111), and children's daily The score assignment for the hand washing and HFMD knowledge.
## Factors
Variables Score
The average number of daily washing Each time is 1 point Washing their hands after they go home?
Always (3 points
## Table 2
Comparison of reported risk factors, hand washing and HFMD knowledge scores between intervention and control groups in the baseline survey.
## Items
Intervention group Control group x 2 /t # P cleaning habits scores (2.2 for intervention group vs 2.2 for control group; t = À0.177, P = .860) . Before the intervention, the positivity rates of coliform bacteria in hand swabs for the children and their caregivers did not differ significantly between the intervention group and the control group (7.3% vs 9.3%, x 2 = 0.529, P = .467).
## Scores of hygienic habits and knowledge of hfmd
The result of multivariable linear regression analysis is shown in Tables 3 to 6. Take the scores of hand-wasing habit of children at the end of year 1 for example, in the case of adjusting other variables, compared to the control group, the probability of intervention group's score reduced at least 1 level was 0.158 times (exp(À1.847)). It means that interventions can improve and maintain high scores of hand-wasing habit of children. This explanation applies to other coefficients in and [fig_ref] Table 5: Hand-washing habits of parents of multiple linear regression at the end of... [/fig_ref]. For the HFMD knowledge's score of parents, control group seems more likely to get a high score, compared to the intervention group.
## Respiratory symptoms, gastrointestinal symptoms, and hfmd
At the end of year 1, the incidence rates of respiratory-related symptom including fever(6.4%), headache (3.0%), runny nose (6.6%), throat (3.8%), and cough (6.1%) in the intervention group were all significantly lower than those in the control group (19.9%,5.9%,16.5%,8.9% and 17%, respectively). the incidence rates of gastrointestinal-related symptom including bellyache (1.2%), vomit (1.2%), and diarrhea (1.5%) in the intervention group were all significantly lower than those in the control group (3.0%,4.9%, and 6.2%, respectively). The incidence of HFMD was 1.2% in intervention group, a little lower than that in control group (1.3%)(P = .706). At the end ofyear2 the incidence rates of respiratory-related symptom including fever (8.3%), headache (3.8%), runny nose (6.6%), throat (4.4%), and cough (6.2%) in the intervention group were all significantly lower than those in the control group (20.7%,5.3%,18.1%,10.0%, and 18.6%, respectively). the incidence rates of gastrointestinal-related symptom including bellyache (0.8%), vomit (1.3%), and diarrhea (2.2%) in the intervention group were all significantly lower than those in the control group (2.5%,6.4%, and 6.7%, respectively). The incidence of HFMD in intervention group was significantly lower than that in the control group (2.1% vs. 4.2%, P = .000) [fig_ref] Table 7: Incidences in intervention group and control group at the end of year... [/fig_ref].
## Positive rate of coliform bacteria
The positive rates of Coliform bacteria were significantly lower in the intervention group than in the control group during (3.4% vs 9.0%, P = .288) and at the end of year 1 (1.9% vs 9.2%, P = .001). Similarly, the positive rates in the intervention group were significantly lower than in control group during (4.5% vs 9.9%) and at the end of year 2 (2.0% vs 9.5%) .
# Discussion
We conducted an intervention study to evaluate the effect of intensive education on hand hygiene on prevention HFMD in Handan, Hebei province, China from 2010 to 2013. The result showed that the intervention measures could improve the hand The Children's daily cleaning habits of multiple linear regression at the end of year 1 and year 2. washing habit in both children and parents, children hygiene habit, HFMD knowledge in parents after intervention period. It also decreased the incidences of respiratory and intestinal related symptoms among children and their family members. The hands in intervention group were less contaminated with col-form bacteria than I control group after intervention. Importantly, through repeated intervention, the incidence of HFMD was markedly reduced. HFMD could cause severe disease such as central nervous system, respiratory system damage which caused widespread concern in the community. Because there is no effective vaccine, therefore, strengthening the HFMD knowledge for children and parents, and promoting them to develop healthy behavior is necessary. We showed the effect of the intensive education on hand hygiene on preventing HFMD in this study from the knowledge, behavior, disease incidence, and hand contamination status. These measures are easy to be implemented and could be used widely in communities in the future.
## 95% confidence interval
This study included 2 stages. At the end of year 1, the HFMD knowledge, hand washing habits, and hygiene habit were all improved, but the HFMD incidence did not decrease significantly. However, at the end of year 2, the HFMD incidence in the intervention group was lower significantly than that in control group. That means the ongoing intervention was important to achieve the objective of decreasing HFMD among children. We suggested expanding the intervention measures in community to prevent HFMD. On the other hand, it was estimated that the incidence rate of HFMD were122.54/100 000 in 2010, 34.90/ 100 000 in 2011 and 97.01/100 000 in 2012 in Handan City, respectively. It was possible that any intervention will not expect Our study had some limitations. One major limitation was that this study used various interventions, that is, hand-washing and other hygienic improvement measures. Therefore, our study could not demonstrate whether it was hand-washing alone that reduced the symptoms, and it was not possible to know which intervention worked. It was difficult to use blind method in this community trial, which was the second limitation. And the control group might receive intervention measures in some degree, that is, posters, broadcasted over the radio station from the neighboring, intervention group villagers, which would reduce the study effects. The results would show bigger difference if the control group did not receive any intervention measures. The positive rate of Coli-form bacteria in hand swab specimens before, during and after intervention.
[table] Table 4: Hand-washing habits of children of multiple linear regression at the end of year 1 and year 2. [/table]
[table] Table 5: Hand-washing habits of parents of multiple linear regression at the end of year 1 and year 2. [/table]
[table] Table 6: Knowledge of HFMD of parents of multiple linear regression at the end of year 1 and year 2. Sewage ditch around the house (ref. for NO) HFMD = hand, foot, and mouth disease. [/table]
[table] Table 7: Incidences in intervention group and control group at the end of year 1 and year 2. HFMD = hand, foot, and mouth disease. [/table]
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SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes
H., and J.M.V. contributed new reagents/analytic tools; P.P. and J.A.S. analyzed data; and P.P. and J.A.S. wrote the paper.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease , continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2-infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). Its abundance ranges from low to moderate as compared to host miRNAs and it associates with Argonaute proteins-core components of the RNA interference pathway. We identify putative targets for CoV2-miR-O7a, including Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which participates in interferon signaling. We demonstrate that CoV2-miR-O7a production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.
micoRNA j SARS-CoV-2 j noncoding RNA C oronaviruses are large single-stranded positive-sense RNA viruses that infect various animals; in humans coronaviral infection results in mild to severe respiratory disease. β-coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), cause very severe disease in humans, while the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in coronavirus disease characterized by wide array of symptoms from mild to serious illness. SARS-CoV-2 is the cause of the COVID-19 pandemic due to its high transmissibility and emergence of novel variants. It is crucial to understand all aspects of SARS-CoV-2 pathogenesis in order to develop multiple complementary tools to combat the constantly adapting virus. microRNAs (miRNAs) are ∼22-nucleotide (nt)-long small noncoding RNAs (ncRNAs) involved in posttranscriptional regulation of gene expression in animals and plants. During canonical miRNA biogenesis, RNA polymerase II transcribes primary miRNAs (pri-miRNAs) characterized by formation of strong hairpins that are recognized and cleaved by the endonuclease Drosha, resulting in ∼70-nt-long precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported to the cytoplasm, where they are subjected to yet another cleavage by Dicer, giving rise to ∼22-nt-long miRNA duplexes, which are then loaded onto Argonaute (Ago) proteins. The passenger strand is removed, and the mature miRNA guides Ago proteins to partially complementary target sequences, usually located in the 3 0 untranslated region (UTR) of messenger RNAs (mRNAs). miRNA binding leads to translation inhibition and/or mRNA decay by decapping and deadenylation. In the special case where the miRNA is perfectly complementary to its target mRNA, it directs Ago2 (one of four Ago proteins in humans) to cleave the mRNA target, a function typically associated with small interfering RNAs (siRNAs). Both of these modes of target repression belong to the RNA interference (RNAi) pathway, but Ago2-mediated cleavage is more potent in target silencing and requires lower intracellular copy numbers than the canonical miRNA action mode. Importantly, miRNA profiles differ between cell types and developmental stages, and a single miRNA can down-regulate multiple transcripts, precisely regulating gene expression dependent on the cell's needs. In humans, most mRNAs are regulated by miRNAs, and aberrant miRNA levels are linked to disease.
Viruses often hijack the miRNA pathway, either by depleting host miRNAs or by producing their own miRNAs (reviewed in refs. . Some examples of host miRNA regulation include selective host miRNA decay mediated by certain herpesviral transcripts in a process called target-directed miRNA degradation (reviewed in refs. 6 and 7) and widespread miRNA polyadenylation by poxvirus poly(A) polymerase, which results in miRNA decay. Viruses can produce their own miRNAs, often via noncanonical biogenesis pathways; cytoplasmic viruses especially have devised multiple strategies to bypass the requirement for nuclear Drosha. It has been proposed Significance We discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral noncoding RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). CoV2-miR-O7a associates with the cellular RNA interference machinery and has the potential to regulate host transcripts, likely via target slicing. The production of CoV2-miR-O7a relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly described CoV2-miR-O7a may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.
that coronaviruses, SARS-CoV and SARS-CoV-2, produce small viral RNAs (svRNAs) involved in viral pathogenesis independent of the RNAi pathway. Recent studies suggested the existence of miRNA-like RNAs produced by SARS-CoV-2, which might regulate inflammation and interferon (IFN) signaling.
In this study, by using a small RNA-sequencing (smRNAseq) library preparation protocol that selectively enriches for miRNAs, we unexpectedly discovered an miRNA-like viral small RNA, named CoV2-miR-O7a, expressed by SARS-CoV-2. We show that it associates with Ago proteins and has the potential to regulate host transcripts, likely via target slicing. We demonstrate that CoV2-miR-O7a production relies on cellular machinery but is independent of Drosha proteins and is enhanced by the presence of a strong hairpin formed within ORF7a sequences. This newly described viral ncRNA may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.
# Results
## Sars-cov-2 infection
Has Minimal Impact on the miRNA Population of Its Host Cell. To explore how SARS-CoV-2 affects host miRNA populations, we infected three lung-derived human cell lines either at high multiplicity of infection (MOI of 5) to detect the possible direct impact of the incoming virions on host miR-NAs or at low MOI (0.05) to assess the effect of viral replication on these small RNA species. We used non-small-cell lung cancer cells Calu-3, lung adenocarcinoma cells A549 transduced with the human viral receptor, angiotensin-converting enzyme 2 (ACE2), and lung adenocarcinoma cells PC-9, all of which we found to support SARS-CoV-2 replication (red, yellow, and blue dots, respectively, in. SARS-CoV-2 replicated better in Calu-3 and PC-9 cell lines than in the A549-hACE2 cell line, possibly because the viral receptor is greatly overexpressed in the last cell line (SI Appendix,, which could sequester budding virions at the cell surface.
Small RNA sequencing was performed at both 6 h and 24 h postinfection (hpi) at high MOI and 48 hpi at low MOI, as well as for the uninfected controls (at the 24-h time point). The vast majority (84% ± 5%) of obtained reads mapped to host miRNAs (red bars inwith an average read length of 22 or 23 nt (SI Appendix,. Overall, only a small fraction of small RNA reads mapped to the viral genome (0.01 to 1.63% depending on conditions; blue bars in. This number is in agreement with similar studies of small viral RNAs in other RNA viruses.
SARS-CoV-2 infection resulted in minimal alteration of some host miRNA levels, but no miRNA displayed a uniformly significant change across all three cell lines (red and blue dots inand SI Appendix,. To identify any consistent shifts in host miRNA levels across the three cell lines, we generated a heat map of fold changes for miRNAs that were significantly altered, by at least twofold, in at least one condition (SI Appendix,. Some of these trends were further examined by Northern blotting of RNA from Calu-3 cells infected with SARS-CoV-2 at low MOI. The most reproducibly changed (up-regulated) miRNA, although not reaching statistical significance, was miR-210-3p (SI Appendix,. Interestingly, Gene Ontology term analyses for validated miR-210-3p targetsrevealed that this miRNA is involved in regulating responses to cellular stimuli, such as hypoxia (SI Appendix,. In addition, in two datasets obtained from lung biopsies of COVID-19 patientsmiR-210-3p targets were downregulated to a greater extent than other human mRNAs (SI Appendix,.
Of note, the changes in miRNA levels observed by smRNAseq were not detected by TaqMan RT-qPCR from nasopharyngeal swabs from SARS-CoV-2-positive individuals. Interestingly, analysis of unnormalized Ct values revealed that after SARS-CoV-2 infection the overall miRNA abundance increased, while the levels of ncRNAs from two other small RNA classes (small nuclear RNA U6B and small nucleolar RNA U44) decreased (SI Appendix,, suggesting that miRNAs might escape the viral host shut-off effect. This agrees with the notion that miRNAs can be very stable, e.g., in circulating exosomes. Thus, it may be possible to devise small RNA-mediated treatments to treat COVID-19. Overall, we did not find any major impact of SARS-CoV-2 infection on host-cell miRNA populations.
SARS-CoV-2 Expresses an miRNA-Like Small RNA Derived from the ORF7a Sequence. Of the reads derived from SARS-CoV-2, 5.2% ± 2.8% mapped to a single peak within the ORF7a sequenceand SI Appendix,. ORF7a encodes a SARS-CoV-2 accessory protein, a putative type-I transmembrane protein involved in antagonizing the type I IFN response (reviewed in ref.. The reads mapping to the ORF7a sequence are ∼20 to 25 nt long. Because the small RNA libraries were dominated by host miRNAs and a U [the preferred 5 0 -terminal nucleotide for Ago loading ] is present at the 5 0 end of this RNA, we interrogated whether it could represent a viral miRNA-like small RNA. Since miRNA biogenesis relies on formation of RNA hairpins, we asked whether such an RNA structure could be found within the viral ORF7a sequence. Indeed, the putative viral miRNA together with its downstream region forms a strong hairpinwhose folding energy is unusually low given the AU-rich nucleotide composition (ΔG = À19.7 kcal/mol), suggesting that it arose through an evolutionarily driven process. Indeed, the thermodynamic Z-score is À5.5 (meaning that the wild-type sequence is more than 5 SDs more stable than random). In addition, a comprehensive analysis of RNA structural motifs in SARS-CoV-2 found that this hairpin is one of the few (nine) that show evidence of statistically significant sequence covariation-a feature of evolutionary conservation. The formation of the hairpin is also in agreement with previously reported structure probing reactivity datasets, where the only reactive nucleotides occur in loop regions. Since the newly identified small RNA is derived from this hairpin-which is a hallmark of miRNA biogenesis (27)-we named it CoV2-miR-O7a (for SARS-CoV-2 miRNAlike ORF7a-derived small RNA). In addition, there exists sequence and structure similarity of the RNA hairpin between SARS-CoV-2, bat coronavirus RaTG13, and a pangolin coronavirus (SI Appendix,. CoV2-miR-O7a and its pre-miRNA-sized precursor are detectable by Northern blotting of RNA from Calu-3 cells infected with SARS-CoV-2 at low MOI. Interestingly, the most prevalent species of CoV2-miR-O7a observed by Northern blotting are 25 to 27 nt long, longer than those identified by small RNA sequencing. Since the small RNA library was size-selected, this could mean that CoV2-miR-O7a is in fact more abundant than when quantified by smRNA-seq. However, because miRNAs are usually ∼22 nt long, CoV2-miR-O7a or its precursor could have an additional RNAiindependent function. Importantly, CoV2-miR-O7a was detected in individuals infected with SARS-CoV-2and its abundance correlated positively with viral load/genomic RNA (as measured by the levels of RNA-dependent RNA polymerase, RdRp).
[formula] M K I I L F L A L I T L A T C E L Y ORF7a: G A A C A [/formula]
## T g a a a a t t a t t c t t t t c t t g g c a c t g a t a a c a c t c g c t a c t t g t g a g c t t t a t c
[formula] U U U U C U U G G C A C U G A U A A C A C U C G C U A C U U G U G A G C U U U A U C A C U A C C A A G A G U G 27,409 27,328 27,459 5' ΔG = -19.7 kcal/mol ΔG Z-score = -5.5 CoV2-miR-O7a CoV2-miR-O7a CoV2-miR-O7a-3p CoV2-miR-O7a-3p A C D E CoV2-miR-O7a mock 24h 48h miR-O7a- [/formula]
CoV2-miR-O7a Binds Ago and Can Repress Human mRNAs. Since the miRNA program is executed by Argonaute proteins, we asked whether these host proteins bind CoV2-miR-O7a. We performed anti-pan-Ago RNA immunoprecipitation (RIP) on extracts from Calu-3 and A549-hACE2 cells infected with SARS-CoV-2 at high MOI, followed by small RNA sequencing. In both cell lines, CoV2-miR-O7a associates with Ago proteins, although to a lesser extent than most host-cell miRNAs; its expression levels are comparable to low or moderately expressed host miRNAs. To obtain enough material for Northern blotting experiments, we performed an anti-hemagglutinin (HA) RIP from Calu-3 cells transduced with either empty vector or FLAG-HA-tagged human Ago2 . Although weak, Ago2 selectively binds CoV2-miR-O7a, but not CoV2-miR-O7a-3p. In these experiments, we observed slight RNA degradation likely arising during the SARS-CoV-2 inactivation step (30-min incubation at room temperature). Perhaps this is the reason why CoV2-miR-O7a bands inmigrate somewhat faster than those in.
The role of Ago-associated small RNAs is to repress targeted mRNAs via sequence complementarity. We thus computationally searched the repository of human mRNA transcripts (obtained from GENECODE) for putative CoV2-miR-O7a targets. Considering the moderate expression levels of CoV2-miR-O7a as compared to those of host miRNAs, we hypothesized that CoV2-miR-O7a might rely on Ago2's ability to cleave its mRNA target. Since Ago2-mediated target cleavage tolerates a limited number of mismatches, we searched for mRNAs with high complementarity to CoV2-miR-O7aandand selected some of these for further validation. CoV2-miR-O7a CoV2-miR-O7a-3p miR-16 probes:were measured by RT-qPCR 24 h later. Means with SD are shown. *P < 0.05, **P < 0.01 as calculated by two-tailed paired t test.
[formula] A C D B vmiR-5p 5' UUCUUGGCACUGAUAACACUC ||||||||||||||| ||||| HSPG2 3'GAGGGGCCGUGACUGU-GUGAGG vmiR-5p 5' UUCUUGGCACUGAUAACACUC ||||||||||||| |||||| BATF2 3'GAAGAACCGUGACUUUUGUGAUG [/formula]
Due to the widespread host-shutoff effect after infection with SARS-CoV-2 (17), we were unable to perform meaningful quantification of mRNAs from infected cells. We thus transfected synthetic CoV2-miR-O7a (annealed to a passenger strand) into HEK293T cells and assayed transcript levels of predicted target mRNAs. Two mRNAs, Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2) and Heparan Sulfate Proteoglycan 2 (HSPG2), were significantly downregulated. BATF2 has been linked to INF-gamma signaling via association with Irf1 (31). HSPG2 is the core protein of a large multidomain proteoglycan that binds and cross-links many extracellular matrix components; interestingly, it participates in the attachment of some viruses, including the coronavirus NL63. We hypothesize that CoV2-miR-O7a has the potential to repress human mRNAs to evade the host IFN response and perhaps to inhibit viral superinfection.
## Production of functional cov2-mir-o7a depends on cellular
Machinery. To assess whether CoV2-miR-O7a biogenesis can occur in a virus-free system, we transfected in vitro-transcribed RNA into HEK293T cells. A 100-nt-long viral RNA sequence (WT), alongside with its counterparts containing mutations either in the 3 0 portion (m3p) or in the 5 0 portion (m5p) of the hairpin, were transfected into cells and RNA samples collected after 6 h were analyzed by Northern blotting. The WT sequence was processed inside cells to produce a 21-nt-long band of CoV2-miR-O7a, while the m3p did not yield efficient production of an RNA of such size.
To investigate whether the produced RNA functionally associates with Ago proteins, we transfected in vitro-transcribed RNA together with luciferase reporters containing 3 0 UTR sequences complementary to either CoV2-miR-O7a or scrambled controls. The luciferase activity of the reporter containing viral sequences was significantly decreased in the presence of the WT ORF7a RNAbut not by the other constructs. Finally, we used Drosha knockout cell linesto show that the production of CoV2-miR-O7a occurs independently of this enzyme's activity. These results clearly demonstrate that CoV2-miR-O7a is processed from a hairpin independently of viral proteins and of cellular Drosha, and that it can be functionally loaded on Ago.
# Discussion
Here, we began by investigating the impact of SARS-CoV-2 infection on host miRNA populations using three human lungderived cell lines (at various MOIs) and found no consistent changes detected in all three cell lines. We also assessed the levels of selected miRNAs in patient samples and again did not detect major differences after SARS-CoV-2 infection. Other groups have also ventured into examining miRNA profiles during SARS-CoV-2 infection, but there is little overlap between these prior studies (34-36) and our results. Differences between the outcomes likely stem from the use of different methods of smRNA-seq library preparation. Our libraries were prepared with the NEXTflex v2 kit, which has been shown to be highly selective for miRNAs, and contained ∼84% host miRNAs, while libraries that rely on template switching used by others (34) yield only ∼17% miRNAs. The presence of additional reads in the latter libraries could result in nonspecific alignment to miRNA loci. For example, miR-155-3p, which is believed to represent a host miRNA passenger stand, was reported to increase upon SARS-CoV and SARS-CoV-2 infection (34), but we were unable to detect this miRNA either by sequencing or by Northern blotting. Similarly, miR-4485 was described to be up-regulated in another study, but it did not pass the abundance threshold (>1 counts per million [CPM]) in our smRNA-seq analysis. Also, other studies assessing the impact of various coronaviruses on host miRNA levels do not support the notion that coronaviruses have major impact on host miRNA populations. We cannot rigorously exclude the possibility that some host miRNAs levels are altered during SARS-CoV-2 infection, but if so these changes are likely minor.
Interestingly, in samples from individuals infected with SARS-CoV-2 all assessed host miRNAs seemed to be stabilized, whilesimilar to what is known about host mRNAs in infected cells (17)-U6B small nuclear RNA and U44 small nucleolar RNA levels decreased (SI Appendix,. These results suggest that miRNAs might be resistant to the viral host-shutoff effect. Indeed, miRNAs can be very stable because of their association with Ago proteins, especially when secreted in exosomes. It is tempting to speculate that, because of their high stability, small RNAs could be successfully used as therapeutic agents against SARS-CoV-2. Regardless, we caution against examining miRNA profiles during viral infection using RT-qPCR and normalizing to ncRNAs from different classes, such as small nuclear RNAs and small nucleolar RNAs.
In this study, we discovered that SARS-CoV-2 expresses an miRNA-like small RNA, which we call CoV2-miR-O7a. Of note, Singh et al. have independently reported the existence of CoV2-miR-O7a. Interestingly, this small RNA was also detected by Meng et al. but was not selected for further validation. These independent studies indicate that CoV2-miR-O7a is a functional svRNA. There are multiple examples of svRNAs expressed by RNA viruses, such as influenza (43), enterovirus 71, hepatitis C (45), hepatitis A (46), polio, Dengue, vesicular stomatitis (45), West Nile, coronaviruses, and retroviruses. It has been suggested that some svRNAs function through the host-cell RNAi pathway (12, 45, 46), but most proposed ))))))))))))).))))). À31.5 ))))))))))..)))))) À31. ))))))))))).)))))). )))))))))))).)))))). À29.9 CDS SLC30A3
CGGGATACGCTGTTGTCGGTGCCAGGAGT ))))))))))))))))))) À28.9 CDS LAMA3 GGAGTGTGCCAGTGCCGAGAG Yes . )))))))))...)))))). )))))))))))))))))). À28. ))))))))))))..)))))) À28.4 ))))))))))..)))). ))))))))))..)))))) À28.3 ))))))))))))))))). ))))))))))).))))). À28 CDS CDS, coding DNA sequence. UTR, untranslated region.
## Biochemistry
Pawlica et al. SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes roles are independent of this machinery. In this study, we investigated the RNAi-related function of CoV2-miR-O7a. CoV2-miR-O7a is derived from the beginning of the coding sequence of the ORF7a transcript, which is the most abundant subgenomic viral RNA detected during SARS-CoV-2 infection. The sequence of CoV2-miR-O7a is preserved in all SARS-CoV-2 isolates and variants of concern and is related to those of two other coronaviruses (SI Appendix,. Interestingly, ORF7a deletions, which lead to decreased innate immune suppression, occur frequently in SARS-CoV-2 but affect almost exclusively the C terminus of the protein, while the N terminus-where the CoV2-miR-O7a sequence is locatedis preserved. These data and the observed conservation of the hairpin containing CoV2-miR-O7a (22) support the notion that this sequence is functional.
The abundance of CoV2-miR-O7a ranges from low to moderate as compared to host miRNAs, similar to other svRNAs, and is consistent with the idea that its functionality relies on high complementarity to target mRNA, likely inducing mRNA cleavage. Indeed, we have shown that CoV2-miR-O7a associates with human Ago proteins and that it can repress human targets. Using bioinformatics, we identified two host transcripts that are significantly downregulated in the presence of a synthetic CoV2-miR-O7a, BATF2 and HSPG2and. In addition to these, there are likely more targets that may be identified by crosslinking ligation and sequencing of hybrid [CLASH] methodology. To address the functionality of CoV2-miR-O7a in the context of viral infection, we attempted to use luciferase reporters, but due to the host-shutoff effect there was almost no luciferase signal in infected cells. We avoided using antisense
## Firefly
Renilla oligonucleotides to block CoV2-miR-O7a because they would undoubtedly also suppress production of the ORF7a protein and inhibit viral replication. Upcoming studies will focus on the construction of mutant viruses in which the ORF7a coding sequence and hairpin formation are preserved. Another intriguing possibility is that SARS-CoV-2 uses CoV2-miR-O7a to regulate the level of its own negative-sense subgenomic RNAs, or of antigenomic RNAs. It is also plausible that the viral hairpin, in addition to being the source of a viral miRNA, could have an additional function independent of the RNAi pathway. RNA viruses have been shown to express regulatory svRNAs. For example, svRNA1 from enterovirus-71 binds to a viral internal ribosome entry site to regulate translation of viral proteins. Another example is svRNAs from influenza that associate with viral RdRp, possibly enabling the switch from transcription to replication.
[formula] 4 x BS to CoV2-miR-O7a SV40 HSV-TK 4 x scrambled sequence U U U U C U U G G C A C U G A U A A C A C U C G C U A C U U G U G A G C U U U A U C A C U A C C A A G A G U G 35 84 5' U U U U C U U G G C A C U G A U A A C A C U C G C U A C U U C U C U C C U U A U A G A C U A C C A A G A G U G 35 84 U U U A G A A C G C A C U C U A U U C A C U C G C U A C U U G U G A G C U U U A U C A C U A C C A A G A G U G [/formula]
Finally, we addressed CoV2-miR-O7a biogenesis and were able to demonstrate that its processing occurs via cellular machinery and relies on the formation of an RNA hairpin but is independent of Drosha protein. Yet, it is possible that viral genes enhance this processing pathway. Many viruses bypass Drosha to produce their pre-miRNAs, e.g., by utilizing RNase Z (56, 57), Integrator complex (58), or a viral protein (such as HIV-1 TAT). As shown by Singh et al., Dicer, which is responsible for production of the majority of host and viral miRNAs, is involved in CoV2-miR-O7a biogenesis. Future efforts will aim to uncover how the pre-CoV2-miR-O7a hairpin is cleaved out from viral transcripts.
In summary, viruses develop multiple ways to suppress host gene expression, and various overlapping mechanisms often evolve. SARS-CoV-2 regulates host mRNA expression on many levels: stability, export, translation, and splicing. It is not surprising that yet another strategy, which utilizes the RNAi pathway, to selectively target host-and perhaps viral-transcripts could have evolved.
# Materials and methods
Cells. Calu-3 (ATCC) cells were cultured in Eagle's minimal essential medium (EMEM; ATCC) with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (Pen/Strep; GIBCO). PC-9 cells (kind gift from Craig Wilen, Yale University, New Haven, CT) were cultured in RPMI medium (Gibco) with 10% FBS and Pen/ Strep. A549 (ATCC) cells were transduced as described previously (28) with hACE2 plasmid (kind gift from Benjamin Goldman-Israelow, Yale University, New Haven, CT) and cultured in F-12 medium (Gibco) with 10% FBS, Pen/Strep, and 1 μg/mL of puromycin (Gibco). Vero-E6 (ATCC), Huh7.5, and Huh7.5 Drosha knockout [kind gift from Charles Rice, Rockefeller University, New York, NY (33)] cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and Pen/Strep. Calu-3 cells were transduced either with FLAG-HA-Ago2 (28) or pLVX (for empty vector control) and cultured in the presence of 1 μg/mL of puromycin. Viral Stocks. To generate SARS-CoV-2 stocks, Vero-E6 were inoculated with P2 of SARS-CoV-2 isolate USA-WA1/2020 (kind gift from Craig Wilen, Yale University, New Haven, CT) at 0.01 MOI for 1 h, after which the inoculum was replaced with DMEM with 5% FBS. After 3 d the supernatant was harvested and clarified by centrifugation, concentrated on Ultra-15 Centrifugal Filters (Amicon), aliquoted, and stored at À80°C. Virus titers were determined by plaque assay using Vero-E6 cells. All infectious growth was performed in a Biosafety Level (BSL) 3 laboratory and was approved by the Yale University Biosafety Committee.
Plaque Assays. Vero-E6 cells were seeded at 4 × 10 5 cells/well in 12-well plates. The next day, medium was removed, and cells were incubated for 1 h at 37°C with 100 μL serially diluted sample; plates were rocked every 15 min. Next, 1 mL of the overlay media (DMEM, 2% FBS, and 0.6% Avicel) was added to each well. Three days later, the plates were fixed with 10% formaldehyde for 30 min, stained with crystal violet solution (0.5% crystal violet in 20% methanol) for 30 min, and then rinsed with water to visualize plaques.
Samples for Small RNA Sequencing. For small RNA sequencing, 5 × 10 5 of Calu-3, 5 × 10 5 of PC-9, or 1 × 10 5 of A549-hACE2 cells were plated per well in six-well plates. Cells were infected with SARS-CoV-2 at MOI of either 5 or 0.05 for 1 h in 200 μL of inoculum and incubated for 1 h at 37°C. After that, the cells were rinsed with phosphate-buffered saline to remove the unbound virus and fresh media were added. Cells were incubated for either 6 h and 24 h (MOI 5) or 48 h (MOI 0.05). In addition, mock-treated controls were collected after 24 h. Cells were harvested by removing the media and adding TRIzol at each time point; supernatants were kept to assess viral titer by plaque assay. RNA isolation and library preparation was performed under BSL2+ confinement; RNA concentration was measured by Qubit Fluorimeter (Thermo Fisher) inside a biosafety cabinet. One microgram of total RNA was used for library preparation using NEXTflex v2 kit (PerkinElmer) according to the manufacturer's instructions. Libraries were amplified for 16 cycles and the complementary DNA (cDNA) libraries were sequenced on the HiSeq 2500 Illumina platform.
Small RNA Data Processing. The reads were trimmed of adaptors using Cutadapt (65) with the following settings: -u 4 -O 7 -a Nf4gTGGAATTCTCGGGT GCCAAGG -q 10 -m 18 -M. The reads were mapped with bowtie2 (66) (-verysensitive-local) to an index containing human and SARS-CoV-2 genomes. miRNAs were counted by using featureCountsand annotations obtained from miRBase. For Ago immunoprecipitation (IP) counts, CoV2-miR-O7a was added to the annotation file and treated as a host miRNA. Differential expression was determined using edgeR. Track visualization was performed using an IGV browser (70) of generated with BEDtools (71) bed files.
The initial experiments were done in two biological replicates at high MOIs (series A and B); subsequently, the experiment was repeated for the third time, also adding a low MOI condition (series 1, 2, and 3). Because the series A and B and 1 through 3 were sequenced separately, for differential expression analysis, we used mock samples from series A, B, 1 and 2; all other conditions had three replicates.
Patient Samples and RT-qPCR. The human study reported here from which nasopharyngeal swab samples were obtained was approved by the Yale Human Research Protection Program (Protocol 2000027971). Subjects from whom nasopharygeal swabs were collected were enrolled in a randomized, placebo-controlled, double-blind clinical trial for early treatment of SARS-CoV-2 infection (https://clinicaltrials.gov/, NCT04353284). Each provided written informed consent both for enrollment in the trial and for additional uses of the samples including work described herein.
The nasopharyngeal swab samples were extracted using MagMAX mir-Vana Total RNA Isolation Kit (catalog no. A27828), with the script A27828_FLEX_Biofluids for miRNA extraction from biofluid samples on the KingFisher FLEX-96 Magnetic Particle Processor. Briefly, each 100-μL specimen of nasopharyngeal swab in transportation medium was processed and the miRNA-enriched RNA was collected in 50 μL of elution buffer. miRNA RT-qPCR was performed using TaqMan MicroRNA Assay (Applied Biosystems); for miR-16, miR-210-3p, miR-31-3p, miR-193-5p, miR-193-3p, U6B, and U44 predesigned assays were obtained (catalog no. 4427975), and for miR-O7a a custom assay to detect the sequence UUCUUGGCACUGAUAACAC was ordered (catalog no. 4398987). RT-qPCR was performed according to the manufacturer's guidelines; briefly, cDNA was prepared separately for each miRNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and qPCR was performed using the TaqMan Fast Advanced Master Mix (Applied Biosystems). For detection of viral RdRp, cDNA was prepared using random primers and SuperScript III (Invitrogen) and qPCR was performed according to the protocol (72) using oligonucleotides and a probe ordered from IDT (sequences are given in SI Appendix, . All work was done in BSL2+ conditions. Northern Blot Analysis. Calu-3 cells (5 × 10 6 ) were infected with SARS-CoV-2 at MOI 0.05 in T75 flasks for 1 h, after which the inoculum was replaced by fresh media, and cells were incubated for either 24 h or 48 h (mock-treated samples were collected after 48 h). Samples were inactivated for 30 min in 5 mL of TRIzol and RNA was isolated in BSL2+ conditions. Ten to 15 μg of total RNA was separated by 15% urea-PAGE, electrotransferred to Hybond-NX membrane (Amersham), and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). miRNAs were detected using 32 P 5 0 -radiolabeled DNA probes. Densitometry was performed by using Quantity One. IP. For anti-pan Ago IP, 5 × 10 5 Calu-3 cells or 1 × 10 5 A549-hACE2 cells were infected with SARS-CoV-2 at MOI 5 for 24 h. Cells were detached with trypsin-EDTA (ethylenediaminetetraacetic acid), pelleted, and resuspended in 500 μL of NET-2 buffer (50 mM Tris [pH 7.5], 150 mM NaCl, and 0.05% Nonidet P-40). After 30 min of cell lysis inside the biosafety cabinet (BSC), the nuclei were pelleted and supernatants were transferred to 2.0-mL screw-top tubes with O-rings containing magnetic beads (SureBeads Protein G Magnetic Beads; NEB) coupled to antibodies (anti-pan Ago antibody, clone 2A8; Sigma Millipore). Ten percent of supernatants were kept for input (in TRIzol). The tubes were sealed, decontaminated, and placed inside 15-mL screw-top falcon tubes. The 15-mL falcon tubes were decontaminated and transferred to the refrigerator containing the rotator where immunoprecipitation took place. After 6 h, the beads were washed inside BSC seven times with NET-2 by gentle pipetting and with using a magnetic stand. Finally, the magnetic beads were resuspended in 1 mL of TRIzol. Described procedures were performed in a BSL 3 laboratory and were approved by the Yale University Biosafety Committee. RNA isolation and library preparation (as described above) was performed under BSL2+ confinement.
For anti-HA IP, 5 × 10 6 of Calu-3 cells, transduced with either EV or Ago2, were infected with SARS-CoV-2 at MOI 0.1 for 48 h. Cells were detached with trypsin-EDTA, pelleted, and resuspended in 2 mL of Pierce IP Lysis Buffer (Thermo Fisher). Cells were incubated at room temperature for 30 min (approved by the Yale University Biosafety Committee SARS-CoV-2 inactivation method). After this time, the samples were moved to the BSL2+ laboratory, sonicated using a Diagenode Bioruptor Pico sonication device and IP was performed using anti-HA Magnetic Beads (Pierce). Beads were resuspended in TRIzol and RNA was extracted and analyzed by Northern blot as described above.
Western Blot Analysis. IPs were done as described above (excluding SARS-CoV-2 infection), and supernatants were mixed with 4× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Typically, 25 μL (corresponding to ∼40 μg total protein) were separated on a 10% SDS-PAGE gel and electrotransferred to a poly(vinylidene difluoride) membrane (Bio-Rad). After blocking with 5% milk in 1× TBST (20 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), the membrane was probed with the appropriate antibodies and detected with Western Lightning Plus-ECL (PerkinElmer) using a Gbox (Syngene). Primary antibodies used were anti-FLAG M2 (Sigma Millipore), anti-Ago2 (MA5-23515; Invitrogen), and anti-GAPDH (Cell Signaling).
Target Predictions. Custom Perl scripts that use the RNAduplex algorithmpart of the Vienna RNA Package (53)-were used to hybridize the CoV2-miR-O7a sequence to mRNA transcripts obtained from GENCODE (v38)fragmented into 50-nt windows with 5-bp steps. Fragments with the lowest hybridization energy (ΔG) were chosen for further analysis.
Synthetic RNA Transfections and qPCR. Synthetic RNAs (see SI Appendix, for sequences) were annealed by heating equimolar concentrations for 1 min at 90°C in siRNA buffer (Horizon, 60 mM KCl, 6 mM Hepes [ pH 7.5], and 0.2 mM MgCl 2 ) and then incubating for 1 h at 37°C. HEK293T cells (5 × 10 5 ) were transfected with 30 μM of either CoV2-miR-O7a or control siRNA by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). Cells were collected 48 h later, RNA extractions were performed using TRIzol, and samples were treated with RQ1 DNase (Promega). cDNA was made using SuperScript III and random primers (Invitrogen), and qPCR was performed using FastStart Essential DNA Green Master (Roche). Primer sequences are given in SI Appendix, . Results were analyzed using the comparative Ct method.
In Vitro RNA Transcription and Processing. The sequences were PCR-amplified from templates containing desired mutations by using flanking primers; the forward primer contained the T7 promoter (see SI Appendix, for sequences). The PCR product was purified and used for in vitro transcription at a final concentration 25 ng/μL. The reaction was carried out for 4 h at 37°C and contained 400 mM Hepes [pH 7.5], 120 mM MgCl 2 , 200 mM dithiothreitol, 10 mM spermidine, 4 mM of each ribonucleoside triphosphate , 20 U RNase Inhibitor (Roche), and 5 U of laboratory-made T7 RNA polymerase. The product was purified by 8 M urea 6% PAGE, extracted in G-50 buffer (20 mM Tris [pH 7.5], 0.3 M NaOAc, 2 mM EDTA, and 0.1% SDS), and phenol-extracted. To ensure hairpin formation, RNAs were annealed by heating in siRNA buffer for 1 min at 90°C and then incubating for 1 h at 37°C.
HEK293Ts, Huh7.5, or Huh7.5 knockout (KO) cells (5 × 10 5 ) were transfected with 30 μM of in vitro-transcribed RNAs by using Lipofectamine RNAi-MAX Transfection Reagent (Invitrogen). Cells were collected 6 h later, RNA extractions were performed using TRIzol, and samples were processed for Northern blotting as described above.
Luciferase Reporter Assays. Four sites complementary to CoV2-miR-O7a (or scrambled sequence as control) separated by 20-nt-long spacer sequences were PCR-amplified using four primers overlapping at the unique spacer sequences (listed in SI Appendix, . The PCR products were cloned downstream of Renilla luciferase of psiCHECK(TM)-2 vector (Promega) using XhoI and NotI sites.
HEK293Ts, Huh7.5, or Huh7.5 KO cells (5 × 10 5 ) were transfected with 30 μM of in vitro-transcribed RNAs by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). Cells were collected 6 h later, RNA extractions were performed using TRIzol, and samples were processed for Northern blotting as described above. Twenty-four hours later, 10 ng psiCHECK reporters and 2 μg pBlueScript II (Stratagene) were transfected using TransIT-293 Transfection Reagent (Mirus). After an additional 24 h, Firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega) on a GloMax-Multi+ Microplate Multimode Reader (Promega) according to the manufacturer's instructions.
Data Availability. The RNA sequencing data generated in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database (accession no. GSE183280). All other data are included in the article and/or supporting information.Custom Perl scripts (available in the SI Appendix) that use the RNAduplex algorithm-part of the Vienna RNA Package part of the Vienna RNA Package (53)-were used to hybridize the CoV2-miR-O7a sequence to mRNA transcripts obtained from GENCODE (v38) (75) fragmented into 50-nt windows with 5-bp steps. |
Guy's Hospital Reports
We have been more than usually pleased with this Number. The papers are just what Hospital Reports, in our opinion, ought to be ; and we shall therefore present our readers, in this article, with a pretty full analysis of their contents. I. Cases selectedfrom the ward-hooks of Petersham House, with remarks; by John C. W. Lever, m.d. This is an interesting paper illustrating several forms of disease. The first group of cases consists of uterine polypi, the special character of the growth being vesicular in six of the cases, fibrous in two, and malignant in two.Dr. Lever's object in narrating the former of these, has been to draw attention to the symptoms which they usually produce, rather than to enter into any explanation of their nature, origin, and seat. The continued irritation of the surface of the womb which is in contact with the polypus produced central uneasiness, and pains in the loins, groins, and thighs. The neck of the uterus becomes affected with subacute or chronic inflammation ; its glands are enlarged; there is sometimes hypertrophy, with induration, and, in most instances, superficial ulceration. The discharge is very like the decoction of quince-seed ; and when ulceration is present, the glutinous discharge is usually mixed with one of a mucopurulent character, and occasionally streaked with blood. In some instances, as in Case iv here narrated, these growths coexist with polypi of a fibro-cellular structure. From one patient Dr. Lever removed seven in the course of eight years. They are equally common in the married and the unmarried, and frequently remain long undetected. The treatment is simple?removal of the growth either by the forceps or scissors; reduction of inflatnmation, if such be present, by local depletion ; washing away discharges, and relief.of pain by medicated injections, as the decoct, papav., with liquor plumbi diacet., and the cure of superficial ulcerations by the occasional application of lunar caustic.The two cases of fibrous polypi do not call for any remarks. They were both removed by ligature. Dr. Lever observes that he prefers using the 1850.] Dr. Lever's Cases. 75
single tube whenever it is possible, as it is not so likely to injure the patient if she should make any sudden movement during sleep, nor is there the same necessity to keep her on her side as when the double canula is employed. In all cases where this operation is performed, the bladder should be closely watched, as much pain and fear may be occasioned by overlooking its distension.
In one of the cases of malignant polypus, the tumour was removed, to the great temporary relief of the patient. In doing this Dr. Lever advises the surgeon to use the largest whipcord that will pass through the canula, and only to tighten the ligature every other day instead of daily. If there be much hemorrhage at the time of operation, it will be advisable to employ the tampon, confining this by a compress placed over the vulva, and keeping all in position by means of the T bandage. Dr. Lever prefers the ligature, where practicable, to tlie application of nitric acid, or the nitrate of silver.
Our author next narrates six cases of pelvic cellulitis, a disease which is very frequently overlooked or mistaken. In the majority of instances it is not caused by mismanagement or neglect during labour. It may follow abortion and labour, either premature or at the full term. It will come on after preternatural, complex, or flooding delivery, and is sometimes very prevalent during an epidemic of puerperal fever. The child born may be small or large; but in cases where there has been a want of proportion between the foetal head and the pelvis, and where, consequently, the labour has been lingering, Dr. Lever has observed that the part of the pelvis affected corresponded with the position of the occiput, the left side being, therefore, most frequently involved. Patients of a strumous constitution are most susceptible of the disease. The symptoms usually supervene on the second or third day after delivery; in some instances they come on actively, in others insidiously ; quickness of pulse, and anxiety of countenance being the most constant attendants. In all cases the secretion of milk, and the lochia are diminished, and in some they are arrested. Pain is a constant symptom, at first confined to the part inflamed, but afterwards radiating along the nerves of the region. The decubitus of these patients is most marked ; they lie on the back or on the sound side, with the thighs flexed, and the affected extremity resting on the sound limb, and rotated inwards, precisely as in cases of diseased hip-joint. The disease terminates either in resolution, suppuration, or absorption of the inflammatory deposit. The abscess may burst externally or internally into the vagina and neighbouring viscera. Sometimes the matter burrows deep into the pelvis, and the bone becomes diseased.
In the treatment, local depletion, repeated according to the severity of the disease and the state of the patient's strength; warm poultices, simple or medicated ; tepid anodyne injections carefully thrown into the vagina; attention to the state of the bowels, which are prone to irregularity; and the maintenance of a gentle mercurial action, are the most advisable means to be employed. The diet must be bland and nutritious, and the powers of the patient must be supported. When matter is formed it should be evacuated, as soon as that can be done with safety. Dr. Lever has great faith in blisters for the purpose of hastening the external progress of the pus, and promoting the absorption of the induration which foflows suppuration. For the latter purpose the iodide of potassium is also useful. Case x-viii is an example of monocystic ovarian dropsy, which is chiefly interesting as illustrating the advantage of not entirely emptying the sac when paracentesis is performed. The patient first came under Dr. Lever's care in 1844, and the last tapping took place in May 1849, there being throughout the whole of that period an entire absence of constitutional symptoms. On each recurrence of the operation however, a larger quantity of fluid was withdrawn, and the final result we suspect is not doubtful.
Case xix is a good specimen of the aphthous condition of the vagina, which was cured by the use of a lotion of argent, nitrat., gr. ij ad 5J.
Case xx is rare. The nymplue presented an appearance as of spots ot eceliymosis, and upon introducing the speculum, the mucous membrane 76 Guy's Ilosjrital [Jan.
of the vagina, as well as that covering the cervix uteri, was found to be similarly affected. The patient was a healthy young woman, married ten years, and having two children, the last three years old. She complained of severe pain at the lower part of the back, shooting up from the lower part of the abdomen, where she had also a bearing-down forcing pain.
She passed her water with tolerable freedom, as also her motions, but she suffered severely from bearing-down pains when the bowels were relaxed.
The uterus was retroverted, heavy, and tender.
Case xxi is an example of abnormal formation of the sexual organs. The vulva was placed more anteriorly than usual; the genital fissure was of the ordinary extent, but on inspecting the perineum, no anal aperture was discoverable, although in its place there was a slight depression of the integuments, of a paler colour than the surrounding parts. On passing the finger within the vulva, and pressing it slightly against the symphysis pubis, it readily entered the vagina, and could be carried along the canal to the os uteri, which was situated anteriorly, small, and its lips imperfectly developed. When the finger still within the vulva, was withdrawn to within one inch of the external parts, and then pressed downwards and backwards, it passed into the rectum ; so that the two canals, vagina and rectum, were merely separated by a thin partition, entirely concealed within the labia ; the termination of both being in one common cloaca. This is followed by the record of three cases of tumour of the labium. The first, a simple fibrous tumour, does not require any notice. The second was an encysted tumour cured by seton. The third was a large sanguineous tumour, suddenly produced during an epileptic fit, the patient being six months advanced in pregnancy. In this respect it is remarkable, giving striking evidence of the violence of the muscular contractions during the fit.
Case xxv is an instance of division of extensive cicatrices of the vagina, resulting from severe and long protracted labour. The operation was successfully performed without injury to the neighbouring viscera, but the patient was seized with peritonitis on the third day, and the attack proved fatal. A low form of erysipelas was very prevalent in the hospital at the time.
The case affords another instructive illustration of the danger attendant upon operations on and wounds of the vagina. The next two cases are examples of chorea occurring during pregnancy. The irregular movements were in a measure controlled by treatment, but did not cease in either case until after delivery.
Case xxviii is an instance of suppuration in the lymphatic glands of the neck, and in several of the large joints, proving fatal in a few days. The woman had resided in a cellar contiguous to the common sewer, which, for some days previous to the attack, had emitted a most offensive smell.
The last two cases in this paper are interesting, rather morally than medically. In one pregnancy and delivery were feigned for the purpose of revenge; in the other the more common falsehood of denial was exemplified.* * There is a very funny misprint in the narrative of this case; the abdomen is represented as being found " placid, but marked with lines crescenting towards the groins"?a contrast to the turbulence of labour ! We may take this opportunity of observing, thai the editors would do well to attend a little more closely to the correction of the press. There are many other errors which should not have been left unnoticed.
## 1850.]
Mr. France's Case of II. Case of Ptosis ; by John F. France. > The regular readers of these Reports will doubtless remember Mr. France s former paper on this disease, in which he demonstrated its cerebral origin. The case now recorded is particularly interesting as being a rare example of purely functional disorder. The patient was a female, aged 19, of moderately healthy aspect, but of a highly excitable temperament. She was a barmaid, and had been able to attend to her duties until the 20th July. On the morning of tliatdayshe experienced a continued quivering?"life-blood ?in the left upper eyelid, but was able to open the eye well. In the course of some hours, during which the above symptom persisted, the eye became painful on exposure to light; towards evening it became entirely closed, and could not be opened even in the dark, and a sensation of giddiness was the result. The intolerance of light continued, together with the inability to elevate the upper lid, either in the dark or the light, until her application at the hospital on the 23d. The right eye, meanwhile, was quite free from ailment of any kind. Her previous health had not been very good ; the catamenia were habitually irregular, and had been absent for three months. Some months before she had been ill, retention of urine and flatulent distension of the abdomen being promineiit features in her disease. She had suffered from globus and palpitation, but had neverany hysterical fit. When first seen, the left upper eyelid was completely closed, and could not be separated from the lower by the effort of its proper muscle; the loss of power extended also to the anterior portion of the occipito-frontalis. The orbicularis, however, retained its contractility ; and, owing to a great degree of morbid irritability which existed in the neighbouring integument and conjunctiva, was thrown into spasmodic action when any attempt was made to raise the lid. The globe of the eye, when examined, was found quite healthy, and vision good ; but considerable intolerance of light. There was no other paralysis of the muscles of the face ; nor was motion or sensation impaired in any other part.
The case was successfully treated as hysterical. We quote Mr. France's account of the way in which he arrived at this diagnosis, because it appears to us a very admirable specimen of the right method of proceeding in circumstances of such a nature. " Before examining the real pathology of the foregoing case, it may be as well to notice the circumstances attending it, which altogether exclude the supposition that the dropped eyelid was a simulated complaint. I would not lay too much stress on the moral evidence forbidding that supposition; such as the desire of the patient to retain her situation and get well (which was attested through a different channel), and the fact that the disease was one which she had never seen an example of, and therefore could not imitate. Independently of these considerations, there was enough in the objective symptoms of the case to annul the idea of deception. Thus, it is a physical impossibility for any one cither to create that partial muscular spasm, commonly known as the c life-blood,' which was obvious to the bystanders at the outset of this case ; or to cause that peculiar abnormal excitability of the pupil which marked its later stage. Nor, further, was the closure of the lid the mere instinctive result of photophobia ; cases in which it is so apply by hundreds, and hardly admit of misconception. They are invariably relieved in direct proportion to the individual's seclusion from light, and both eyes invariably participa e m the affection. The present, however, stood in obvious contrast to sucii cases m both these respects, and therefore could have no pathological relation to tnem.
## 78
Guy's Hospital Reports.
[Jan.
" Again, that it was not of identical nature with the ordinary instances of paralytic ptosis was proved, by the general healthiness of the patient, by the entire absence of premonitory or concomitant cerebral symptoms; by the immunity of all the muscles supplied by the third cerebral nerve, except the levator of the lid; by the non-dilatation of the pupil; by the perfect state of vision, &c. " The above considerations, by successively negativing the different hypotheses which might probably suggest themselves as to the essential character of this case, go far towards revealing its true nature, and at least clear the ground for a valuation of the more direct and positive evidence. The substantive proofs that the affection was hysterical are based on its history, its symptoms regular and anomalous, and the process of cure. " As respects the history, the facts of most important bearing upon the case were ascertained, in great measure, from a collateral source, after the formation of the diagnosis, and the prescription of corresponding treatment. The facts referred to are those of the excitable habits of the patient; the irregularity of the catamenial flow; her frequent suffering from globus and flatulence; and the distinct attack of retention of urine. As respects symptoms, one directly indicative of hysteria was the intense morbid irritability of the integuments about the orbit and of the conjunctiva, so that the least touch was shrunk from with apprehension; that irritability not being in relation with any apparent adequate cause, inflammatory or otherwise. Other symptoms, indirectly pointing out the same conclusion (from their anomalous character irreconcileable with any more ordinary form of the complaint), were, besides those already noticed, the one-sided intolerance of light, and exalted susceptibility of the iris,?the former symptom not found among, the latter opposed to, the phenomena of the common paralytic affection; and lastly, the simultaneous privation of power of the levator palpebral and adjacent portion of the occipito-frontalis, which are functionally allied, but anatomically quite unconnected.
The force of this sign is derived from the circumstance, that the other muscles of the groups, to which these respectively belong, all, without exception, retained their usual energy; and thus, in departing from the type of true paralysis, which always corresponds with the distribution of the nerves, the disease betrayed that remarkable capriciousness so characteristic of hysterical complaints." III. Contribution to the anatomy of double monsters; by Alfred Poland. An analysis of this paper could not be made either interesting or instructive. We must therefore refer our readers to the original.
IV. Considerations connected with the pathology and surgery of urinary
[formula] concretions ; by B. B. Cooper, f.r.s. [/formula]
The chief peculiarity in this paper is the advocacy of the doctrine that carbonic acid is the natural solvent of the earthy salts in the urine, and that their deposition upon foreign bodies in the bladder is due, not so much to the irritation produced on the mucous lining of the bladder, as to an action set up by them in the urine itself.
"It is well known," Mr. Cooper observes, "that any solid body, having an uneven surface, and offering points and asperities, will cause the rapid evolution of carbonic acid from any fluid impregnated with that gas: thus, for example, if into soda water, in which the effervescence from the spontaneous liberation of the gas has completely ceased, we throw a few pieces of cuttings of metal, or even bread or wood, gas will again begin to be given off from the fluid. If, then, we consider (and I think it scarcely admits of doubt) that carbonic acid is, at least in some measure, the solvent for the earthy constituents of the urine, it will at once be seen that the presence of a foreign "body of irregular figure may cause the gradual but continuous discharge of the carbonic acid from the urine; and as the liberation of this gas takes place from the points presented by the foreign substance, it fol-lows that the earthy matter, thus deprived of its solvent, is precipitated upon that body. This precipitation takes place, however, with extreme slowness; so much so, that the particles aggregate as they arc deposited upon the nucleus. In addition to this natural aggregation of particles (which may always be observed when solid matter is deposited from a solution by slow degrees, even when regular crystals are not formed), the vesical mucus seems to operate as a kind of cement, assisting in the consolidation of the calculus during its formation If, on the other hand, the foreign substance be not of a nature to produce great irritation of the bladder, or to induce the liberation of gas; and if at the same time the fluid portion of the urine be from any cause diminished, or uric acid generated in excess ; the deposit would probably consist of uric acid or the alkaline urates." (pp. 270-1.) Several cases are narrated which seem to bear out these views, especially some instances of calculi partially inclosed in cysts, and presenting layers of the earthy phosphates on that side only which had been exposed to the action of the urine.
Still, we think that more is to be attributed than Mr. Cooper seems to allow to the influence of the mucus, poured out under the irritation setup by the presence of the foreign body; that mucus exciting a catalytic action like a ferment, and thus producing decomposition of the urea and formation of carbonate of ammonia, which neutralizes the dissolving acid and precipitates the phosphates.
Mr. Cooper's views of the pathology of the various urinary deposits are quite in accordance with those held by the best observers, and his rules for treatment appear to us highly judicious.
V. Select cases of hernia. Third series ; by E. Cock.
The first case narrated illustrates the danger to which a patient is exposed, who is the subject of irreducible omental hernia. The subject of this case was admitted four times with symptoms of strangulation, the last attack proving fatal after operation. In commenting upon it, Mr. Cock correctly observes that a portion of irreducible omentum, so far from forming a plug to the abdominal outlet, offers actual facilities for the future descent of intestine, at the same time that it, in great measure, renders abortive the usual means employed to prevent the recurrence of a hernia. It also renders the diagnosis and the treatment of every fresh protrusion more obscure, uncertain, and dangerous. Besides this, the surgeon must not forget that there is the probability of mischief from bands of omentum extending from the abdomen through the rings, and giving rise to fatal and irremediable obstruction. Mr. Cock appears, therefore, inclined to advise an operation in these cases, as the best and safest measure. He is a strenuous advocate for the external division of the stricture. When the patient will not submit to an operation, it is, of course, necessary that some mechanical means should be employed to prevent, as far as may be, the descent of intestine. For this purpose, instead of a truss, Mr. Cock places a pad, closely stuffed with wool, over the tumour, and secures it in its position by a roller of caoutcliouc-webbing, a few turns of which are passed round the loins and the thigh, so as to form a figure of 8 crossed over the groin.
The succeeding cases afford additional proof of the value of calomel and opium in the treatment after operation, and of the advantage of abstaining from aperient medicines. In one case, the bowels were not relieyec for nine days, during the whole of which time the patient steadily improved. Free evacuations were then procured by a simple enema. [Jan.
The same case is also very instructive in another respect. When the sac was opened, the intestine was found black, with a narrow band of omentum, black and gangrenous, coiled round it. The parts were carefully separated, the omentum excised, and the bowel returned into the abdomen. In doing this, Mr. Cock remarks : " I followed a principle of which experience has taught me the value,?that the safest place for an intestine not actually perforated, but which is in a doubtful condition, and of which any portion is likely to lose its vitality, or to become opened by ulceration, is the abdomen. By restoring the gut to the peritoneal cavity, we afford a fair opportunity for the mending of any subsequent breach in its coats and for the preservation of the integrity of its canal, by the adhesions which it may contract to the opposite serous surfaces. I believe the patient will thus have a better chance of recovery than that afforded by trusting to the forlorn hope of leaving the bowel in the hernial sac. I some time ago operated on a young woman, in her fifth month of pregnancy, and returned the bowel without opening the sac. She went on favorably for a week, when she miscarried, and died of hemorrhage. On examining the portion of intestine which had been strangulated, I found a piece about the size of a shilling, including the whole thickness of the gut, had been completely separated, although not yet thrown off, by a process of sloughing. The breach, however, was accurately patched by adhesions to the neighbouring intestiue, and no reasonable doubt could be entertained, but that, had she lived, the reparative process here exerted by nature would have been perfectly successful." (pp. VI. On mania, coexisting with uterine disease; by John C. W. Lever, m.d. This paper contains the history of two very interesting cases, illustrative of the following propositions, of the truth and importance of which we are deeply persuaded :
1. That mania, developing itself in the female, is sometimes associated with, and depends upon, organic disease and irritation of the sexual organs.
2. That, unless remedial measures be applied to relieve such disease or irritation, the symptoms of insanity will be permanent.
3. That, in selecting the proper treatment, attention must be specially directed to the nature of the local disease. 4. That in most cases there is a diminution or cessation of the catamenial flux.
5. That at the close of the treatment counter-irritation in the neighbourhood of the sacrum is a valuable auxiliary. 6. That by pursuing this plan of treatment, before the symptoms of insanity become confirmed, in many cases it will be attended by success.
VII. Description of some of the tumours removed from the breast, and preserved in the museum of Guy's Hospital; by E. Z. Birbett, M.D. An analysis of this paper would be of little use. It is illustrated by six plates.
VIII. Case of complete 'placental presentation, treated by separation of the placenta and the application of electro-galvanism ; by This was a dangerous case brought to a successful issue by the means adopted. IX. A case of labour, complicated with a double state of the internal sexual organs ; and a report of other cases of defective development of the female sexual organs ; by Henry Oldham, m.d. This is a communication replete with interest and instruction, and to which we would desire specially to direct the attention of our readers.
Dr. Oldham's assistance was requested in a first labour, the surgeon in attendance being unable to find the os uteri. The patient was 21 years ?f age, of diminutive stature, and girlish look. She had been in labour for some hours, and the pains were strong. On examination, a globular body, resembling a foetal head, was found in the pelvic cavity, but completely covered over by the sac which contained it, and which had no aperture. Dr. Oldham' observed, however, that this body was more on the left than on the right side of the pelvis, and that the finger could not pass entirelv around it at an equal height. An external exploration of the abdomen also showed that the uterus occupied, in a very marked manner, the left side, while the right was flattened and resonant. These discoveries led to the supposition that there might be a double vagina ; and by another digital examination, directed more particularly to ascertain this, another passage was discovered, with a smaller external aperture, leading directly to the foetal head. The labour was completed naturally. Six weeks afterwards Dr. Oldham again examined the sexual organs. The septum, which formed the bipartite vagina, was found complete throughout; it was a dense, well-organized tissue, sufficiently loose and elastic to stretch to either side of the vagina, without causing pain, so that both canals were equally capacious, and no feeling of constriction, or indeed any notable peculiarity, would have been observed in either of them. The os uteri on the left side was rather large, and fissured in its anterior lip ; the uterine sound curved very distinctly to the left side, and passed rather more than two inches and a half. The right os uteri was of virgin smallness, and its edges smooth and even. The sound passed to about two lines short of the two inches and a half, and curved very perceptibly to the right side.
There had been no impediment to sexual intercourse, and during pregnancy the catamenia had been completely suspended. Two preparations of a similar abnormal formation are preserved in the museum of Guy's Hospital. It is interesting to observe, in the above case, that when one uterus was impregnated, the catamenial functions of the other were suspended at the same time.
Dr. Oldham also relates two cases of double uterus, with a single vagina, which were diagnosed during life. In the first a septum of the cervix was first detected, and the sound revealed the rest. In the second, the separate cornua could be felt through the abdominal parietes ; the patient lay upon her back, with the knees drawn up, the index-finger of the right hand then fixed the cervix, and the body was felt by pressing the fingers of the left hand into the inlet of the pelvis. This is a method of examination which Dr. Oldham has found extremely useful in many cases. This is followed by a case of congenital absence of the internal sexual organs.
The undersized womb. Dr. Oldham has met with many cases in which the uterus has retained some of the characteristics of the infantile condition, being smaller and thinner than usual, with a diminutive cervix, and corresponding small aperture. He has also found the longitudinal striae in the cavity of the body of the organ, which in the healthy, full-sized, 9-v. virgin uterus are confined to the cervix. The vagina in these cases varies a good deal. Sometimes it is well formed, and in full proportions, but generally it is short and shallow, and rather contracted at its orifice. The principal signs of such a condition are scanty and generally painful menstruation, and, in the married woman, sterility. Amenorrhcea, for two or three months or longer, without any known cause, and without chlorosis, generally forms a part of the history of these cases.
It is of importance that this condition of parts should be well known ; for it is quite apparent that the adoption of mechanical means, to relieve the dysmenorrhcea, or to cure the sterility, would only be productive of mischief.
Dr. Oldham has noticed two complications, which are deserving of attention. The first, of which he has seen one example, is a disease of the cavity ; we quote the case : " A married woman, an out-patient of mine at the hospital, who had the characteristics and history of this organic defect of the sexual system, began, three years after marriage, to have a thick, white discharge, sometimes streaked with blood. She came under my care after five weeks' spotting of the red discharge, having all the aspect and emaciation of phthisis. Her lungs were carefully and frequently examined, but without detecting any tubercular disease. On examination by the speculum, I found that the discharge was not yielded from the vagina or cervix, which was healthy; but, as I pressed with the speculum, a small quantity oozed out from the os uteri. This was not the mucus from the cervix, but presented the characters of broken-up tubercle, imperfectly mixed with blood." (p. 367.) This disease, a true uterine phthisis, is not confined to the above type of womb. Dr. Oldham has carefully examined four specimens, and believes?
"That the glandular system of the cavity of the body of the uterus becomes filled with scrofulous matter. This at first swells out the lining membrane; then it' begins to break up, and, with the escape of the matter, the inner surface becomes ragged and deeply furrowed, and the uterine walls consume; that is, become thinner and thinner. This goes on equally over the whole surface; but in one specimen the breaking-up process had gone on so deeply towards one horn, as to form a cavity there, and, had the patient lived, I think it would have gone through the uterine peritoneum, unless its progress had excited inflammatory adhesion around it. This disease is strictly confined to the cavity of the body of the womb ; it does not touch the cervix; but a clear line, separating the diseased from the healthy structure, exists at the os internum. In one specimen in our museum, the vagina is attached, but the cervix is free." (p. 367.)
The other complication, to which reference was made above, is an anteversion of the body of the uterus, caused by a weakened state of the tissue of the organ, which renders it quite flexible. Dr. Oldham has never seen a true retroversion of the undersized womb.
How, then, are the dysmenorrhoea and the sterility to be cured? Dr. Oldham believes that much may be done by hygienic means and proper constitutional treatment, and lays down very judicious rules to this effect.
He is, as is well known, most strongly opposed to all mechanical treatment.
Where the womb is undersized, it is perfectly clear that neither dilatation nor supporting can do anything to remedy the defect, and no prudent practitioner would, we think, attempt either. We also most perfectly coincide in the opinion, that it is quite impossible to fix a standard size for the inlet of the womb; and that, to attempt forcible dilatation, merely because, in any particular instance, tlie aperture is small, is mischievously meddlesome practice, which may prove fatally injurious, as we shall show immediately by most lamentable proofs. In judging of the existence or non-existence of morbid contraction, respect must be had to the relation of parts. A small orifice in a small cervix is natural; the same sized opening in a long, bulky cervix would be abnormal. Cases of the latter kind do, every now and then, occur, and have been effectually and safely remedied by mechanical means, either dilatation or incision. The last operation is the most speedy, and perhaps the most effectual, but we have known foi'midable hemorrhage result from it.
We agree, again, with Dr. Oldham, in doubting the frequent relation of cause and effect between displacements of the uterus and sterility : because we cannot see that these curvatures of the body of the womb are sufficiently decided, at least in the great majority of instances, to prevent the passage of the seminal fluid towards the ovum. Nor, even granting that such may sometimes be the result, can we have any confident hope of cure from the use of the uterine stem-supporter. We remember a case, in which an instrument of that kind had been worn for many months, but the uterus immediately fell back into its old place, whenever the support was withdrawn. It could not, therefore, have remedied sterility. But though, in the instance just named, and in others which have come to our knowledge, the instrument in question was productive neither of suffering nor injury, it is now too plain that such is not always the case ; that these mechanical means, if inoperative for good, are not invariably so as regards danger of the most formidable kind. And therefore we feel it a duty to lift our voice in solemn warning, and to urge, at the very least, the extremest caution in the employment of such expedients. To arrive at a doubtful good by means that have been proved to be fatally dangerous, even in well-skilled hands, is scarcely consistent with right principle. But example is ever more effective than mere precept, and therefore we shall quote in extenso the two most melancholy cases which Dr. Oldliam has here put on record. " On April 7th, 1849, I received from Dr. Golding Bird the uterus and appendages of a lady who had died from peritonitis, excited by attempts to cure sterility by mechanical dilatation, whose history, as furnished to me by Dr. Bird, is as follows, and with whose concurrence I publish it:?' A lady of dark complexion, aged 36 ; married several years, and never pregnant; resided in Jamaica. From youth she suffered intense dysmenorrhea, and always had pains during sexual intercourse. She was nervous, hysterical, and excitable to the last degree, and was supposed to have suffered from every possible form of inflammation; these attacks obviously being neuralgic, so common in hysterical women. In June last, by the advice of her physician in Jamaica, she came to London for the express purpose of having the os uteri dilated, which had already been attempted by wax dilators.
The obstetric physician who was consulted in London coincided in this opinion, and thought the sterility and dysmenorrhoea depended on a stricture ol the os uteri. He divided the os uteri with a cutting instrument, and introduced silver dilators. This produced horrible suffering; and although at first she fancied the pains of menstruation were rather better, they soon became as bad as ever, and she did not experience the slightest relief. She left off the treatment for a time, but was soon again inclined to resume it; and silver canulse were passed into the os, and lett there. Again she suffered frightfully. On Saturday, March 31st, a gentleman, the assistant of the physician, passed in another tube, but the distress was intolerable; and sickness and shivering coining on, she urgently begged her sistei to try and remove it, which she succeeded in doing. Getting worse, a neighbouring surgeon was summoned, and he found her labouring under what he regarded as peritonitis masked bj hysteria. She had scarcely any fever, collapse coming on almost immediately, and she continued sinking until Tuesday, when I (Dr. G. Bird) was summoned to her. I found her at her sister's residence, at T Park, presenting almost the collapse of cholera : pulse 200, and a mere thread; distended abdomen; vomiting of black fluid; intense irritability. All treatment was useless, and she soon sank. On examining the body, and raising the omentum, no appearance of disease of any kind was found above a line connecting the anterior superior spinous processes of the ilia. Below this line there was intense peritonitis -. the convolutions of the intestines, covered with butter-like lymph, and the pelvis filled with pus-like fluid; the right ovary and broad ligament covered with the same butter-like lymph, but so feebly adherent, that it washed away by dipping it in water; the cavity of the uterus was lilled with bloody mucus. There was no other disease.' " The uterus and appendages examined by Dr. Oldham : " The uterus had been opened by a single oblique division of the anterior wall, directed from the cervix to the left angle of the womb. The uterus was larger than usual for the virgin; it was rounded on its anterior surface, and a bulging convexity of the posterior wall, which, with the general softness of the tissue, showed it to have been the seat of recent engorgement. The blood-vessels over the entire surface of the uterus and appendages were injected with blood, especially the fimbriated extremity of the tubes, the ovaries, the broad and round ligaments. On the anterior surface of the body of the uterus were two small, projecting, fibrous tumours, the size of a large and small pea; the serous investment of them was highly vascular, the blood-vessels rising over them just .like the calyx of the ovarian ovum of the bird. There was a similar, more flattened growth in the posterior wall. The divided surface of the anterior wall showed its proper structure to be much enlarged (it measured in the body eight lines); the muscular structure was soft, and the veins large: a probe easily ran through them. The length of the united cavities was two inchcs and ten lines, the canal of the cervix being one inch five lines. The mucous membrane of the cavity of the body was soft, slightly raised, and of a vermilion hue. Agitation in the water was sufficient to loosen and separate it. At [the os uteri internum there was a zone of highly-injected blood-vessels, broken only at one point; the circumference of this aperture was eight lines. The os externum had a clean, smooth edge, without any break or mark of division; its circumference measured one inch one line. The cervix had its characteristic markings, and the glands were empty of mucus. On the right side of the divided cervix, which would have formed the front wall, the ribbings were stretched upwards, enlarging the mesh-like appearance; and towards the os internum some were lacerated transversely, and from this to the os externum the structure was more ragged than usual.
" The right Uibe. The extremity of this tube was almost entirely closed as a congenital' formation, the aperture being very small. "When opened, the fimbriated end showed its characteristic rich folds of mucous membrane, which were much injected, and were covered with bloody mucus. The remaining two thirds of the tube was apparently healthy, not vascular, and pervious throughout.
" The right ovary, which was almost covered with lymph, was soft and large. There was a cyst large enough to hold a small nut 011 the uterine end of the ovary. The stroma was gorged with blood. There was only one puckered Graafian follicle.
The surface of the ovary was thick and corrugated. The left ovary was irregular in its shape, a projecting mamillary portion coming out from its outer end. This, on being cut into, was hard and vascular, like the commencement of malignant disease; the ovarian tunic was thick and wrinkled; the stroma vascular; a few remains of Graafian vesicles, with puckered tunics, and some clots of different colours, black and brownish. The left tube vascular at its fimbriae, healthy in its mucous membrane, and its canal pervious throughout. This 1850.] Drs. Daniell and Mackinnon on Tropical Ilygibie. 85 tube passed into the uterus more directly than its fellow, which was more curved. Tiie veins healthy; the arteries healthy; the right round ligament large and vascular ; vagina healthy. " It is unnecessary to comment at length on this case. _ It affords a most instructive example of the dangerous effects of dilatation, even in experienced hands, and the great caution with which it should be undertaken. It is important, too, as showing the difficulty of detecting the cause of sterility. I am sure that there was no kind of morbid contraction in this case, and that the os and cervix uteri, which were alone treated, had nothing whatever to do with the dysmenorrhcea or sterility. Both of these, no doubt, were dependent on the atrophy of the ovary; and the congenital obliteration of the end of the right tube would have been sufficient to exclude the corresponding ovary from any share in the function of feproduction."
The next case exemplifies the dangers attendant upon the use of the uterine stem-supporter. It was related to Dr. Oldham by Mr. Bransby Cooper: " A young married lady, of great personal attractions, was attended by Mr.
Cooper, for a very painful fissure in the anus, which he divided and speedily cured.
She then spoke to him of what had been to her a very distressing social trouble, namely, her sterility, which she associated with a perfect indifference to sexual intercourse. Mr. Cooper examined the sexual organs ; but as he did not discover any defect which could be remedied by surgery, he referred her to a physicianaccoucheur. Tliis gentleman detected the uterus in a retroverted state, which he looked upon as the probable cause of the sterility. For the cure of this displacement, he introduced a uterine stem-supporter, which set up peritonitis, of which she died in three days." |
Similar early characteristics but variable neurological outcome of patients with a de novo mutation of KCNQ2
Background: Early onset epileptic encephalopathies (EOEEs) are dramatic heterogeneous conditions in which aetiology, seizures and/or interictal EEG have a negative impact on neurological development. Several genes have been associated with EOEE and a molecular diagnosis workup is challenging since similar phenotypes are associated with mutations in different genes and since mutations in one given gene can be associated with very different phenotypes. Recently, de novo mutations in KCNQ2, have been found in about 10% of EOEE patients. Our objective was to confirm that KCNQ2 was an important gene to include in the diagnosis workup of EOEEs and to fully describe the clinical and EEG features of mutated patients.Methods:We have screened KCNQ2 in a cohort of 71 patients with an EOEE, without any brain structural abnormality. To be included in the cohort, patient's epilepsy should begin before three months of age and be associated with abnormal interictal EEG and neurological impairment. Brain MRI should not show any structural abnormality that could account for the epilepsy. Results: Out of those 71 patients, 16 had a de novo mutation in KCNQ2 (23%). Interestingly, in the majority of the cases, the initial epileptic features of these patients were comparable to those previously described in the case of benign familial neonatal epilepsy (BFNE) also caused by KCNQ2 mutations. However, in contrast to BFNE, the interictal background EEG was altered and displayed multifocal spikes or a suppression-burst pattern. The ongoing epilepsy and development were highly variable but overall severe: 15/16 had obvious cognitive impairment, half of the patients became seizure-free, 5/16 could walk before the age of 3 and only 2/16 patient acquired the ability to speak.Conclusion: This study confirms that KCNQ2 is frequently mutated de novo in neonatal onset epileptic encephalopathy. We show here that despite a relatively stereotyped beginning of the condition, the neurological and epileptic evolution is variable.
# Background
KCNQ2 encodes a channel subunit carrying the neuronal Im current whose inherited mutations were first described in autosomal dominant benign familial neonatal epilepsy (BFNE, OMIM#121200) [bib_ref] A potassium channel mutation in neonatal human epilepsy, Biervert [/bib_ref] [bib_ref] A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy..., Singh [/bib_ref]. Patients affected by a BFNE displayed stormy phase of motor seizures during the neonatal period, lasting 2 to 6 weeks in average. Interictal EEG was normal or slightly modified. Subsequently, seizure frequency quickly decreased and the vast majority of patients became seizure free before the age of three months. Motor and cognitive outcome were usually normal. Recently, de novo mutations of KCNQ2 have been described in early onset epileptic encephalopathies (EOEEs; OMIM#613720) [bib_ref] Neonatal convulsions and epileptic encephalopathy in an Italian family with a missense..., Dedek [/bib_ref] [bib_ref] Whole exome sequencing identifies KCNQ2 mutations in ohtahara syndrome, Saitsu [/bib_ref]. EOEEs are a group of devastating epilepsies beginning before three months of age, with frequent seizures and abnormal interictal EEG leading to a rapid deterioration of motor, cognitive and sensori-neuronal functions. Patients carrying de novo KCNQ2 mutations displayed abnormal interictal EEG that could reveal multifocal spikes or a suppressionburst pattern, and all had poor neurological outcome [bib_ref] Whole exome sequencing identifies KCNQ2 mutations in ohtahara syndrome, Saitsu [/bib_ref]. This dramatic form of KCNQ2-related epilepsy, with very poor neurological outcome, was unexpected. In order to assess the importance of KCNQ2 screening for the molecular diagnosis of early onset epilepsies, and mostly to describe the outcome of the sporadically mutated patients, we have analyzed a cohort of 71 patients with an early onset, severe epilepsy, without any familial history of epilepsy.
# Methods
This study was approved by CPP Sud Méditerannée (Comité de protection des personnes). Seventy one patients were included in a cohort of subjects who displayed an early onset epileptic encephalopathy. All the patients or their parents gave their informed consent to join the cohort. Inclusion in the cohort was decided according to the following criteria; (1) epilepsy onset within the first 3 months of age; (2) abnormal interictal EEG (3) brain MRI without obvious cortical malformation or hypoxic lesion; (4) normal metabolic screening (exclusion of nonketotic hyperglycinemia, hyperammonemia, urea cycle defect, organic aciduria, hyperlactacidemia, pyridoxine-dependent and pyridoxal-dependent seizures);No mutation of STXBP1, a major gene involved in early onset epileptic encephalopathy with or without suppression-burst [bib_ref] De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile..., Saitsu [/bib_ref] ; [bib_ref] Neonatal convulsions and epileptic encephalopathy in an Italian family with a missense..., Dedek [/bib_ref] No mutation of ARX [bib_ref] A longer polyalanine expansion mutation in the ARX gene causes early infantile..., Kato [/bib_ref] in male patients (n=35); (7) patients must be regularly followed till now. All the girls that displayed early onset epileptic spasms and/or tonic seizures without any suppressionbursts were tested for CDKL5 (n=36). The epilepsy began during the neonatal period for 47/71 patients, the EEG showed a suppression-burst or discontinuous traces in 33 of them (Groupe A), and multifocal spikes in the remaining 14 (Groupe B). Epilepsy began between 1 and 3 months for the 24 patients of groupe C. The 18 coding exons (including alternative exons) of KCNQ2 were sequenced. Primer sequences are available upon request. The identified mutations were numbered according to the KCNQ2 reference sequence NM_172107.2.
# Results and discussion
We found heterozygous mutations in KCNQ2 in 16/71 patients [fig_ref] Table 1: KCNQ2 mutations and main features of the patients Tonic and tonic-clonic, cyanosis [/fig_ref]. All of them have occurred de novo. Typically, the first seizure was observed before the 5 th day of life (n=14/16), taking the form of clonic and/or tonic seizures resembling those observed in BFNE (12/16). These seizures were very frequent, rapidly leading to obvious neurological impairment before the end of the first week (10/16). Eight patients carrying a KCNQ2 mutation were initially diagnosed with an Ohtahara syndrome, with a typical suppression-burst pattern on the EEG (Table 1, . The first EEG did not show any suppression-burst pattern, but discontinuous traces in the remaining patients (Table 1, . In three cases, EEGs evolved into a hypsarythmic pattern, but the majority quickly developed into a continuous pattern with multifocal asynchronous spikes and/or slowing of the traces (13/16). The outcome of epilepsy was highly variable: 9/16 patients became seizure free during the followup, 6 of them before the end of the first year of life, while 7/16 patients were still epileptic, three of them had only myoclonic jerks, two of them had recurrent generalized tonic clonic seizures and two had focal seizures [fig_ref] Table 1: KCNQ2 mutations and main features of the patients Tonic and tonic-clonic, cyanosis [/fig_ref]. Fifteen patients had obvious developmental delay: 4/15 could walk but 3/4 had no language and 1/3 had autistic features; 11/15 were profoundly impaired with poor or absent head control and eye contact (8/15) or global/ axial hypotonia with poor or absent hand use (3/15). One patient had a good evolution with normal neurological evaluation at age 6. The initial brain MRI was normal or showed very slight and transitory brain signal abnormalities in 12/16 patients, while in 3 patients, abnormal signal intensity were found, as previously described [fig_ref] Table 2: Data on initial evaluation and treatment, EEG evolution and brain MRI Suppression-burst [/fig_ref]. Only one patient had extra-neurological features: congenital left hip luxation and cleft palate (patient 2). Fifteen patients had a mutated KCNQ2 in the group A (45%, n=33), one patient had a mutation of KCNQ2 in the group B (7%, n=14) and none of the patient had a KCNQ2 mutation in the group C. Thus, KCNQ2 was mutated in half of patients with a neonatal onset epileptic encephalopathy and an EEG showing either discontinuous or suppression-burst pattern. Here, we confirm that, besides well described entity BFNE, KCNQ2 mutations can also be associated with severe epileptic and cognitive phenotypes defining early onset epileptic encephalopathies. Hence, it should be considered in the diagnosis workup of neonatal onset epilepsies especially those beginning during the first week of life with stormy clonic and/or tonic seizures, whatever the presence or absence of a familial history. If cognitive outcome is relatively reliable and good in familial cases of BFNE, it is not the case in sporadic ones, where neurological outcomes range from dramatic to normal. We did not find any relationship between the initial history of the epilepsy and the severity of outcome. For example, patients 1 and 12 had relatively similar features at the beginning and displayed very different outcomes (Tables 1, 2 and . Since KCNQ2 is now implicated in various forms of epilepsies, from the most benign to the most dramatic, additionnal data on phenotype/genotype correlations would be particularly relevant. Interestingly, none of the mutation reported in neonatal epileptic encephalopathies had previously been reported in BFNE, and the severe mutations that have been found in several patients (p.G290A, p.T274M and p.A294V(present study)) lead to relatively similar features in terms of initial EEG and development, however different in terms of evolution of the epilepsy. The different epileptic features in patients carrying the same mutation of KCNQ2 may be due to genetic modifiers or non genetic factors. Overall, this cohort of patients highlights the heterogeneous evolution of the neurological phenotypes associated with de novo heterozygous mutations in KCNQ2. This heterogeneity could be at least partially related to the impact of the mutations on the Im current. Analysis of the functional consequences of "benign" versus "severe" mutations in KCNQ2 should be of paramount importance to better understand the molecular and cellular mechanisms involved in the emergence of an epileptic encephalopathy. This has recently been tested with two mutations of KCNQ2 affecting the same residue in the S4 domain of the protein KV7-2 but associated with either a benign phenotype, or with a neonatal epileptic encephalopathy with severe drug-resistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct
[formula] Fp2-T4 T4-O2 Fp2-C4 C4-O2 Fp1-C3 C3-O1 Fp1-T3 T3-O1 BREATH ECG Fp2-T4 T4-O2 Fp2-C4 C4-O2 Fp1-C3 C3-O1 Fp1-T3 T3-O1 BREATH ECG T4-Fp2 Fp2-Fp1 Fp1-T3 T4-C4 C4-C3 C3-T3 T4-O2 O2-O1 O1-T3 BREATH ECG T4-Fp2 Fp2-Fp1 Fp1-T3 T4-C4 C4-C3 C3-T3 T4-O2 O2-O1 O1-T3 BREATH ECG A B 1s [/formula]
100µV Representative early EEGs of patients carrying a de novo KCNQ2 mutation. A. Interictal EEG (Day 3, patient 1), showing a typical suppression-burst pattern, with burst of spikes and slow waves alternating with periods of electric silence (left panel). Sometimes, the burst should be much longer than the periods of suppression, leading to a discontinuous pattern (Right panel). B. EEG displaying the same features (Patient 12, suppression-burst in left panel, discontinuous pattern in right one), with a very different outcome (normal development at 5 years old, see [fig_ref] Table 1: KCNQ2 mutations and main features of the patients Tonic and tonic-clonic, cyanosis [/fig_ref]. neuroradiological features [bib_ref] Genotype-phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor..., Miceli [/bib_ref]. The authors showed that, while both mutations destabilized the open state of the channel causing a reduction of the voltage sensitivity, the functional changes were more pronounced in the "severe" mutation than in the benign one. In both cases, the functional impairment could be fully restored by the neuronal Kv7 activator retigabine. This study suggested that the clinical disease severity may be related to the extent of the mutation-induced functional K + channel impairment and set the preclinical basis for the potential use of Kv7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates. However, since two patients carrying the same KCNQ2 mutation do not have the same epileptic outcome, correlations between Im impairment and the severity of the encephalopathy should be made with caution. Other unknown factors may be involved in the occurrence of the epileptic encephalopathy. Moreover, the vast majority of the mutations we described here were localised on segment S6 and should have different consequences on Im current that those which have been previously described, affecting segment S4 [bib_ref] Genotype-phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor..., Miceli [/bib_ref]. These consequences still have to be studied. Overall, it is not known whether ongoing brain dysfunction that is observed in several patients is due to the Kv7-2 channelopathy or if it is a sequel of neonatal epilepsy. This question would be of paramount interest.
# Conclusions
KCNQ2 is frequently found mutated de novo in early onset epileptic encephalopathies, especially if the epilepsy begins within the first week of life. Despite relatively stereotyped initial phenotype, the neurological and epileptic outcomes were highly variable, overall severe.
[table] Table 1: KCNQ2 mutations and main features of the patients Tonic and tonic-clonic, cyanosis. 2-6 weeks: Tonic and tonic-clonic seizures in clusters. 2 m: seizure stop Poor activity. Prolonged periods of flatness of the traces. Generalized spikes predominating on the left hemisphere. Then suppressionburst. No sit, no speech (11 y). Multiple focal seizures: tonic contractions of one or several limbs, cyanosis. 2-9 years: seizure-free. > 9 years: monthly GTC seizures.Bursts of multifocal spikes and periods of poorness of the activity. [/table]
[table] Table 2: Data on initial evaluation and treatment, EEG evolution and brain MRI Suppression-burst. 2-6 m: General slowing of the traces, no spike. 6 m-2 y: Rare spikes in the right central region, Normal background. >2 y: normal traces. Day 7: T2 hyperintensity of the basal ganglia 2 y: Normal 3y: Normal Patient 13 Full term. Fetal distress. BW, HC: ND [/table]
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An Exploration of Domain-Specific Sedentary Behaviors in College Students by Lifestyle Factors and Sociodemographics
# Introduction
Emerging epidemiologic evidence suggests an independent and positive association between high levels of sedentary time and increased risk of chronic conditions, such as cardiovascular disease, hypertension, and type 2 diabetes. When examined by age, adolescents and young adults are one of the most sedentary subgroups, with estimates of over 8 h of sedentary time per day. Despite the high levels of sedentary behavior among young adults, there has been little research examining characteristics of sedentary time within this population.
Insights into the sedentary behavior of college students are of particular interest given the sizeable proportion of young adults who attend collegeand the nature of the undergraduate experience which incorporates so many sedentary activities. Accelerometery data estimate that college students spend nearly 10 h per day engaged in sedentary behavior, with a majority of that time spent sitting in class or studying. This is particularly worrisome given evidence that lifestyle habits developed during the transition from adolescence into adulthood track well into later life; therefore, sedentary college students are likely to become sedentary adults, and experience all the negative health sequalae associated with sedentary patterns.
Despite the apparent high levels of sedentary behavior among undergraduate students and the risks associated with sedentary patterns, few studies have characterized the domains in which college students spend their sedentary time and identified subgroups of undergraduates at highest risk of elevated sedentary time. For example, there is evidence of racial differences in sedentary behavior in adultsand suggestions that screen time and homework time may vary by sex, but a more robust exploration of the full range of sedentary behavior domains across subgroups of college students would aid in parsing out those individuals to whom sedentary behavior reduction interventions might be targeted.
Furthermore, there is growing interest in how sleep quality, physical activity and mood are associated with sedentary time across the relevant domains. Research in other populations indicates that screen time is the most common sedentary activity, and screen time has been associated with poor sleep quality, decreased physical activity, and higher body weight among adolescents, as well as poor sleep qualityand higher body mass index (BMI)among adults. There are initial indications that stress, poor sleepand low levels of physical activitymight be associated with sedentary behavior among college students. However, no studies of which we are aware have examined the range of sedentary behavior domains among college students relative to these other important lifestyle factors.
Thus, the purpose of this study was to characterize the extent and domain-specific patterns of sedentary behavior among college students, to explore sedentary profiles across sociodemographic subgroups, and to examine relationships between sedentary patterns and other lifestyle factors. We hypothesized that total sedentary time would be higher among minority students, individuals with overweight or obesity, and individuals with a lower physical activity level. In addition, we hypothesized that screen time would be associated with poorer sleep quality, weight status, and lower levels of physical activity.
# Materials and methods
## Study design
This cross-sectional study assessed self-reported sedentary behavior, stress, sleep and physical activity among undergraduate students at a major public southeastern university.
## Participants
Current undergraduate students were eligible to participate without any other inclusion criteria. Participants were recruited in the fall semester of 2018 through flyers, listservs, and announcements made by course instructors in various colleges. Interested students were directed to a study website, where they completed the online informed consent and an online survey via a unique and secure link (REDCap, Vanderbilt, TN, USA). The 272 students who completed the survey were entered into a gift card drawing. All participants gave their informed consent before inclusion in the study, which was approved by the University of South Carolina Institutional Review Board.
## Measures
Sedentary Behavior. The Sedentary Behavior Questionnaire (SBQ) is an 8-item selfreport measure of time spent engaged in sedentary activities on weekdays and weekend days separately across a range of domains. It has been shown to be reliable and validand was adapted to reflect current technologies likely to be most relevant to college students (e.g., replacing videocassette recorder (VCR) with smartphone or tablet), to expand descriptions to include activities common to college students (e.g., attending class, doing coursework, school-related computer time) and to add an item about sedentary socializing (i.e., at coffeeshop, sports event, bar or house). Mean daily sedentary time was calculated using a weighted average of weekday and weekend day sedentary time. In addition to quantifying total sedentary time, domain-specific sedentary time was calculated for leisure screen time, educational/work time, and socializing (on phone and in person). Any total sedentary time values greater than 1440 min (i.e., 24 h), were truncated to 1440 min.
Physical Activity. Physical activity was assessed using the International Physical Activity Questionnaire (IPAQ) short form, which has been shown to be valid and reliable for young adults. The IPAQ quantifies physical activity accrued in bouts of at least ten minutes over the previous week, measuring frequency and duration of vigorous and moderate intensity physical activity, walking, and sitting time. This allows the calculation of metabolic equivalent (MET) minutes per week and gives an indication of energy expenditure. It also allows the classification of individuals as low active, moderately active, or high active using established metrics. However, because there were few low active individuals in the sample of this study, low and moderately active students were combined into a single group for analysis purposes.
Sleep was assessed using the valid and reliable Pittsburgh Sleep Quality Index (PSQI). The PSQI is a 19-item questionnaire that evaluates seven components of sleep, including sleep duration, sleep disturbance, sleep latency, daytime dysfunction due to sleepiness, sleep efficiency, overall sleep quality and sleep medication use. The Global PSQI score (range 0-21) allows classification into individuals with poor sleep quality (score > 5) and good sleep quality (score ≤ 5). The measure has been used in studies which examine relationships between sleep and physical activity among college students.
Stress was assessed using the 10-item Perceived Stress Scale (PSS)to determine the degree to which individuals perceived their lives as stressful over the previous month. Scores range from 0 to 40 and are categorized into low (0-13), moderate, or highstress.
Demographic variables assessed included age, sex, race/ethnicity, and whether their parent(s) went to college. Height and body weight were self-reported and body mass index was then calculated (kg/m 2 ) and categorized according to CDC guidelines into underweight, normal weight, overweight and obese. Students with overweight or obesity were grouped together and compared with underweight and normal weight students.
## Statistical analyses
Descriptive statistics for continuous variables were calculated using means and standard deviations. Categorical variables were described using frequencies and percentages. Independent t-tests were used to examine whether sedentary behavior differed by sex, race/ethnicity, parental college attendance, weight category, physical activity level category or sleep category. One-way ANOVA was conducted to examine whether there were differences in sedentary behavior across the stress categories. Pearson's correlation was used to examine whether there were correlations between sedentary behavior domains and weight, physical activity, sleep and stress variables. Correlation strength was defined as small (0.1-0.3), medium (0.3-0.5), and large (0.5-1.0). All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).
# Results
A total of 354 young adults consented to participate in the study. A total of eightytwo individuals were excluded from analyses because they did not complete the survey (n = 78), were enrolled in a graduate program (n = 1) or were not currently enrolled at the university (n = 3). This left a sample of 272 eligible undergraduates. Overall, participants were predominately white females (79%) of normal weight (71%), reporting high levels of physical activity (2514 MET minutes/week), moderate perceived stress and poor sleep quality. Sample characteristics can be found in. Overall and domain-specific sedentary behavior patterns differed by demographic characteristics (see. Although female students reported similar amounts of total time spent sedentary to males, and comparable time in sedentary educational pursuits and recreational screen time, female undergraduates reported significantly more sedentary time socializing than males. Minority students reported significantly greater overall sedentary time than white students. Furthermore, minority undergraduates reported more time engaged in sedentary educational activities and significantly greater recreational screen time than did white college students. Indeed, the only domain in which there were no differences between minority and white students in sedentary time was sedentary socializing. Bolded numbers and *: statistically significant differences between groups, p < 0.05.
Among students reporting lower parental education, total sedentary time was significantly greater than was reported by students with higher parental education. Students with ≤1 parent who attended college also spent significantly more time engaged in sedentary educational/work-related activities than did students who had two parents that attended college. However, leisure screen time and sedentary socializing were similar between the two groups.
## Sedentary time and other health variables
Weight category (e.g., underweight/normal weight vs. overweight/obese) was significantly associated with sedentary time among undergraduates, with students with overweight or obesity reporting significantly greater total sedentary time than students who were normal weight or underweight. Those who were classified as overweight or obese also reported significantly greater time in sedentary socializing. However, there was no difference between students of differing weight groups with respect to educational sedentary time or screen time. Similarly, students who were classified as low/moderate active had significantly greater total sedentary time and significantly greater social sedentary time compared with those who were high active but did not differ in screen time or educational sedentary time.
Total sedentary time and screen time were both significantly higher among individuals with poor sleep quality compared to those with good sleep quality. However, no differences in sedentary time were apparent among students with differing levels of stress.
To explore the strength of associations between sedentary behavior and these other lifestyle factors, we examined the correlations between total sedentary time, domainspecific sedentary time and the lifestyle factors of interest. There were significant but small positive correlations between BMI and overall sedentary time, screen time, educational sedentary time and socializing time. Total MET minutes had a small but significant negative correlation with social sedentary time, but not any of the other sedentary behavior domains. Global PSQI sleep score had small significant positive associations with both total sedentary time and screen time. There were no significant associations between stress level and any of the sedentary behavior parameters.
# Discussion
College students engaged in substantial amounts of sedentary time, reporting an average sitting time of over 12 h a day. The majority of sedentary time was accrued in recreational screen time, with students reporting 4.5 h of daily leisure screen time. This is double the 1.5-2.4 h per day of screen time reported in older studies of college populations. Differences between these previous studies and the current findings may reflect how screen time was operationalized; some previous studies included only TV viewing as screen timeand others combined multiple screen-based activities into screen time. The comprehensive range of screen formats noted in survey items for the current study may have served as a prompt to remind students of the spectrum of activities to consider when responding about screen time, and that might have contributed to the higher report of sedentary time. The current results, however, are comparable to a recent study which reported 4 h of screen time among college students in Spain. Importantly, these data were confirmed by objective measurement. Moving forward, to fully capture the time young adults spend engaged in recreational screen-related activities, it will be critical to include a comprehensive list of all possible screens; the evolution of technology has created the ability to access content on multiple types of screens and not just TVs. Thus, asking only about time spent viewing TV runs the risk of missing a considerable amount of screen time.
Education and work-related sedentary time accounted for 3.4 h of daily sedentary time, which is more than double the homework time reported a previous study of undergraduates. This too might reflect how educational sedentary time was operationalized in the current study. We included not only doing homework, but also time sitting in class and doing course-related work on the computer to capture the full range of sedentary educational pursuits. There are emerging data that it is feasible to reduce sedentary time in the college classroom settingthrough the implementation of environmental changes, such as installing standing desks or encouraging standing in lectures, which may help reduce educational sedentary behavior without decreasing the time spent in educational pursuits. This is one of the first studies we are aware of to have examined sedentary time spent socializing. Undergraduates reported engaging in 3 h a day of sedentary socializing, indicating that this domain merits greater attention. Our results showed that female undergraduates spent more time engaged in sedentary socializing than males. Others have observed that college females are more likely to spend time using social media than males, but the current study expands socializing to include both media-based and in-person socializing. Other subgroups that indicated greater sedentary socializing in the current study included those who were overweight or obese and those who engaged in low levels of physical activity, which are subgroups that likely overlap. Since socializing is a significant part of the lifestyle of college students, and is often accomplished while sedentary, it may represent a "hidden" aspect of undergraduate sedentary time which requires greater quantification and characterization to determine whether it is amenable to modification to reduce overall sedentary time.
Minority students were particularly likely to spend substantial amounts of time engaged in sedentary behaviors, with 6 h a day engaged in recreational screen time, which is almost 2 h more than the screen time reported by white students. This was accompanied by higher amounts of sedentary time spent engaged in educational activities as well, resulting in significantly higher overall sedentary time among minority undergraduate students. Although this is not the first study to report greater screen time among minorities, it is the first of which we are aware to highlight racial differences in education-based sedentary time. These data echo studies noting racial differences among adults in sedentary behavior, and suggest that sedentary habits develop early in young adulthood and track into later life. Interventions to reduce sedentary behavior designed to reach minority undergraduate students could interrupt this pattern and reduce known health disparities in conditions related to sedentary behavior, such as cardiovascular disease and type 2 diabetes.
First-generation college students and those with lower parental education were another group that emerged from these analyses as a likely sedentary population and thus worthy of consideration for health promotion efforts. Students with lower parental education had greater overall sedentary time than students with higher parental education, as well as greater sedentary time spent engaged in educational/work activities. This is consistent with studies examining the role of parental education on screen time that report an association between lower parental educational attainment and higher screen time. However, this is the first study we are aware of that examined educational sedentary time among students with low parental education. Undergraduates with lower parental education are less likely to graduate and to have other indicators of academic distress. The current findings that they spend a greater amount of time engaged in educational and work-related sedentary behaviors may signal that they are either struggling with their coursework and thus spending more time sedentary while doing this coursework or that they spend more time working at sedentary jobs than do students with higher parental education. Since young adults with lower parental education tend to have a worse health profile than do students from families with higher parental education, sedentary behavior reduction may offer a novel lifestyle behavior to target to ameliorate some of that elevated risk.
Sleep quality, but not perceived stress, was associated with elevated sedentary behavior. Of particular note is the relationship between elevated recreational screen time and poor sleep quality seen in this study. Associations between poor sleep and both objectively measured sedentary timeand screen timehave previously been reported among college students. In this cross-sectional study, it cannot be ascertained whether increased screen time resulted in poor sleep or vice versa. If excessive screen time were a determinant of poor sleep, reducing screen time may be an important and attractive behavioral intervention target for undergraduates given the association between poor sleep and decreased academic performanceand impaired mental health.
Although this study advances our understanding of sedentary patterns among undergraduate students and points to specific groups at risk of greater time spent sedentary, the study is not without limitations. Foremost is the reliance on a self-reported measure of sedentary behavior. Self-report offers the advantage of elucidating domains of sedentary behavior, but self-report can underestimate time spent sedentary, particularly among college students. Objective measurement coupled with self-report is considered the optimal approach to ascertaining sedentary patterns. Furthermore, there exists the possibility that students over-reported their time engaged in sedentary behaviors, as the current generation of undergraduates tends to multi-task with media, and thus, time spent in sedentary activities could be counted twice across multiple domains (e.g., texting while watching TV). However, these considerations would impact the precision of the measurement rather than the comparative patterns which emerged, suggesting that results highlighting particular subgroups at risk for elevated sedentary time who might benefit from targeted sedentary reduction interventions to mitigate associated negative health consequences are likely robust. Other study limitations are the cross-sectional design and lack of sufficient sample size within individual minority subgroup populations to explore comparisons within specific racial-ethnic groups to pinpoint those subgroups at highest risk for a deleterious pattern of sedentary behavior. A larger sample would also enable the examination of low and moderate active students separately, as well as the full range of weight categories. Future studies could also build upon the t-tests used in this study and employ multivariate analyses to examine differences within each of the identified subgroups, which would provide informative next steps for this research. Finally, whether physical activity negates sedentary behavior's effects on health(or vice versa) and how sedentary behavior should be addressed within the broader context of health promotion are both areas for continued discussion.
# Conclusions
College students engage in a substantial amount of sedentary behavior, with sedentary time accrued across several important domains, including recreational screen time, educational activities, and socializing. Subgroups that are at particular risk for elevated sedentary time (and therefore higher risk of the negative health consequences associated with sedentary time) include minority students, those with low parental education and students with overweight/obesity. Targeted interventions to reduce sedentary time might be considered for these subgroups. However, it is important to note that although sedentary behavior was elevated in these subgroups, it was also quite high in their counterparts. In all pairs of subgroups compared, even the "healthier" of the two groups engaged in at least 12 h of sedentary behavior each day. Therefore, all undergraduates would likely benefit from sedentary behavior reductions. Designing interventions that meet the unique needs of undergraduates and address sedentary behavior across the range of domains would likely be most effective. Informed Consent Statement: All participants gave their informed consent before inclusion in the study.
Data Availability Statement: Research data are not available because participant consent did not include sharing of data.
## Conflicts of interest:
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. |
Repetitive Transcranial Magnetic Stimulation: A Potential Treatment for Obesity in Patients with Schizophrenia
# Introduction
The prevalence of obesity in patients with schizophrenia is as high as 60%and the odds of obesity is thrice that of the general population. Furthermore, the high prevalence of obesity in patients with schizophrenia is linked to a high rate of metabolic syndrome. Obesity is also associated with cardiovascular disease (CVD) and patients with schizophrenia die from CVD complications 15-20 years earlier and at 2-3 times the rate in the general population. While patients with schizophrenia might be genetically predisposed to obesity and other metabolic derangements (independent of psychiatric medications), psychotropic medications (especially certain antipsychotic medications) are a significant contributor to high obesity rates in schizophrenia.
So far, pharmacologic, and behavioral interventions have not been effective in stemming the tide of obesity in schizophrenia. It is therefore important to identify novel interventions for the treatment and prevention of obesity in schizophrenia, which will ultimately reduce morbidity and premature death in this patient population. One such intervention is noninvasive neuromodulation-specifically, repetitive transcranial magnetic stimulation (rTMS)-a promising intervention for obesity. While still in the preliminary phase, emerging data suggest that rTMS can reduce food craving and food Behav. Sci. 2021, 11, 86 2 of 15 consumption, and lead to weight loss in obese individuals. However, the utility of rTMS for weight loss in obese patients with schizophrenia has not been evaluated. The unavailability of data on rTMS for treatment of obesity in patients with schizophrenia is related to the fact that patients with schizophrenia were excluded from the few controlled studies of rTMS for obesity. Therefore, as a first step in filling the void in the literature, we have carried out this narrative review to stimulate research in the field by highlighting the potential utility of rTMS for reducing food craving and food consumption, and inducing weight loss in obese patients with schizophrenia.
In this review we will first discuss brain mechanisms implicated in obesity, emphasizing how these mechanisms have also been implicated in the neurobiology of schizophrenia. We will then describe the basics of rTMS, review the literature on the use of rTMS for treating obesity in non-schizophrenia samples, and explain why it is plausible to hypothesize that rTMS will be effective for reducing food cravings and excessive food consumption, promoting weight loss, and treating obesity in individuals with schizophrenia. In the final section, we will discuss deep TMS and why it is reasonable to speculate that, relative to conventional rTMS, deep TMS will have a larger effect size in the treatment of obesity in schizophrenia.
## The brain and obesity
The brain is involved in the control of appetite, energy intake, and expenditure. The critical role of the brain in metabolic homeostatic control is reflected by the fact that obesity is now conceptualized as a brain disease. While the hypothalamus acts as the key regulator of appetite and satiety (and hence, food consumption), the mesolimbic reward systemand the prefrontal cortexare also involved in the regulation of food consumption. Since weight gain occurs when energy intake exceeds expenditure, any impairment in the brain's ability to optimally regulate energy intake will ultimately result in obesity.
## Brain mechanisms of appetite regulation and food consumption
## Hypothalamic appetite-regulating system
The arcuate nucleus (ARC) of the hypothalamus contains two groups of neurons: (1) Neurons expressing proopiomelanocortin (POMC) and cocaine and amphetamineregulated transcript (CART); (2) neurons expressing neuropeptide Y (NPY) and agoutirelated protein (AgRP). POMC is a prohormone that is post-translationally cleaved into alpha-melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), and beta-endorphin. POMC/CART and NPY/AgRP neurons project to second order neurons in the paraventricular nucleus (PVN) of the hypothalamus. Both the ARC and PVN neurons possess melanocortin receptors 3 and 4 (MC3R and MC4R); α-MSH is an agonist, while AgRP is an antagonist at both receptors. PVN neurons project to the nucleus tractus solitarius (NST), an important brain region where sensing and integration of signals relevant for the control of feeding behavior occur.
POMC/CART neurons reduce appetite (i.e., they are anorexigenic) and promote increased energy expenditure, while NPY/AgRP neurons promote hunger (i.e., orexigenic). It is important to note that there is crosstalk between the brain, gut, and other peripheral metabolic organs (e.g., liver, pancreas, and adipose tissue) via neurohormones and sympathetic and parasympathetic innervation, respectively. The crosstalk between the brain and periphery is illustrated by the fact that food ingestion causes the secretion of peripheral signals of excess nutrients/energy stores such as leptin and insulin, which stimulate POMC/CART neurons while simultaneously inhibiting NPY/AgRP neurons. As a result of the stimulation of POMC/CART neurons, α-MSH binds MC3R and MC4R receptors on the PVN neurons which in turn lead to satiety signals being sent to the NTS to ultimately reduce food intake and increase energy expenditure. By contrast, fasting results in the production of hunger signals such as ghrelin, which stimulate NPY/AgRP neurons to increase food intake. In summary, the POMC/CART and NPY/AgRP neurons function in a 'Yin and Yang' fashion to regulate food intake.
## The mesocorticolimbic reward system and food consumption
The reward circuitry in humans consists of dopaminergic neurons projecting from the ventral tegmental area (VTA) in the midbrain to the nucleus accumbens (NAc) (the mesolimbic pathway) and the prefrontal cortex (PFC) (the mesocortical pathway), respectively. In addition to the primary projections to the NAc and PFC, VTA neurons also project to the cingulate cortex, hippocampus, amygdala, and olfactory tubercle. The mesolimbic pathway to the NAc is involved in the positive reinforcing effects of natural rewards, drugs of abuse, as well as certain foods (described in the next paragraph below), while the mesocortical pathway to the PFC is important for planning motivated behaviors (e.g., seeking and acquiring food, street drugs, and sex) and emotional responses. Notably, the dopaminergic neurons in the VTA (which project to the NAc and PFC) possess receptors for hunger/satiation-mediated peptides including leptin, ghrelin, and orexin, findings that link the reward pathways to homeostatic processes of energy regulation.
Food ingestion can produce pleasurable, positively reinforcing, and rewarding effects. In fact, appetitive, hyperpalatable, calorie-dense, high-carbohydrate, high-fat foods are very salient, rewarding stimuli which (like drugs of abuse) can elicit the release of dopamine in the NAc which can reinforce consumption of these foods. Therefore, food-related activation of the brain reward circuitry, the reinforcement of behaviors, and the production of memories can lead to food craving and consumption of larger amounts of food than was intended.
## The prefrontal cortex and food consumption
The most anterior part of the frontal cortex, i.e., the prefrontal cortex, contains the cortical networks that support behavioral regulation (i.e., the integration of cognitive processes including attention, working memory, and inhibitory control). Specifically, dietary self-regulation refers to an individual's ability to exert conscious control over food choice and consumption. As previously mentioned, certain foods provoke robust activation of neurons in the brain's reward system (the VTA and NAc). Importantly, the dorsolateral prefrontal cortex (DLPFC) mediates appropriate cognitive strategies necessary to inhibit food-evoked visceral cravings (e.g., secondary to exposure to palatable food and food cues such as food advertisements), thus preventing overindulgence in the absence of physiological energy deficit/hunger. In addition, the DLPFC implements cognitive control by modulating dopamine neurotransmission in the ventromedial PFC (VMPFC), VTA, and NAc, resulting in the inhibition of inappropriate responses, devaluation of immediate appetitive rewards, and the implementation of goal-directed behaviors. In summary, the DLPFC is a critical functional node for the downregulation of the rewarding properties of energy-dense foods, inhibiting impulsive food consumption in the absence of a physiological energy deficit, thereby enabling individuals to exert control over their consumptive behaviors.
## Brain abnormalities implicated in dysregulation of energy homeostasis and obesity
## Hypothalamic inflammation and obesity
Animal models and human studies have shown that inflammation of the hypothalamus leads to dysregulation of energy homeostasis, which subsequently results in obesity, glucose intolerance/diabetes, and hyperlipidemia. Importantly, the observed hypothalamic inflammation is chronic, low-grade, nonsuppurative, and occurs without evidence of foreign substances in the brain. Imaging studies have also provided evidence in support of the existence of obesity-related hypothalamic neuroinflammation. However, it is yet to be determined conclusively whether the hypothalamic inflammation seen in obese individuals is secondary to diet or whether it is independent of diet, and a part of the mechanism of disease. Nevertheless, studies have shown that high-fat diet induces activation of glial cells (astrocytes and microglia), which then mount inflammatory responses which ultimately produce inflammation of the hypothalamus, specifically the POMC neurons in the ARC. Moreover, obesity is associated with chronic low-grade peripheral inflammation, which can promote the migration of peripheral immune cells into the brain, further activating microglia, and contribute to hypothalamic inflammation. In addition to activating glial cells, a high-fat diet also reduced the response of ARC neurons to exogenous leptin in miceresulting in leptin resistance, a phenomenon associated with obesity. While hypothalamic inflammation seems to perpetuate obesity, a limitation of the current studies on hypothalamic inflammation and obesity is that they do not clarify whether hypothalamic inflammation is a precursor (i.e., precedes weight gain) or strictly a consequence of overeating and obesity.
## Dysregulated mesocorticolimbic reward system and uncontrolled food consumption
Food ingestion (especially appetitive, hyperpalatable, calorie-dense foods) produces pleasurable and rewarding effects elicited by the release of dopamine in the NAc. However, obesity maybe associated with blunting of the pleasurable and rewarding effects of food (the reward deficiency hypothesis) such that greater consumption of food might be required for an individual to experience the same levels of pleasure as non-obese individuals. In the same vein, impaired dopamine receptor (D2/D3) binding in the ventral striatum was associated with obesity in some studies, suggesting that lower dopamine receptor binding probably led to excessive food consumption to compensate for blunted responses in the reward neural pathway. However, prospective functional magnetic resonance imaging (fMRI) studies did not confirm an association between dopamine occupancy in the ventral striatum and the risk of obesity in non-psychiatric samples.
Another construct which is biologically mediated by the mesocorticolimbic reward system is food reward sensitivity (the tendency to seek and derive pleasure from food), which has also been positively correlated with food addiction, uncontrolled eating, and obesity. Additionally, impaired mesocorticolimbic reward circuitry may predispose an individual to the overconsumption of hyperpalatable calorie-dense foods due to an increase in the reward values assigned to such foods. In conclusion, dysregulated mesocorticolimbic reward function may be associated with uncontrolled food intake and obesity, and this association could be related to impaired dopaminergic transmission in the ventral striatum, but well-designed prospective studies are needed to clarify the role of mesocorticolimbic dopamine neurotransmission in the pathogenesis and perpetuation of obesity.
## Impaired prefrontal cortex function and food consumption
Neuroimaging studies have demonstrated a negative relationship between body mass index and DLPFC activation, suggesting that individuals with obesity may have greater difficulty utilizing the DLPFC when making food consumption decisions. In addition, experimentally induced inhibition of the left DLPFC using noninvasive brain stimulation techniques resulted in increased appetitive snack food consumption. Importantly, the participants in the DLPFC inhibition study were of normal weight, which suggests that impaired DLPFC function precedes the onset of obesity and could have a causal role in obesity. In addition, obese individuals have been shown to exhibit reduced grey matter volume in the DLPFC when compared with lean individuals. The DLPFC is also a prominent brain region involved in cognitive control, one hallmark of which is the ability to easily disengage from one train of thought or activity to seek an alternative. Therefore, people with impaired DLPFC function may find it difficult to disengage from food cravings and ultimately end up overconsuming food. Finally, impaired DLPFC function is associated with impulsivity, which (along with poor cognitive inhibitory control) is associated with delay discounting (a tendency to choose small, immediate rewards over larger, delayed rewards); impulsivity and delay discounting have been associated with overconsumption of highly palatable foods.
## Brain abnormalities implicated in dysregulated energy homeostasis and obesity have also been implicated in the neurobiology of schizophrenia
Interestingly, the previously described brain abnormalities linked to impaired energy homeostasis and obesity have been described as part of the neurobiology of schizophrenia, which suggests that patients with schizophrenia might have an underlying vulnerability to excessive food consumption and obesity .
of which is the ability to easily disengage from one train of thought or activity to seek alternative. Therefore, people with impaired DLPFC function may find it difficu disengage from food cravings and ultimately end up overconsuming food. Finally, paired DLPFC function is associated with impulsivity, which (along with poor cogni inhibitory control) is associated with delay discounting (a tendency to choose small, mediate rewards over larger, delayed rewards); impulsivity and delay discoun have been associated with overconsumption of highly palatable foods.
## Brain abnormalities implicated in dysregulated energy homeostasis and obesity have a been implicated in the neurobiology of schizophrenia
Interestingly, the previously described brain abnormalities linked to impaired ene homeostasis and obesity have been described as part of the neurobiology of schizop nia, which suggests that patients with schizophrenia might have an underlying vuln bility to excessive food consumption and obesity . ` . Three overlapping brain mechanisms in obesity and schizophrenia. Impaired prefrontal cortex function, hypothalamic inflammation and dysregulated mesocorticolimbic reward system have been implicated in the pathogenesis of obesity and have also been described as part of the neurobiology of schizophrenia.
## Figure 1.
Three overlapping brain mechanisms in obesity and schizophrenia. Impaired prefrontal cortex function, hypothalamic inflammation and dysregulated mesocorticolimbic reward system have been implicated in the pathogenesis of obesity and have also been described as part of the neurobiology of schizophrenia.
## Hypothalamic inflammation in schizophrenia
Immune activation and inflammation have been found in the periphery and in the brain of patients with schizophrenia. The chronic low-grade inflammation in the brain (neuroinflammation) of patients with schizophrenia is thought to be related to microglial activation. However, neuroinflammation might be relevant for only a subset of patients and not across the board, an idea consistent with the heterogeneity found in the illness. Of note, post-mortem examination revealed chronic low-grade inflammation of the hypothalamus in patients with schizophrenia, although there was insufficient information to specifically link the inflamed hypothalamus to obesity in the patients. Additionally, atypical antipsychotics (now the mainstay of treatment of schizophrenia) have been causally linked to hypothalamic inflammation in animal models (in vitro and in vivo)and it is therefore reasonable to speculate that hypothalamic inflammation in patients with schizophrenia might be linked to treatment with atypical antipsychotic medications. Moreover, the two atypical antipsychotic medications with the highest liability for increasing appetite (clozapine and olanzapine) and inducing obesity in patients are also the ones with the strongest evidence for inducing hypothalamic inflammation in animal models [53-55].
## Dysregulated mesocorticolimbic reward system in patients with schizophrenia
Dopamine neurotransmission plays a critical role in reward processingand is also the neurotransmitter system consistently implicated in the neurobiology of schizophrenia. Moreover, all antipsychotic medications currently approved for schizophrenia target dopamine. Specifically, abnormal mesolimbic and mesocortical brain connectivity involving dopamine neurotransmission are longstanding models posited to explain positive (e.g., delusions and hallucinations) and cognitive/negative (e.g., executive function deficits, amotivation, and alogia) symptoms of schizophrenia. Considering the role of the mesocorticolimbic reward system in controlling food intake (please refer to the earlier sections of this review), abnormalities of the reward circuitry may predispose patients with schizophrenia to abnormal eating behaviors that can lead to obesity. Interestingly, patients with schizophrenia and comorbid metabolic syndrome (truncal obesity, hypertension, dyslipidemia, and glucose intolerance) were found to have smaller reward-related brain structures than patients without metabolic syndrome, a finding that supports a role of the brain reward circuitry in the development of obesity in patients with schizophrenia.
Moreover, blockade of dopamine receptors in the mesocorticolimbic neural pathway by antipsychotic medications could also contribute to impairments that result in unhealthy eating behaviors in patients with schizophrenia. Indeed, it has been posited that blockade of dopamine receptors in the nucleus accumbens by antipsychotics could reduce the pleasurable and rewarding effects of food, causing patients to compensate by increasing their food intake, especially calorie-dense, hyperpalatable food. In this vein, studies have shown that, relative to second-generation antipsychotics, first-generation antipsychotics (which have stronger dopamine-blocking activities) result in less activation of the ventral striatum using a monetary incentive delay task (MID). While MID is not a food-related reward paradigm, it is a validated measure of reward function. However, brain reward abnormalities might be intrinsic to schizophrenia, as unmedicated patients with schizophreniaand their healthy first-degree relativesexhibited reduced activation of the ventral striatum during reward anticipation. Finally, the magnitude of ventral striatal activation during reward anticipation negatively correlated with antipsychotic-induced weight gain in patients with schizophrenia such that those with the least striatal reward activity at baseline, gained the most weight after six weeks of antipsychotic treatment.
## Impaired prefrontal cortex (pfc) function in patients with schizophrenia
Individuals with schizophrenia have been shown to exhibit structural and functional impairments in their PFC. PFC abnormalities in patients with schizophrenia are related to cognitive function (working memory and other executive functioning) deficits. Specifically, neuroimaging studies have shown that the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia function less efficiently in comparison with healthy controls, similar to reports of dysfunction in the same brain region of individuals with obesity. In addition, dysfunction in the PFC is linked to poor decision-making and impulsivity in individuals with schizophrenia. Poor decision-making and lower levels of inhibitory control secondary to impaired DLPFC function in patients could then extend to decision-making about food such that patients excessively consume unhealthy foods that are likely to result in obesity.
Although we have a made a distinction between the prefrontal cortex (cognitive function) and reward circuitry, emerging data suggest a dysfunctional interaction between prefrontal cortex and reward circuits in patients with schizophrenia. For example, patients with schizophrenia exhibit delayed discounting deficits, i.e., relative to healthy controls, they will choose a significantly smaller immediate reward over a larger delayed rewardwhich might be related to prefrontal cortex-related inability to represent the value of outcomes and plans. Importantly, cognitive function, specifically episodic and working memory, inversely correlated with the degree of discounting of the value of future rewards in patients. It is therefore possible that patients with schizophrenia choose appetitive, hyperpalatable, calorie-dense, high-carbohydrate, high-fat foods over healthier foods because of the immediate hedonic response (i.e., immediate reward) of appetitive, unhealthy foods but attach less value to the larger, delayed health rewards of healthy foods.
## Repetitive transcranial magnetic stimulation (rtms) as a potential treatment for obesity in schizophrenia
## The basics of rtms
From a historical perspective, the prototype of the modern day transcranial magnetic stimulation (TMS) machine was designed by Barker and his colleagues in 1985 at the University of Sheffield, England. Transcranial magnetic stimulation (TMS) involves the use of an alternating magnetic field to induce electric current in cortical brain tissue using a coil placed on the scalp. TMS is based on the principle of induction of a magnetic field by an alternating electric current flowing through a coil (ampere's law), and the induction of an electric current by an alternating magnetic field (Faraday's law). The electric current induced in the cortical tissue by the magnetic field emanating from the TMS coil will cause the depolarization of neurons which will produce an action potential and firing of neurons which will subsequently have behavioral effects. In general, the magnetic fields generated by commercially available figure-of-eight (figure-8 coils) TMS coils, which is on average 1.5 to 4 Tesla, do not extend into the brain beyond 2.5 cm from the scalp, although H-coils penetrate deeper to levels up to 6 cm. TMS can be delivered as single pulses or in a repeated rhythmic fashion referred to as repetitive TMS (rTMS). Moreover, the frequency of the pulses in rTMS is said to be "low" if ≤ 1 Hz or "high" if ≥ 1 Hz. A single pulse of TMS can result in quick, immediate behavioral changes, such as involuntary movement or perceived flashes of light, depending on the area of the brain being targeted. However, rTMS results in changes in neuronal activity and brain function that last well beyond the initial stimulation and is used to induce longer-lasting changes in behavior and cognitive processing. In psychiatry, rTMS (usually high frequency) is approved for treatment-resistant major depressive disorder. Deep TMS, which uses the H-coil, has been approved for obsessive-compulsive disorder.
For the treatment of major depressive disorder, the procedure for most protocols involves daily treatment sessions (usually Monday to Friday) lasting 20-30 min for a total of 30 treatment sessions with the TMS coil placed on the left DLPFC. The commonest side effect of rTMS is local scalp/facial pain or headache, related to the stimulation of peripheral scalp/facial muscles at the point of contact of the TMS coil; the pain or headache can usually be treated with acetaminophen or aspirin. The continuous clicking sound of the TMS machine could potentially affect a person's auditory threshold and for this reason, it is standard practice for patients and treaters to wear earplugs during treatment sessions. A rare but more serious side effect of rTMS is seizure, which is self-limiting and does not result in epilepsy (i.e., seizure disorder). The risk of seizure increases with higher stimulaton intensity. Since rTMS involves the application of a magnetic field to the brain, it is contraindicated in individuals with ferromagnetic implants.
## Efficacy of rtms for reducing food craving, food consumption and treating obesity in non-psychiatric samples
The awareness of the DLPFC's role in dysregulated eating behaviors (discussed in earlier sections of this review), has resulted in increased interest in rTMS targeting the DLPFC for modulating eating behaviors, reducing food cravings, and secondarily treat obesity. Of note, rTMS applied to the DLPFC of smokers and cocaine users reduced subjective craving for cigarettes and cocaine, and based on the similar appetitive qualities of both substance use disorders to those of food in individuals with dysregulated eating, suggest that the same methods could be applied to control food cravings. Indeed, meta-analyses have shown that single-, and multi-session rTMS reduce food craving and food consumption with multi-session rTMS being associated with larger effect sizes. While there is good evidence supporting the efficacy of rTMS in reducing food craving and consumption, there is a relative paucity of studies specifically evaluating the efficacy of rTMS for weight loss in obese individuals. To our knowledge, only three randomized, sham-controlled studies of high frequency rTMSand one study of deep TMSare currently available and all four studies resulted in significant weight loss in the active treatment group relative to the sham treatment group.
## Rtms effects that we hypothesize to be relevant for reducing food craving, food consumption and inducing weight loss in obese patients with schizophrenia
We hypothesize that rTMS will reduce food cravings, which will secondarily lead to a reduction in calorie intake, and ultimately result in weight loss in obese patients with schizophrenia via three mechanisms, namely: (1) anti-inflammatory effect, (2) modulation of mesocorticolimbic reward circuitry, and (3) modulation of pre-frontal cortex function .
## Food consumption and inducing weight loss in obese patients with schizophrenia
We hypothesize that rTMS will reduce food cravings, which will secondarily lead to a reduction in calorie intake, and ultimately result in weight loss in obese patients with schizophrenia via three mechanisms, namely: (1) anti-inflammatory effect, (2) modulation of mesocorticolimbic reward circuitry, and (3) modulation of pre-frontal cortex function . . rTMS effects that we have hypothesized to be relevant for treating obesity in patients with schizophrenia. The documented effects of rTMS, including its anti-inflammatory effects, modulation of dopaminergic neurotransmission in the mesocorticolimbic system, and modulation of prefrontal cortex function, all have the potential to be beneficial for reducing food cravings and food consumption and lead to weight loss in patients with schizophrenia comorbid with obesity. rTMS: Repetitive transcranial magnetic stimulation; Nrf2: Nuclear factor erythroid-2-related factor 2. . rTMS effects that we have hypothesized to be relevant for treating obesity in patients with schizophrenia. The documented effects of rTMS, including its anti-inflammatory effects, modulation of dopaminergic neurotransmission in the mesocorticolimbic system, and modulation of prefrontal cortex function, all have the potential to be beneficial for reducing food cravings and food consumption and lead to weight loss in patients with schizophrenia comorbid with obesity. rTMS: Repetitive transcranial magnetic stimulation; Nrf2: Nuclear factor erythroid-2-related factor 2.
## Anti-inflammatory effect of rtms
rTMS could potentially be effective for treating obesity in schizophrenia by reducing hypothalamic inflammation associated with schizophreniaand implicated in the pathogenesis of obesity. In vitro animal and human studies have demonstrated the anti-inflammatory effects of rTMS in the periphery and in the brain. Pre-clinical studies indicate that the anti-inflammatory effect of rTMS is mediated by increased translocation of the transcription factor, nuclear factor erythroid-2-related factor 2 (Nrf2) into the nucleus of brain cells. Interestingly, ziprasidone (an antipsychotic medication approved for the treatment of schizophrenia and also considered to possess the least risk of weight gainalso increased the translocation of Nrf2 from the cytoplasm to the nucleus, suggesting that this shared mechanism with rTMS could be responsible for its relatively low liability for weight gain in patients. Theoretically, rTMS could also potentially be anti-inflammatory via its effect on glial cells, since high frequency rTMS was shown to inhibit the proliferation of astrocytes and reduced microglial activation in rodent CNS; reactive astrocytes and activated microglia can contribute to neuroinflammation and neuronal deathand have been implicated in the pathophysiology of schizophrenia.
## Modulation of the mesocorticolimbic reward circuitry by rtms
rTMS targeting the frontal cortex has a local effect on the neurons directly under the TMS coil but can also modulate subcortical networks, including the striatum, and hence influence the mesocorticolimbic reward network. In rodents, high frequency rTMS applied to the frontal cortex, resulted in increased dopaminergic neurotransmission in the mesocorticolimbic tract. Similarly, in human subjects without schizophrenia, TMS applied to the anterior frontal cortex modulated reward-related processing in the striatum, and high-frequency rTMS to the DLPFC increased dopamine neurotransmission in the striatum. Therefore, considering the hypothesized blunted reward response secondary to hypodopaminergia in the reward circuitry of patients with schizophrenia, we argue that rTMS applied to the DLPFC of obese individuals with schizophrenia could ameliorate the hypodopaminergia and reduce excessive food consumption and subsequently result in weight loss. Furthermore, it is plausible to speculate that the enhancement of dopamine neurotransmission in the striatum by rTMS could act in a similar manner as stimulants, which also enhance dopamine neurotransmission in the striatum and decrease appetite, and consequently weight loss. However, the potential to worsen psychosis secondary to increased dopamine neurotransmission in the striatum, must be taken into consideration when planning to administer rTMS for obesity in patients with schizophrenia. The risk of worsening psychotic symptoms notwithstanding, it is important to note that high frequency rTMS applied to the left DLPFC has also been found to reduce psychotic symptoms in patients with schizophrenia.
## Modulation of pre-frontal cortex function by rtms
We also hypothesize that rTMS applied to the left DLPFC of obese individuals with schizophrenia would result in reduced food craving, consumption and ultimately, weight loss, via rTMS modulatory effect on the pre-frontal cortex. Specifically, rTMS could reduce impulsivity, and translate to reduced impulsive and compulsive eating in obese patients with schizophrenia. For example, in patients with cocaine use disorder who also exhibit impulsive behavior, rTMS applied to the left DLPFC increased the functional connectivity between the left DLPFC and ventromedial prefrontal cortex and reduced impulsivity in the patients. rTMS applied to the left prefrontal cortex also reduced behavioral impulsivity in patients with methamphetamine use disorder. In addition, bilateral high frequency rTMS applied to the DLPFC reduced craving for cigarettes in patients with schizophrenia, a finding that further highlights the need to test the efficacy of rTMS for reducing food craving in patients with schizophrenia. Moreover, since cognitive function inversely correlated with the degree of discounting of the value of future rewards in patients with schizophrenia, rTMS might also reduce food consumption by improving overall cognitive function; of note, adjunctive high frequency rTMS applied to the left DLPFC of patients with schizophrenia improved multiple domains of cognition in the patients. In summary, based on similarities between substance use disorders and obesity, we argue that the observed efficacy of rTMS in reducing impulsivity and craving in substance use disorders is likely to be replicable in obese patients with schizophrenia and the cognitive-enhancing properties of rTMS in patients with schizophrenia is also likely to result in healthier food consumption.
## Deep tms
Unlike conventional TMS (with , which does not modulate cortical excitability beyond a depth of 2.5 cm from the scalp, deep TMS (dTMS) with H-coil can modulate cortical excitability up to a maximum depth of 6 cm and is therefore preferred for modulating the activity of deeper neuronal circuits. Moreover, conventional TMS misses the cortical target in 27-32% of patients if neuronavigation is not used but dTMS is unlikely to miss the target because it stimulates larger areas of brain tissue (approximately 17 cm 3 for dTMS vs. 3 cm 3 for conventional TMS). Therefore, based on the properties of dTMS, it is reasonable to postulate that dTMS would have a larger effect size when used for the treatment of obesity in patients with schizophrenia. Consistent with our hypothesis of greater effect size of obesity treatment with dTMS vs. conventional TMS, the only available study (to our knowledge) of dTMS for weight loss in a non-psychiatric samplerevealed a larger decrease in weight (−7.83 ± 2.28 kg) when compared to weight loss in the three studiesthat used conventional TMS (−1.35 ± 2.31 kg, −2.75 ± 2.37 kg, and −1.31 ± 1.3 kg, respectively)
## Concluding remarks and future directions
Based on certain shared neurobiological mechanisms between obesity and schizophrenia and data (although limited) showing the effectiveness of rTMS for obesity in nonpsychiatric samples, we propose that rTMS is a potential treatment for obesity in patients with schizophrenia. However, there is currently no data on the effectiveness of rTMS for reducing food craving and consumption and inducing weight loss in obese individuals with schizophrenia. There is therefore an urgent need for clinical trials of rTMS for obesity in patients with schizophrenia, considering the epidemic of obesity in this patient population. Since our goal is to stimulate research on rTMS for obesity in patients with schizophrenia, a list of some of the questions that must be answered by future studies include: (1) What would be the optimal stimulation parameters? (2) Should rTMS be administered only to the left side, only to the right side, or bilaterally to the brain? (3) What would be the optimal duration of treatment? (4) What safety issues would arise? (5) Would rTMS in combination with another obesity intervention be superior to rTMS alone?What would be the specific neurobiological substrates of the potential clinical benefits of rTMS for obesity in patients with schizophrenia? |
Safety and tolerability of 6-month supplementation with a vitamin D, calcium and leucine-enriched whey protein medical nutrition drink in sarcopenic older adults
Aims Safety and tolerability of prolonged supplementation with a vitamin D, calcium and leucine-enriched whey protein medical nutrition drink (WP-MND) was evaluated in sarcopenic older adults. Methods A 13-week double-blinded, randomized, isocaloric placebo-controlled trial (PROVIDE study; n = 380) was extended with a voluntary 13-week open-label extension (OLE). OLE participants were randomized to receive daily 1 or 2 servings of WP-MND (21 g protein, 3 g leucine, 10 µg vitD and 500 mg calcium per serving). Gastro-intestinal tolerability, kidney function and serum levels of calcidiol, parathyroid hormone (PTH) and calcium were evaluated at week 0, 13 and 26. Results and discussion In response to the high daily protein intake (median1.5; IQR: 1.3, 1.7 g/kg BW/day), the estimated glomerular filtration rate (eGFR) increased in the test group during the RCT (p = 0.013). The same trend was observed for those participants with moderate chronic kidney disease. During OLE no eGFR change was observed in any of the groups. Serum calcidiol and calcium reached a plateau after 13-week WP-MND supplementation. As expected, PTH significantly changed in the opposite direction, decreasing during RCT in the test group (T vs C: p < 0.001) and during OLE in former control groups. During RCT, 20/366 participants with normal baseline calcidiol reached levels ≥ 100 nmol/L (T: n = 18; C: n = 2) and 6 developed albumin-corrected calcium levels > 2.55 mmol/L (T: n = 3; C: n = 3), without associated adverse events. Conclusion A 6 months intervention with up to 2 servings of WP-MND did neither result in kidney function deterioration nor symptoms of vitamin D or calcium toxicity. The product was overall well tolerated.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
# Introduction
Sufficient dietary intake of protein, and an adequate vitamin D and calcium status has been proposed as a way to attenuate age-related loss of muscle and bone [bib_ref] Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient..., Chanet [/bib_ref] [bib_ref] OH) 2-vitamin D3 enhances the stimulating effect of leucine and insulin on..., Salles [/bib_ref] [bib_ref] A randomized study on the effect of vitamin D(3) supplementation on skeletal..., Ceglia [/bib_ref] [bib_ref] Low vitamin D and high parathyroid hormone levels as determinants of loss..., Visser [/bib_ref] [bib_ref] The 2011 report on dietary reference intakes for calcium and vitamin D..., Ross [/bib_ref] [bib_ref] Nutrition, frailty, and sarcopenia, Cruz-Jentoft [/bib_ref] [bib_ref] The role of dietary protein and vitamin D in maintaining musculoskeletal health..., Rizzoli [/bib_ref] [bib_ref] Dietary strategies for mitigating osteosarcopenia in older adults: a narrative review, De Rui [/bib_ref]. This is especially important for frail, sarcopenic and osteoporotic older adults who are at risk of falls, fractures and subsequent hospitalization or institutionalization [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref] [bib_ref] Falls in the elderly: a prospective study of risk factors and risk..., Graafmans [/bib_ref] [bib_ref] The role of parathyroid hormone (PTH) and vitamin D in falls and..., Dretakis [/bib_ref]. The European Society for Clinical Nutrition and Metabolism (ESPEN), the European Union Geriatric Medicine Society (EUGMS), and the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) recommend a protein intake of 1.0-1.2 g protein/kg bodyweight (BW)/day (d) for healthy older individuals and 1.2-1.5 g/kg BW/day for geriatric patients with acute and/or chronic diseases [bib_ref] The role of dietary protein and vitamin D in maintaining musculoskeletal health..., Rizzoli [/bib_ref] [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref] [bib_ref] Protein intake and exercise for optimal muscle function with aging: recommendations from..., Deutz [/bib_ref]. The Institute of Medicine in the USA and ESCEO recommend a daily intake of 800 IU (= 20 µg) vitamin D 3 for people of 71 years and older to sustain 25-hydroxyvitamin D (calcidiol) levels above 50 nmol/L [bib_ref] The 2011 report on dietary reference intakes for calcium and vitamin D..., Ross [/bib_ref] [bib_ref] The role of dietary protein and vitamin D in maintaining musculoskeletal health..., Rizzoli [/bib_ref] [bib_ref] Vitamin D and fracture prevention, Bischoff-Ferrari [/bib_ref] [bib_ref] A pooled analysis of vitamin D dose requirements for fracture prevention, Bischoff-Ferrari [/bib_ref]. Calcidiol is the precursor of the active, but short-lived 1,25-hydroxyvitamin D (calcitriol), and represents an integrated marker of vitamin D status as a result of endogenous synthesis from sun exposure and dietary intake [bib_ref] The 2011 report on dietary reference intakes for calcium and vitamin D..., Ross [/bib_ref]. The recommended daily allowance (RDA) for calcium for people of 71 years and older is 1200 mg/day [bib_ref] The 2011 report on dietary reference intakes for calcium and vitamin D..., Ross [/bib_ref]. Despite the recommendations above, insufficient intake of protein, vitamin D and calcium is still common in communitydwelling older adults [bib_ref] Macronutrient intake and inadequacies of community-dwelling older adults, a systematic review, Borg [/bib_ref] [bib_ref] Micronutrient intakes and potential inadequacies of community-dwelling older adults: a systematic review, Borg [/bib_ref]. This may also partly explain the observed 45% prevalence of vitamin D deficiency, i.e. calcidiol < 50 nmol/L, in a large cohort of community-dwelling older (≥ 65 years) individuals [bib_ref] The association between 25-hydroxyvitamin D concentration, physical performance and frailty status in..., Vaes [/bib_ref].
Previously, an intervention with a vitamin D, calcium and leucine-enriched whey protein medical nutrition drink was shown to enhance muscle mass and improve lower-extremity function in sarcopenic older adults [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref]. To obtain these beneficial effects, two servings of the medical nutrition drink and thereby high amounts of protein (42 g), leucine (6 g), vitamin D (40 µg) and calcium (1000 mg) per day were consumed on top of the regular diet. However, this may cause safety concerns in this vulnerable older target population with regard to kidney function and potential vitamin D or calcium toxicity. Older adults experience kidney function decline with age [bib_ref] Levels of serum creatinine and estimated creatinine clearance over the age of..., Fastbom [/bib_ref] and those with moderate or severe chronic kidney disease are advised to restrict protein intake and/or have their kidney function monitored on a regular basis [bib_ref] The role of dietary protein and vitamin D in maintaining musculoskeletal health..., Rizzoli [/bib_ref] [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref] [bib_ref] Protein intake and exercise for optimal muscle function with aging: recommendations from..., Deutz [/bib_ref]. Vitamin D or calcium toxicity related hypercalcemia may facilitate the formation of kidney stones and calcification of soft and vascular tissues, and result in acute symptoms such as nausea, vomiting, increased thirst and depression [bib_ref] Effectiveness and safety of vitamin D in relation to bone health, Cranney [/bib_ref].
The aim of this post-hoc evaluation was to determine the effect of 6 months supplementation with a vitamin D, calcium and leucine-enriched whey protein medical nutrition drink (WP-MND) on kidney function and on vitamin D, PTH and calcium levels in sarcopenic older adults. For this purpose, we extended the 13-week randomized placebocontrolled trial [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] , with a subsequent voluntary week open-label study period with WP-MND supplementation in both the former test and control groups. In addition, general safety was evaluated with parameters of liver function and registration of adverse events and tolerability was evaluated by a questionnaire addressing symptoms of gastrointestinal discomfort.
# Methods
## Participants
As described in detail before [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] [bib_ref] Thirteen weeks of supplementation of vitamin D and leucine-enriched whey protein nutritional..., Liberman [/bib_ref] , participants were recruited from 18 study centers in 6 European countries. Older adults (≥ 65 years) were eligible when they suffered from sarcopenia, defined as low skeletal muscle mass index (SMI) combined with mild to moderate limitations in physical performance (for definitions applied see reference [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref]. People with liver or kidney failure were excluded from participation. Known kidney failure was defined in the published protocol as estimated glomerular filtration rate (eGFR) below 30 mL/min/1.73 m 2 , what means that participants with moderate chronic kidney disease (eGFR between 30 and 60) were eligible for participation in the study. For this group, Bauer et al. recommend that they adhere to general protein recommendations for older people (1.0-1.5 g protein/kg BW/day), but have their GFR monitored twice a year [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref]. Other exclusion criteria relevant were: (a) intake of a high protein diet 3 months before start of or during the study, (b) intake of a protein-or amino acidcontaining nutritional supplement 3 months before start of or during the study, (c) consumption of more than 22 µg/ day vitamin D from vitamin supplement use, (d) consumption of 11.25-22 µg vitamin D daily from vitamin supplement use in combination with a serum calcidiol concentration ≥ 50 nmol/L, (e) consumption of more than 500 mg calcium daily from mineral supplement use.
## Study design
The 13-week double-blinded RCT (known as the PROVIDE study) involved consumption of 2 servings of a vitamin D, calcium and leucine-enriched, whey protein medical nutrition drink (WP-MND) in the test group or 2 servings of an iso-caloric drink with only carbohydrates, fat and some trace elements in the control group [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref]. This RCT was extended with a 13-week open-label extension (OLE) study period. Participants in the RCT who volunteered to participate in the OLE part of the study, were randomized to receive either 1 or 2 servings of the WP-MND (FortiFit, Nutricia N.V., the Netherlands). One serving of WP-MND contained 21 g total protein, including 20 g whey protein and 3 g total leucine, 9 g carbohydrates, 3 g fat, 800 IU (20 µg) vitamin D 3 , 500 mg calcium, and a mixture of vitamins, minerals and fibers (for detailed composition see online supplementary material-Supplemental [fig_ref] Table 1: Baseline [/fig_ref] of Bauer et al. [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref]. The one-serving group took the WP-MND before breakfast. In the two-serving group, the regimen was the same as in the intervention group of the RCT, i.e. before breakfast and lunch [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref].
## Outcome measures
Parameters were assessed prior to the RCT (baseline RCT), at the end of the RCT, which was also the start of the OLE part of the study (week 13, Baseline OLE), and after the OLE study period (week 26). For determination of blood parameters, approximately 25.5 mL of venous blood was obtained at each visit. Blood samples in which creatinine (µmol/L), calcidiol (nmol/L), parathyroid hormone (PTH, pmol/L), calcium (mmol/L) and albumin (g/L) were determined, were left at room temperature for 30 min and then centrifuged. Aliquots of serum were stored at − 20 or -80 °C until analysis. These serum samples were analyzed at the Reinier de Graaf Groep medical laboratory, Delft, the Netherlands. Blood samples obtained for determination of the other parameters were processed and determined at the local laboratories on site, according to local procedures.
## Kidney function
Kidney function was defined by the estimated glomerular filtration rate (eGFR, mL/min/1.73 m 2 ) that was calculated with serum creatinine (Creat, µmol/L), using the Chronic Kidney Disease Epidemiology (CKD-EPI) Equation as described by Levey et al. [bib_ref] A new equation to estimate glomerular filtration rate, Levey [/bib_ref]. Briefly, the CKD-EPI equation includes log [Creat] (modeled as a 2-slope linear spline with sex-specific 0.7 mg/dL in women and a 0.9 mg/dL in men), and gender, race, and age on a natural scale.
## Vitamin d and calcium metabolism
Analytical testing for total serum calcidiol was performed using chemiluminescense micro-particulate immunoassay (Abbott Laboratories, Wiesbaden, Germany). To determine the effects of changes in the serum calcidiol on vitamin D metabolism, serum PTH and calcium were determined as well. Serum intact PTH concentrations were measured in serum using an ELISA [Intact parathyroid hormone, MD Biosciences Inc., St. Paul, MN 55108], with an intra-assay CV of 3.4%. For albumin-corrected calcium, total serum calcium and albumin were measured by an automated system [Instrumentation Laboratories UK Ltd, Cheshire, UK]. The intra-assay CV for serum calcium and albumin was 1.7% and 1.8%, respectively. Serum calcium (Ca) normally ranges between 2.15 and 2.55 mmol/L [bib_ref] The diagnosis and management of hypercalcaemia, Minisola [/bib_ref] [bib_ref] Incidence of hypercalciuria and hypercalcemia during vitamin D and calcium supplementation in..., Gallagher [/bib_ref] ; therefore, hypercalcemia was defined as [Ca] > 2.55 mmol/L [bib_ref] Incidence of hypercalciuria and hypercalcemia during vitamin D and calcium supplementation in..., Gallagher [/bib_ref]. Serum calcium was adjusted for albumin according to the equation described by James et al. [bib_ref] Derivation and internal validation of an equation for albumin-adjusted calcium, James [/bib_ref] :
In participants with calcidiol levels ≥ 100 nmol/L at baseline or during the study, serum calcium levels and adverse effects were closely evaluated.
## General safety and gastrointestinal tolerability
Liver function was assessed at the local laboratories by determination of ALanine AminoTransferase (ALAT, U/L), ASparate AminoTransferase (ASAT, U/L), alkaline phosphatase (U/L), and gamma-Glutamyl Transpeptidase (ɣ-GT, U/L). Number and type of adverse events were registered. An adverse event was defined as any untoward medical occurrence in a clinical trial participant who was administered a study product, not necessarily implying a causal relationship with the intervention. The occurrence of adverse events was also checked between visits with intermittent phone calls. Gastrointestinal (GI) tolerability was assessed using a GI questionnaire at baseline, after 13 weeks and after 26 weeks of intervention. This questionnaire covered a range of GI symptoms: nausea, belching, feeling of fullness, vomiting, abdominal distension, flatulence, diarrhoea, constipation, dry mouth and thirst with a four-point scale (absent/mild /moderate/severe).
# Statistical analysis
All the analysis of the main RCT paper [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] were done on the intention to treat (ITT) data set, while the safety and tolerability evaluation of the current paper was performed on the AST data set (n = 379 subjects), which excludes one subject in the control group who was randomized but did not start the intervention.
Normally distributed data were described as mean (SD or range) and analysed using the two-sample and paired t test for between and within-group differences, respectively. Not normally distributed data were described as median (IQR or range) and analysed using the Mann-Whitney test for between-group differences and the Wilcoxon Signed-Rank test for within-group changes. A p value < 0.05 indicates a significant difference between groups. All statistical analyses were performed using SAS software (version 9.4; SAS, Inc, Cary, NC).
# Results
Participants flow [fig_ref] Figure 1: Participants flow [/fig_ref] At the end of the 13-week RCT, 233 participants in the PRO-VIDE study agreed to participate in the OLE sequel of the study (test (T): n = 103; control (C): n = 130). 133 subjects were randomized into the 1-serving groups (CT1 and TT1) and 100 subjects were randomized into the 2-serving groups (CT2 and TT2). At the end of the OLE study, 214 participants completed the study (former T group receiving 1 serving of WP-MND during OLE (TT1): n = 57, former T group receiving 2 serving of WP-MND during OLE (TT2): n = 39, former C group receiving 1 serving of WP-MND during OLE (CT1): n = 72, former C group receiving 2 serving of WP-MND during OLE (CT2): n = 46), and 19 participants exited the study before week 26.
## Characteristics of the study population
Baseline characteristics of the RCT population are shown in the paper by Bauer et al. [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] and in [fig_ref] Table 1: Baseline [/fig_ref] together with week 13 baseline characteristics of the 4 different OLE groups. Characteristics were not different between groups, neither at baseline for the RCT nor at baseline for the OLE study period, apart from a difference in total protein intake, serum calcidiol and PTH at the start of the OLE study period due to prior intervention in the RCT, which excludes potential selection bias in the OLE extension.
## Protein intake and kidney function
During the RCT, supplementation with WP-MND led to a significant increase in median total protein intake from 1.0 (IQR: 0.9, 1.2) g/kg BW/day to 1.5 (IQR: 1.3, 1.7) g/kg BW/day in the test group (p < 0.001), with no change in the control group (T vs. C: p < 0.001). Median protein intake (g/kg BW/day) increased in the former RCT control groups during the OLE study period from 0.9 (IQR: 0.8, 1.1) to 1.3 (IQR: 1.1, 1.5) in the CT1 (p < 0.001) and to 1.4 (IQR: 1.3, 1.7) g/kg BW/day in the CT2 group (p < 0.001). In the TT1 group that consumed one serving of WP-MND during the OLE study period after consumption of 2 servings during the RCT, median protein intake decreased from 1.5 (IQR: 1.3, 1.7) to 1.2 (IQR: 1.1, 1.4) g/kg BW/day (p < 0.001) versus no change in the TT2 group (TT1 vs. TT2: p < 0.001). n=1240 assessed for eligibility n=860 not included for reasons specified elsewhere [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] n=380 randomized in RCT -ITT set [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] n=184 received 2 servings of WP-MND (AST set) n=195 received 2 servings of control product (AST set) n=40 dropout RCT for reasons specified elsewhere [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] n=38 dropout RCT for reasons specified elsewhere [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref] n=103 randomized in OLE period n=144 completed RCT The study population had a median eGFR of 77 (IQR: 63, 87) mL/min/1.73 m 2 at baseline, prior to the RCT. 13-week supplementation with 2 servings of the WP-MND during the RCT led to a small increase in eGFR in the test group (median change 1.0 (IQR: − 2.8, 4.2) mL/min/1.73 m 2 ; p = 0.013) versus no significant change in the control group (median change − 1.1 (IQR: − 4.9, 2.8) mL/min/1.73 m 2 ; p = 0.067)(T vs. C: p = 0.002) . No change in eGFR was observed during the OLE part of the study . Some of the participants (T: n = 36 and C: n = 39) had an eGFR between 30 and 60 mL/min/1.73 m 2 at baseline, before the start of the RCT, which is defined as moderate chronic kidney disease (CKD). In this vulnerable subgroup, the same trend in eGFR was observed as in the total study population. During the RCT, eGFR increased in participants with moderate CKD at baseline in the test group from median 52 (IQR: 48.1, 56.2) to 56.2 (IQR: 46.5, 63.8; p = 0.007) mL/ min/1.73 m 2 vs. no change in the control group (T vs. C: NS). No further change was observed during OLE within or between the moderate CKD subgroups.
## Leucine intake
Based on the reported protein intake data and the estimation that dietary protein contains on average 7.8% leucine [bib_ref] Association between protein intake and blood pressure: the INTERMAP Study, Elliott [/bib_ref] , the total daily leucine intake from the diet and the WP-MND was calculated. The median baseline intake of leucine from the diet at the start of the RCT was estimated to be around 0.078 g/kg BW/day (1.0 g protein/kg BW/day). At week 13, leucine intake from the diet was estimated to be around 0.070 g/kg BW/day (0.9 g protein/kg BW/day) in 50% (median) and 0.086 g/kg BW/day (1.1 g protein/kg BW/day) in 75% (upper IQR) of the test group. The WP-MND supplement contributed to an additional 6 g leucine per day, representing 0.086 g/kg BW/day based on a median bodyweight of 70 kg. In total, leucine intake in the WP-MND group was 0.171 g/kg BW/day in 75% of subjects, which is well below the TUL for leucine of 0.5 g/kg BW/ day [bib_ref] 2220S) Determination of the tolerable upper intake level of leucine in adult..., Pencharz [/bib_ref] [bib_ref] Determination of the safety of leucine supplementation in healthy elderly men, Rasmussen [/bib_ref] [bib_ref] 2249S) A proposal for an upper limit of leucine safe intake in..., Cynober [/bib_ref].
## Vitamin d and calcium metabolism
After 13 weeks of intervention with 2 servings of the WP-MND in the test group, a significant increase in median serum calcidiol (p < 0.001) was observed versus a significant decrease (p < 0.001) in the control population (T vs. C: p < 0.001) . No further increase was observed in either of the former test groups (TT1 and TT2) during the OLE study period (13 vs. 26 weeks) . On the CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE. # Significant difference in change between Test and Control groups. * Significant change within group other hand, a significant increase in calcidiol levels was observed in the participants who switched from the control product during the RCT to the test product in the OLE study period (both CT1, CT2: p < 0.001) and most in the group that received 2 servings of the WP-MND (CT1 vs. CT2: p < 0.001).
Fourteen participants had a serum calcidiol concentration ≥ 100 nmol/L at baseline, prior to the RCT (T: n = 6; C: n = 8). In 20 participants with normal calcidiol levels at baseline, calcidiol increased above 100 nmol/L during the RCT (T: n = 18, C: n = 2). During the OLE study period, the number of participants with serum calcidiol levels ≥ 100 nmol/L decreased in the former test groups (T: n = 20 → 16) and increased in the former control groups (C: n = 6 → 9). In 13 OLE participants (T: n = 10; C: n = 3) with calcidiol levels ≥ 100 at week 13, serum calcidiol increased further at week 26 (range of increase: 2-38 nmol/L), independent of the number of servings.
During the RCT, serum levels of PTH decreased in the test group (p < 0.001) and increased in the control group (p = 0.006) [fig_ref] Figure 4: Serum levels of parathyroid hormone [/fig_ref]. Serum levels of PTH did not change with continued supplementation of a WP-MND but decreased significantly in participants who switched from control to test product during the OLE study period (p < 0.001) [fig_ref] Figure 4: Serum levels of parathyroid hormone [/fig_ref].
During the RCT, the albumin-adjusted serum calcium concentration increased in the test group from 2.29 to 2.33 mmol/L (p < 0.001) versus no change in the control group (mean: 2.31 mmol/L; T vs. C: p < 0.001) [fig_ref] Figure 5: Albumin-adjusted serum levels of calcium in the RCT [/fig_ref]. During the OLE study period, calcium levels increased in the former control population (CT1: p = 0.017; CT2: p = 0.003) whereas no further increase was observed in the former test population [fig_ref] Figure 5: Albumin-adjusted serum levels of calcium in the RCT [/fig_ref]. Although albumin-corrected calcium levels > 2.55 mmol/L were observed, the proportion of participants with hypercalcaemia was not different between groups. At baseline, 12 subjects already manifested albumin-corrected calcium levels above 2.55 mmol/L (T: n = 3; C: n = 9). During the RCT, a similar number of subjects in the test and control group developed hypercalcaemia (T: n = 3; C: n = 3), while 8 (T: n = 2; C: n = 6) of the 14 subjects with hypercalcemia at week 13 already had elevated levels at baseline. This suggests no direct association between the development of hypercalcemia and supplementation of WP-MND but may point to a surprisingly higher prevalence of hypercalcemia in this sarcopenic older population. In OLE extension with 2 servings per day (CT2), 2 subjects maintained the elevated calcium levels and 3 new subjects were found with calcium levels above 2.55 mmol/L. In the OLE group taking 1 serving a day (CT1), 3 subjects maintained higher calcium levels, while 2 subjects with the elevation of calcium above 2.55 mmol/L were found. The presence of hypercalcemia at baseline or development of hypercalcemia during the study in any of the groups showed no connection to evolvement over time of PTH, calcidiol, creatinine, and eGFR.
At the start of RCT, none of the 14 participants with calcidiol levels above 100 nmol/L had albumin-corrected calcium levels above 2.55 mmol/L. At week 13 (end RCT and start OLE), 3 out of 26 participants with calcidiol above 100 nmol/L (from the former test group) had albumin-corrected calcium levels above 2.55 mmol/L. At week 26 (end OLE), 3 out of 25 participants had both levels elevated (1 from the TT1, 1 from the TT2 and 1 from the CT2 group) of which for one of them (from the TT2 group) this was already the case at baseline OLE.
## General safety, vital signs
Parameters of liver function did not increase during the RCT or OLE study period and values were within the range of reference values [fig_ref] Table 1: Baseline [/fig_ref].
At baseline, heart rate was significantly higher in the test group compared to the control group (p = 0.011), but no significant changes in heart rate, systolic and diastolic blood pressure were observed between the two groups after 13 weeks of intervention [fig_ref] Table 3: Measured vital signs at baseline of the RCT study [/fig_ref] in Appendix). At OLE baseline (week 13), heart rate in TT1 was significantly lower compared to the TT2 group (p = 0.018), but no other significant differences were found between the groups at week 26. Similarly, no significant differences were found in systolic or diastolic blood pressure between the different groups (CT1 vs. CT2; TT1 vs. TT2; CT2 vs. TT2) during the OLE extension in Appendix).
## Adverse events
During the RCT, a total of 557 adverse events were reported among 240 subjects. From those, 193 were assessed as being related to the study products, without a significant difference between the test group (46 subjects; 25.0%) and the control group (54 subjects; 27.7%) (p = 0.562). In the OLE study period, a total of 216 adverse events were reported among 112 subjects. From those, 57 were assessed as being related to the study product. The number of subjects reporting one or more related adverse events was significantly higher in the TT2 group (9 subjects; 20.9%) compared to the TT1 group (only 3 subjects; 5%) (p = 0.026). Since other betweengroups comparisons did not show a significant difference (TT2 vs. CT2 and CT2 vs. CT1), there was no apparent doserelationship and the data did not point to any concern. The most commonly reported related adverse events were gastrointestinal (such as abdominal pain and nausea) or metabolic and nutritional disorders (such as hyperglycaemia). No treatment-related serious adverse events occurred during the RCT and OLE study periods. Moreover, no related adverse (serious or non-serious) events, including nephrolithiasis (kidney stones), were observed in participants with serum calcidiol levels ≥ 100 nmol/L or with albumin-adjusted calcium levels > 2.55 mmol/L, at any of the time points.
## Gastrointestinal tolerability
The incidence and the severity of the self-reported GI symptoms (nausea, belching, feeling of fullness, vomiting, abdominal distension, flatulence, diarrhoea, constipation, dry mouth and thirst) did not differ between the control and the test group at baseline or the end of the RCT in Appendix). Moreover, no difference was observed between the two groups with regards to the changes at week 13 relative to the baseline for any of the GI symptoms. The OLE study also showed no difference in the incidence or severity of the GI symptoms between the groups (CT1 vs. CT2; TT1 vs. TT2; CT2 vs. TT2), except for diarrhoea which was significantly lower in CT2 compared to CT1 (p = 0.01) [fig_ref] Table 6: Incidence of the gastrointestinal symptoms at baseline of the open-label extension [/fig_ref] in Appendix).
# Discussion
6-month intervention with up to 2 servings of this WP-MND in addition to a regular diet neither impaired kidney function nor caused vitamin D and/or calcium toxicity and is not related to impaired liver function and vital signs, increased gastro-intestinal intolerance or -adverse events.
## Protein intake and kidney function
The consistent high protein intake in line with recommendations, especially in participants receiving 2 servings of WP-MND throughout the entire 26-week study period (during RCT and OLE), enabled evaluation of the effect of a prolonged high protein intake on kidney function. Concerns regarding the adverse effect of a high protein diet on kidney function are related to glomerular hyperfiltration and hypertensive effects [bib_ref] Higher protein intake is not associated with decreased kidney function in prediabetic..., Moller [/bib_ref] [bib_ref] High-protein diets and renal health, Marckmann [/bib_ref] [bib_ref] Changes in kidney function do not differ between healthy adults consuming higher-compared..., Devries [/bib_ref]. During the RCT, eGFR increased in the test vs. no change in the control group (p = 0.002). The same trend was observed in the subgroup of participants with moderate CKD at baseline. During OLE, no (further) change in eGFR was observed in any of the (sub)groups. The alteration of eGFR in response to a high protein diet is in line with observations by others and is thought to be an adaptive response to the protein feeding and not the development of CKD per se [bib_ref] Higher protein intake is not associated with decreased kidney function in prediabetic..., Moller [/bib_ref] [bib_ref] Changes in kidney function do not differ between healthy adults consuming higher-compared..., Devries [/bib_ref] [bib_ref] Comparison of high vs. normal/low protein diets on renal function in subjects..., Schwingshackl [/bib_ref]. The observed plateau in eGFR after 13 weeks of treatment with 2 servings of WP-MND during the OLE period, supports this notion of adaptation. Moreover, the observed increase and subsequent plateau of eGFR after 13 weeks of treatment in older people with moderate CKD suggest that even those individuals still tolerate a high protein diet. However, caution is still warranted, and kidney function should be monitored on a regular basis in people on a high protein diet with moderate CKD, as recommended [bib_ref] The role of dietary protein and vitamin D in maintaining musculoskeletal health..., Rizzoli [/bib_ref] [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref] [bib_ref] Protein intake and exercise for optimal muscle function with aging: recommendations from..., Deutz [/bib_ref]. Furthermore, blood pressure did not change over time and was not different between groups, which supports the assumption that the observed increase of eGFR was not an indication of early kidney failure.
## Vitamin d and calcium metabolism
Calcidiol levels increased with the intake of 2 servings of WP-MND, providing 40 µg vitamin D, and reached a plateau at ~ 80 nmol/L after 3 months of supplementation. Moreover, 1 serving of WP-MND providing 20 µg vitamin D also increased calcidiol levels after 3 months, although to a lesser extent (~ 70 nmol/L), hence suggesting a dose-response effect. When considering a baseline diet with a median vitamin D intake of 2.0 (IQR: 1.0, 4.0) µg/day at the beginning of the RCT and 2.1 (IQR: 1.0, 4.3) µg/day at the beginning of OLE [fig_ref] Table 1: Baseline [/fig_ref] , the total intake of vitamin D did not exceed 50 µg/day. This amount is well below the TUL for vitamin D intake at 100 µg per day set by EFSA [bib_ref] EFSA Panel on Dietetic Products Nutrition and Allergies) (2012) Scientific opinion on..., Efsa Nda Panel [/bib_ref]. For adults, EFSA selected hypercalcemia as the indicator of vitamin D toxicity that may occur when plasma levels of calcidiol increase above 100 nmol/L [bib_ref] The 2011 report on dietary reference intakes for calcium and vitamin D..., Ross [/bib_ref] [bib_ref] Vitamin D supplementation reduces insulin resistance in South Asian women living in..., Von Hurst [/bib_ref] [bib_ref] Vitamin D and pancreatic cancer, Stolzenberg-Solomon [/bib_ref] [bib_ref] Serum vitamin D and risk of pancreatic cancer in the prostate, lung,..., Stolzenberg-Solomon [/bib_ref]. In our study, some participants had calcidiol levels ≥ 100 nmol/L with or without hypercalcemia (albumin-corrected calcium levels > 2.55 mmol/L) at baseline or during the study. However, no causal relation was observed between high serum calcidiol and calcium levels and no related adverse events were reported. An explanation for high plasma calcidiol levels may be an enhanced endogenous skin vitamin D production following exposure to sunlight [bib_ref] Vitamin D and skin physiology: a D-lightful story, Holick [/bib_ref] [bib_ref] Seasonal variations in serum 25-hydroxy vitamin D levels in a Swedish cohort, Klingberg [/bib_ref] or clandestine consumption of vitamin D containing food supplements.
Although no relationship was observed between hypercalcidiol and hypercalcemia, the overall increase in serum calcium was significant but marginal in the test group during the RCT, and in the control group during OLE. This increase was observed to plateau after 3 months of supplementation with 2 servings of WP-MND. The total group average serum calcium level at baseline was 2.30 mmol/L (not different between groups), which is within the reference range of 2.15-2.6 mmol/L [bib_ref] The diagnosis and management of hypercalcaemia, Minisola [/bib_ref]. Twelve participants had hypercalcemia at baseline of the RCT, which is a surprisingly higher prevalence than reported in other populations, such as in the community and hospital (0.2-4% [bib_ref] Vitamin D-mediated hypercalcemia: mechanisms, diagnosis, and treatment, Tebben [/bib_ref] [bib_ref] Prevalence of hypercalcaemia in normal and in hospital populations, Frolich [/bib_ref]. This warrants confirmation of potentially unknown hypercalcemia cases among the sarcopenic population in future research. The prevalence of hypercalcemia did not change during the course of the RCT and the OLE study periods, with some participants maintaining hypercalcemia and others developing hypercalcemia, independent of the treatment group. The presence or development of hypercalcemia could not be linked to clinically relevant safety indicators or adverse events. As expected, serum parathyroid hormone (PTH) levels decreased with the intake of the WP-MND. Therefore, hyperparathyroidism was unlikely to be the cause of observed hypercalcemia. Another cause for hypercalcemia could be due to the calcium provided (1000 mg in 2 servings of WP-MND on top of the diet providing on average about 1000 g/day of calcium), leaving a margin of 500 mg before exceeding the TUL as established by EFSA [bib_ref] EFSA Panel on Dietetic Products Nutrition and Allergies) (2012) Scientific opinion on..., Efsa Nda Panel [/bib_ref]. However, the Panel considers that no relationship has been established between long-term calcium intakes at 2.5 g/day from diet and supplements and increased risk of nephrolithiasis.
## General safety and gastrointestinal tolerability
There was no difference in the number and origin of the adverse events between test and (former) control groups. Most of the adverse events were gastrointestinal, but the incidence and severity of gastrointestinal symptoms did not differ between the treatment groups. No potentially treatmentrelated serious adverse events were observed. Moreover, no adverse (serious or non-serious) events were observed in participants with serum calcidiol levels ≥ 100 nmol/L or with albumin-adjusted calcium levels > 2.55 mmol/L, at any of the time points. Finally, vital signs did not change throughout the study, and parameters of liver function were within the reference range and did not increase in any of the groups during the RCT or OLE study period.
# Strengths and limitations
The main strength of this investigation is the evaluation of the safety of an effective medical nutrition drink in a study group of sarcopenic older adults (n = 380) [bib_ref] Effects of a vitamin D and leucine-enriched whey protein nutritional supplement on..., Bauer [/bib_ref]. Detailed information was collected about the impact of 1-2 servings of WP-MND on protein intake, kidney function, vitamin D and calcium metabolism. Furthermore, participants with moderate CKD, supra-normal levels of calcidiol or hypercalcemia were separately analyzed and scrutinized for adverse effects. Another strength is that we observed a plateau after 3-months of WP-MND supplementation for eGFR and plasma levels of calcidiol, calcium and PTH, suggesting that the study period was likely long enough to demonstrate that consumption of two servings of WP-MND is safe in the longer term. A study limitation may be that treatment groups were small during OLE, although observed effects point in the same direction as during the RCT. Another limitation may be that we did not determine plasma ammonia and insulin resistance over time, which are indicative of short-term leucine toxicity and potential negative effects of leucine supplementation in longer term [bib_ref] Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway..., Balage [/bib_ref]. Furthermore, eGFR should perhaps have been monitored over an even longer period to ensure that the observed increase in eGFR in the test population just represented an adaptation to the high protein intake.
In conclusion, a 6-month intake of a vitamin D, calcium and leucine-enriched whey protein medical nutrition drink in addition to a regular diet does not impair kidney function or disturb vitamin D and calcium metabolism in sarcopenic older adults. Furthermore, WP-MND does not affect liver function and vital signs and is well tolerated by the gastrointestinal tract. In line with the protein recommendations, we do recommend monitoring of kidney function in consumers with moderate CKD [bib_ref] Evidence-based recommendations for optimal dietary protein intake in older people: a position..., Bauer [/bib_ref]. For kidney protection in general, sufficient fluid intake is recommended for proper hydration [bib_ref] Gastro-Intestinal tolerance and renal safety of protein oral nutritional supplements in nursing..., Ter Wee [/bib_ref]. Funding This study was financially supported and study products were provided by Danone Nutricia Research, Nutricia Advanced Medical Nutrition. The funding source was involved at all stages of the study.
## Compliance with ethical standards
## Conflict of interests
## Statement of human and animal rights
The study protocol was reviewed and approved by The Research Ethics Boards at each of the locations and was published and registered with the Dutch trials register with the identifier: NTR2329 (https ://www.trial regis ter.nl/trial reg). Study procedures were performed in accordance with the Declaration of Helsinki ethical principles for medical research involving human subjects.
Informed consent All participants gave written informed consent prior to the RCT and volunteered to participate in the open-label extension of the study.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. See [fig_ref] Table 3: Measured vital signs at baseline of the RCT study [/fig_ref] , 5, 6.
[fig] Figure 1: Participants flow. WP-MND, a vitamin D and leucineenriched whey protein medical nutrition drink; T, test group during RCT; C, Control group during RCT; OLE, open-label extension; TT1, former RCT [/fig]
[fig] Figure 2, Figure 3: Estimated glomerular filtration rate (eGFR) in the RCT (a), and OLE population (b). Results are expressed as median (IQR). This figure shows the eGFR in the RCT population at baseline (week 0) and after intervention (week 13) with 2 servings of a vitamin D, calcium and leucine enriched whey protein medical nutrition drink (WP-MND) (Test) or Control product (Control) (a), and in the open-label extension (OLE) population at baseline (week 13) and after inter-vention (week 26) with one or two servings of WP-MND) (b). CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE. # Significant difference in change between Test and Control groups. * Significant change within group Serum levels of 25-hydroxy-vitamin D (calcidiol) in the RCT (a), and OLE population (b). Results are expressed as median (IQR). This figure shows the serum concentrations of calcidiol in the RCT population at baseline (week 0) and after intervention (week 13) with 2 servings of a vitamin D, calcium and leucine enriched whey protein medical nutrition drink (WP-MND) (Test) or control product (Control) (a), and in the OLE population at baseline (week 13) and after intervention (week 26) with one or two servings of WP-MND (b). [/fig]
[fig] Figure 4: Serum levels of parathyroid hormone (PTH) in the RCT (a), and OLE population (b). Results are expressed as median (IQR). This figure shows the serum concentrations of PTH in the RCT population at baseline (week 0) and after intervention (week 13) with 2 servings of a vitamin D, calcium and leucine enriched whey protein medical nutrition drink (WP-MND) (Test) or Control product (Control) (a), and in the OLE population at baseline (week 13) and after inter-vention (week 26) with one or two servings of WP-MND (b). CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE. # Significant difference in change between Test and Control groups. * Significant change within group [/fig]
[fig] Figure 5: Albumin-adjusted serum levels of calcium in the RCT (a), and OLE population (b). Results are expressed as mean (SD). This figure shows the albumin-adjusted serum concentrations of calcium in the RCT population at baseline (week 0) and after intervention (week 13) with 2 servings of a vitamin D, calcium and leucine enriched whey protein medical nutrition drink (WP-MND) (Test) or Control product (Control) (a), and in the OLE population at baseline (week 13) and after intervention (week 26) with one or two servings of WP-MND (b [/fig]
[table] Table 1: Baseline (week 0) characteristics of RCT participants (n = 279, AST data set) and baseline (week 13) characteristics of participants in open-label extension of the study (OLE) (n = 233, AST data set) CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE [/table]
[table] Table 3: Measured vital signs at baseline of the RCT study (week 0) and at the end of the RCT study(week 13) in the control and the test group CT1, former RCT Control group receiving 1 serving of WP−MND during OLE; CT2, former RCT Control group receiving 2 servings of WP− MND during OLE; TT1, former RCT Test group receiving 1 serving of WP−MND during OLE; TT2, former RCT Test group receiving 2 servings of WP−MND during OLE *Significant difference between control and test; p value = 0.011 (t test) Vital signs at baseline of the open-label extension (week 13) and at the end of the OLE study period (week 26) CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE * Significant difference between TT1 and TT2; p value = 0.018 (t test)Table 5 Incidence of the gastrointestinal symptoms at baseline of the RCT study (week 0) and at the end of the RCT (week 13) in the test and the control group CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE [/table]
[table] Table 6: Incidence of the gastrointestinal symptoms at baseline of the open-label extension (week 13) and at the end of OLE study period (week 26) CT1, former RCT Control group receiving 1 serving of WP-MND during OLE; CT2, former RCT Control group receiving 2 servings of WP-MND during OLE; TT1, former RCT Test group receiving 1 serving of WP-MND during OLE; TT2, former RCT Test group receiving 2 servings of WP-MND during OLE. Significant difference between CT1 and CT2; p value = 0.01 (Mann-Whitney test) [/table]
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Risk of Suboptimal Iodine Intake in Pregnant Norwegian Women
Pregnant women and infants are exceptionally vulnerable to iodine deficiency. The aims of the present study were to estimate iodine intake, to investigate sources of iodine, to identify predictors of low or suboptimal iodine intake (defined as intakes below 100 μg/day and 150 μg/day) in a large population of pregnant Norwegian women and to evaluate iodine status in a sub-population. Iodine intake was calculated based on a validated Food Frequency Questionnaire in the Norwegian Mother and Child Cohort. The median iodine intake was 141 μg/day from food and 166 μg/day from food and supplements. Use of iodine-containing supplements was reported by 31.6%. The main source of iodine from food was dairy products, contributing 67% and 43% in non-supplement and iodine-supplement users, respectively. Of 61,904 women, 16.1% had iodine intake below 100 μg/day, 42.0% had iodine intake below 150 μg/day and only 21.7% reached the WHO/UNICEF/ICCIDD recommendation of 250 μg/day. Dietary behaviors associated with increased risk of low and suboptimal iodine intake were: no use of iodine-containing supplements and low intake of milk/yogurt, seafood and eggs. The median urinary iodine concentration measured in 119 participants (69 μg/L) confirmed insufficient iodine intake. Public health strategies are needed to improve and secure the iodine status of pregnant women in Norway.OPEN ACCESSNutrients 2013, 5 425 Keywords: iodine; pregnancy; prospective cohort; food frequency questionnaire; the Norwegian Mother and Child Cohort Study (MoBa)
# Introduction
Iodine is an essential component of the thyroid hormones. It is required throughout the life-cycle and is obtained primarily through the diet. Iodine deficiency is the world's greatest single cause of preventable brain damage, and in the last few decades, there has been remarkable progress in the global effort to eliminate iodine deficiency [bib_ref] Iodine deficiency in Europe and its consequences: An update, Delange [/bib_ref] [bib_ref] Global iodine status in 2011 and trends over the past decade, Andersson [/bib_ref] [bib_ref] Assessment of iodine nutrition in populations: Past, present, and future, Zimmermann [/bib_ref]. Insufficient iodine status is not only a problem in developing countries, but is also characterized as a major public health problem in many countries in Europe and the Western world [bib_ref] Iodine deficiency in Europe and its consequences: An update, Delange [/bib_ref] [bib_ref] Iodine deficiency in pregnancy: The effect on neurodevelopment in the child, Skeaff [/bib_ref] [bib_ref] Prevention and control of iodine deficiency in pregnant and lactating women and..., Andersson [/bib_ref] [bib_ref] The changing epidemiology of iodine deficiency, Li [/bib_ref].
Before 1950, there was endemic iodine deficiency in Norway, with goiter prevalence as high as 80% in certain inland areas. Since then, iodine has been added to cow fodder, resulting in a relatively high concentration of iodine in milk and dairy products. Combined with a high consumption of milk and other dairy products, this led to eradication of endemic goiter [bib_ref] Urinary excretion of iodine in Norway, Frey [/bib_ref]. The same happened in Britain [bib_ref] Iodine, milk, and the elimination of endemic goitre in Britain: The story..., Phillips [/bib_ref] , while other countries introduced national iodization programs by adding iodine to salt [bib_ref] Prevention and control of iodine deficiency in pregnant and lactating women and..., Andersson [/bib_ref] [bib_ref] Update on iodine status worldwide, Zimmermann [/bib_ref].
Pregnant women and infants are exceptionally vulnerable to deficiency, because iodine is essential for normal fetal and child brain development and growth [bib_ref] Iodine deficiency in pregnancy: The effect on neurodevelopment in the child, Skeaff [/bib_ref] [bib_ref] The effects of iodine deficiency in pregnancy and infancy, Zimmermann [/bib_ref]. The estimated average requirement for iodine in non-pregnant adults is 95 μg/day, and the recommended intake of 150 μg/day is derived by adding 2 SD and rounding to the nearest 50 μg/day [bib_ref] Assessment of iodine nutrition in populations: Past, present, and future, Zimmermann [/bib_ref] [bib_ref] Prevention and control of iodine deficiency in pregnant and lactating women and..., Andersson [/bib_ref]. The estimated average requirement is increased by at least 50% during pregnancy and lactation [bib_ref] Assessment of iodine nutrition in populations: Past, present, and future, Zimmermann [/bib_ref] [bib_ref] Iodine deficiency in pregnancy and the effects of maternal iodine supplementation on..., Zimmermann [/bib_ref]. The recommended iodine intake during pregnancy is 175 μg/day in Nordic countries, while WHO/UNICEF/ICCIDD recommends 250 μg/day during pregnancy and lactation. According to the epidemiological criteria for assessment of iodine status in pregnant populations, a median urinary iodine concentration (UIC) below 150 μg/L defines iodine deficiency (insufficient iodine intake), while a median UIC in the range 150-249 μg/L defines adequate (optimal) iodine intake [bib_ref] Assessment of iodine nutrition in populations: Past, present, and future, Zimmermann [/bib_ref] [bib_ref] Iodine-deficiency disorders, Zimmermann [/bib_ref]. Iodine deficiency during pregnancy is of concern when iodine intake falls below 100 μg/day.
Iodine deficiency has not been considered an issue in developed countries, such as Norway, for many decades. However, a sub-study in 119 pregnant women recruited to the Norwegian Mother and Child Cohort study (MoBa) in 2003-2004 revealed low iodine intake [bib_ref] Evaluation of urinary iodine excretion as a biomarker for intake of milk..., Brantsaeter [/bib_ref] [bib_ref] Self-reported dietary supplement use is confirmed by biological markers in the Norwegian..., Brantsaeter [/bib_ref]. Low iodine intake was also seen in a study within a larger sample of MoBa, with 10% of the women having iodine intake below 70 μg/day [bib_ref] Dietary supplements contribute substantially to the total nutrient intake in pregnant Norwegian..., Haugen [/bib_ref]. Studies on iodine intake and iodine status of pregnant women are urgently needed.
There have been major changes in Norwegian food patterns over the last decades. The aims of the present study were to estimate iodine intake, to investigate sources of iodine, to identify predictors of low or suboptimal iodine intake (defined as intakes below 100 μg/day and 150 μg/day, respectively) in a large population of pregnant Norwegian women and to evaluate iodine status in a sub-population of the participants.
# Materials and methods
## Population and study sample
The data set is part of the Norwegian Mother and Child Cohort study (MoBa), a prospective population-based pregnancy cohort conducted by the Norwegian Institute of Public Health [bib_ref] Cohort profile: The Norwegian Mother and Child Cohort Study (MoBa), Magnus [/bib_ref]. Participants were recruited from all over Norway from 1999 to 2008, and 38.5% of invited women consented to participate. The cohort now includes 108,000 children, 90,700 mothers and 71,500 fathers. Blood samples were obtained from both parents during pregnancy and from mothers and children (umbilical cord) at birth. Follow-up is conducted by questionnaires at regular intervals and by linkage to national health registries. Several sub-studies are conducting additional collections of data and biological materials.
The data included in this study are from two questionnaires answered in gestational weeks 15 (Q1) and 17-22 (Q2), respectively. Q2 is a detailed food frequency questionnaire (FFQ), while Q1 is a general questionnaire covering health, exposures, lifestyles and background factors. Pregnancy and birth records from the Medical Birth Registry of Norway (MBRN) are linked to the MoBa database [bib_ref] The medical birth registry of Norway. Epidemiological research and surveillance throughout 30..., Irgens [/bib_ref]. Informed consent was obtained from each participant before the study. The study was approved by the Regional Committee for Ethics in Medical Research and the Data Inspectorate in Norway.
This study uses the quality-assured data files released for research in 2009 (version 4). At the time of this analysis, 89,656 women had answered the first MoBa questionnaire and were recorded in MBRN. Of these, 76,218 had also answered version 2 of the FFQ [bib_ref] Methodological challenges when monitoring the diet of pregnant women in a large..., Meltzer [/bib_ref] , and 74,914 had registered a valid food intake (total energy >4500 kJ and <20,000 kJ per day). The range of acceptable energy intake in MoBa has been evaluated elsewhere [bib_ref] Methodological challenges when monitoring the diet of pregnant women in a large..., Meltzer [/bib_ref]. We excluded 5447 participants with missing information on maternal height, weight, smoking habits, educational attainment or household income and 7563 women who had participated in MoBa with more than one pregnancy, resulting in a study sample of 61,904 women for analysis.
Finally, we included data from a subpopulation of 119 women who participated in a validation study of the MoBa FFQ. While data on urinary iodine levels in this sub-sample have previously been reported as 24 h urinary iodine excretion (UIE, μg/24 h), the current study presents these results as urinary iodine concentration (UIC, μg/L). While UIE was relevant for validating estimated iodine intake, UIC is relevant for evaluating iodine status. Details about the study population, urinary sampling and iodine analysis have been described in detail previously [bib_ref] Evaluation of urinary iodine excretion as a biomarker for intake of milk..., Brantsaeter [/bib_ref] [bib_ref] Self-reported dietary supplement use is confirmed by biological markers in the Norwegian..., Brantsaeter [/bib_ref]. Participants in the validation study sub-sample were not representative of all women in MoBa. However, the estimated iodine intake and sources of iodine were similar to the whole cohort [bib_ref] Evaluation of urinary iodine excretion as a biomarker for intake of milk..., Brantsaeter [/bib_ref].
## Dietary information
The MoBa FFQwas specifically planned for the MoBa study and was completed by participating women in gestational weeks 17 to 22. The dietary data used in this study were collected from February 2002 to November 2008. The MoBa FFQ is a semi-quantitative questionnaire designed to capture dietary habits and intake of dietary supplements during the first four to five months of pregnancy and included questions about intake of 255 food items or dishes [bib_ref] Methodological challenges when monitoring the diet of pregnant women in a large..., Meltzer [/bib_ref]. For each food item, the frequency of consumption was reported by selecting one out of 8-10 frequencies, ranging from never to several times monthly, weekly or daily. Consumption frequencies were converted into food amounts (g/day) by the use of standard Norwegian portion sizes. Food and nutrient calculations were performed with FoodCalcand the Norwegian food composition table . For calculation of dietary iodine, we used available data from analyses of Norwegian milk and food samples [bib_ref] Iodine concentration in Norwegian milk and dairy products, Dahl [/bib_ref] [bib_ref] The iodine content of Norwegian foods and diets, Dahl [/bib_ref]. Food items in the FFQ were combined into non-overlapping groups, and the contribution of the various food groups to iodine intake was computed.
The FFQ did not include any question about the use of table salt or use of iodine fortified table salt. In Norway, few brands of table salt are iodized (maximum 5 μg/g NaCl), and the use of this salt is limited to private households and not allowed in the food industry [bib_ref] The iodine content of Norwegian foods and diets, Dahl [/bib_ref]. Drinking water in Norway contains only negligible amounts of iodine (~2 μg/L) [bib_ref] The iodine content of Norwegian foods and diets, Dahl [/bib_ref].
The last page in the FFQ asked about use of food supplements. Thirteen commonly used vitamin, mineral and cod liver oil/fish oil supplements were pre-coded and followed by 6 open-ended spaces where the women were asked to record the name and manufacturer of the supplement(s) used, but not listed. The frequency was reported as one of 9 options of weekly use (never, <1 and 1-7) and the quantity was reported as one of 3 options for liquid supplements and one of 4 options for number(s) of tablets/capsules (1, 2, 3 and ≥4). For calculating nutrients from dietary supplements, an Access database (Microsoft Office 2003) containing the nutrient value of more than 1000 different food supplements was constructed. Commonly sold food supplements in Norway were registered by information provided by the manufacturer, whereas nutritional information of dietary supplements bought from the Internet or abroad were collected from the Internet, either found on the homepage of the manufacturer or the supplier [bib_ref] Dietary supplements contribute substantially to the total nutrient intake in pregnant Norwegian..., Haugen [/bib_ref].
A validation study showed that, relative to a dietary reference method and several biological markers, the MoBa FFQ produces a realistic estimate of habitual intake and is a valid tool for ranking pregnant women according to high and low intakes of energy, nutrients and foods. The relative validity of iodine intake from food and supplements and the intake of specific food groups, such as dairy products and seafood, was evaluated separately. Total iodine intake by the FFQ showed acceptable agreement with iodine intake by the food diary (r = 0.48, 95% CI: 0.33, 0.61) and with urinary iodine excretion (r = 0.42, 95% CI: 0.26, 0.56). Urinary iodine excretion reflected the important food sources and whether or not iodine was contributed by supplements [bib_ref] Evaluation of urinary iodine excretion as a biomarker for intake of milk..., Brantsaeter [/bib_ref] [bib_ref] Self-reported dietary supplement use is confirmed by biological markers in the Norwegian..., Brantsaeter [/bib_ref] [bib_ref] Exploration of biomarkers for total fish intake in pregnant Norwegian women, Brantsaeter [/bib_ref]. Women were recruited to the validation study between January 2003 and February 2004, and a seasonal difference in urinary iodine excretion reflected a seasonal difference in the iodine content of Norwegian milk [bib_ref] Evaluation of urinary iodine excretion as a biomarker for intake of milk..., Brantsaeter [/bib_ref].
The intake of all types of milk and yogurt, except that used in mixed dishes, was divided in several ways: first, into three categories (<200 mL/day, 200-399 mL/day and ≥400 mL/day); second, into six groups (<100 mL/day, 100-199 mL/day, 200-299 mL/day, 300-399 mL/day, 400-499 mL/day and ≥500 mL/day); and third, into two categories (<200 mL/day or ≥200 mL/day). The intake of seafood was divided first into five groups (<5 g/day, 5-19 g/day, 20-39 g/day, 40-59 g/day and ≥60 g/day) and, second, into two categories (<20 g/day or ≥20 g/day). Egg intake was divided into two categories (<8 g/day or ≥8 g/day). Total energy intake was treated as a continuous variable.
## Other variables
Maternal age at delivery was divided into four categories (<25, 25-29, 30-34 and ≥35 years). Marital status was divided into two categories (married/cohabiting or single). Self-reported pre-pregnancy height and weight were used to calculate body mass index (kg/m 2 ), which was divided into WHO categories (<18.5, 18.5-24.9, 25-29.9 and ≥30 kg/m 2 ), length of education into three categories (≤12, 13-16 or ≥17 years), total household income into three categories (both participant and her partner <NOK 300,000, one ≥NOK 300,000 or both partners ≥NOK 300,000) and pre-pregnant smoking in three categories (non-smokers, occasional smokers or daily smokers). Parity was divided into three categories (nulliparous, primiparous or multiparous).
# Statistical methods
The estimated intakes of iodine from food and supplements were skewed and are presented by the median and the 5th and 95th percentiles (P5, P95). For univariate analyses, we used the Mann-Whitney U test for two group comparisons and the Kruskall-Wallis test for multiple group comparisons of iodine intakes. We used multiple logistic regression analysis to identify predictors of low and suboptimal iodine intake and present crude and adjusted odds ratios (OR) with 95% confidence intervals (CIs). Maternal characteristics and lifestyle variables examined as potential predictors were: maternal age, parity, education, marital status, smoking, pre-pregnancy BMI, household income, iodine containing supplement use and intakes of milk and yogurt, eggs and seafood. As iodine from food increased with increasing food intake, we adjusted for total energy intake. The significance level was set at 5% (two-tailed), and all analyses were performed using the statistical software PASW statistics 17 (SPSS Inc., IBM Company, Chicago, IL, USA).
# Results
The median intake of iodine from food was 141 μg/day, and the median total intake from food and supplements was 166 μg/day. Iodine was obtained from supplements in 19,575 (31.6%) of the women, and the median intake contributed by supplements in this group was 107 μg/day, resulting in a median total intake of 252 μg/day in iodine supplement users. Very low iodine intake (<70 μg/day) was observed for 4.8%, low intake (<100 μg/day) for 16.1% and suboptimal intake (<150 μg/day) was observed for 42% of all participants. The prevalence of low and suboptimal intake was much higher in women who did not obtain iodine from supplements [fig_ref] Table 1: Iodine intake in 61,904 pregnant Norwegian women in the Norwegian Mother and... [/fig_ref]. In the total group, 54.3% had iodine intakes <175 μg/day, while the proportion was 70.5% in non-iodine supplement users. In the upper end of iodine intake, two women had intakes above 1100 μg/day, which is considered the upper limit of intake that is unlikely to cause adverse health effects, while 770 women (1.2%) had excessive intakes (≥500 μg/day). Iodine from food did not differ with regard to maternal age, education, use of supplements or marital status, but was lower in smokers than in non-smokers and higher in multiparous than in nulliparous women. Total iodine intake was also higher in nulliparous women than in parous women. Total iodine decreased with increasing BMI, but the negative association was confounded by total energy intake and disappeared when energy was taken into account (data not shown). The most outstanding differences in iodine intakes were found with regards to differences in milk, seafood and egg consumption and whether or not iodine was obtained from supplements [fig_ref] Table 2: Iodine intake, median and range [/fig_ref]. In women who did not use iodine containing supplements and had low intake of milk/yogurt (<200 mL/day), 66.9% had iodine intake <100 μg/day and 98.6% had intake <150 μg/day. For comparison, in iodine supplement users with low milk/yogurt intake, the corresponding figures were 7.9% and 29.9%. No women had iodine intake <100 μg/day if they obtained iodine from supplements and also consumed at least one daily serving of milk and/or yogurt.
Iodine from supplements contributed 19% and dairy products contributed 52% to the total iodine intake in the whole group. In women who did not obtain iodine from supplements, dairy products contributed on average 64% and seafood contributed 15% to the total iodine intake .
## Figure 1.
The contribution (%) to maternal iodine intake from food groups and iodine containing supplements in iodine supplement (n = 19,575) and non-supplement users (n = 42,329).
The prevalence of inadequate iodine intake decreased with increasing milk and yogurt consumption.
The results indicate that an average milk/yogurt intake of 200-300 mL/day (1-2 servings) in addition to iodine from other foods, but no iodine from supplements, would secure 100 μg iodine daily for most women, while higher milk and yogurt intake is needed to obtain 150 μg iodine daily [fig_ref] Figure 2: The prevalence of suboptimal [/fig_ref]. Likewise, the prevalence of inadequate iodine intake decreased with increasing intake of seafood.
The results indicate that an average seafood intake of 20-40 g/day in addition to iodine from other foods, but no iodine from supplements, would secure 100 μg iodine for most women [fig_ref] Figure 3: The prevalence of suboptimal [/fig_ref]. This amount corresponds to one daily portion of fish or other seafood on bread or 1-2 fish dinners weekly. In the adjusted analysis, taking all maternal characteristic and relevant dietary practices into account, the most important predictors of suboptimal iodine intake were: no iodine supplement use, low consumption of milk and yogurt and low consumption of seafood [fig_ref] Table 3: Predictors of iodine intake <100 μg/day in 61,904 pregnant Norwegian women [/fig_ref]. These variables were identified as the major predictors of low iodine intake, also when inadequate iodine intake was defined as <150 μg/day (data not shown). To examine the possible change in dietary practices over time, we examined sources and intake of iodine by year of delivery for the women in our study [fig_ref] Table 4: Intake of iodine and selected iodine sources by year of delivery, n... [/fig_ref]. The proportion of women having iodine intake below 100 μg/day increased from 14.8% to 17.1% from 2002 to 2008. There was also an increase in the proportion of women who obtained iodine from supplements. However, there was a 12% reduction in the median consumption of milk and yogurt, from 413 mL/day to 363 mL/day, and a corresponding reduction in iodine contributed by milk and yogurt. Urinary iodine concentration was measured in 24 h urine samples collected for 119 MoBa participants in 2003-2004. Median UIC was 69 μg/L in the total sample, 64 μg/L in non-iodine supplement users (n = 84) and 84 μg/L in iodine supplement users. The corresponding median UIE was 130 μg/24 h in the total sample, 110 μg/24 h in non-iodine supplement users and 190 μg/24 h in iodine supplement users [fig_ref] Figure 4: Urinary iodine concentration [/fig_ref]. Only 13 (11%) of the women with available UIC data had UIC ≥ 150 μg/L, while 106 (89%) had UIC < 150 μg/L. The median iodine intake in the 13 women with UIC ≥ 150 μg/L was 150 μg/day, and the median intake in the 106 women with UIC < 150 μg/L was 130 μg/day. However, the WHO reference ranges for defining optimal iodine status based on UIC are for larger population groups. Box plot details: the horizontal line indicates the median; the box indicates the interquartile range (IQR) (IQR: 25th percentile to 75th percentile); the whiskers represent observations within 1.5-times the IQR; and the circles indicate observations more than 1.5-times the IQR away from the box, considered as outliers.
# Discussion
Although there is no screening program for iodine deficiency, Norway has been considered iodine replete for six decades. This study shows that inadequate iodine intake is prevalent in pregnant Norwegian women and suggests that iodine nutrition is a health concern. There are few dietary sources of iodine, and the individual intake of these food groups vary significantly. Pregnant women who do not consume or have low intake of dairy and/or seafood and who do not obtain iodine from supplements are at great risk of having inadequate iodine intake.
The use of an FFQ to assess iodine intake, as well as assigning an average iodine concentration to all milk and milk products, may result in imprecision in the intake estimates. However, in the present study, we based the estimates mainly on iodine concentrations measured in Norwegian food items and a detailed database for iodine content in specified dietary supplements. Data on urinary iodine excretion in a healthy and highly educated subsample of pregnant women supported that inadequate iodine intake should be regarded as a health concern also in Norway.
Low iodine intake in women of child-bearing age has been documented in other studies in Norway [bib_ref] The iodine content of Norwegian foods and diets, Dahl [/bib_ref] [bib_ref] Iodine intake and status in two groups of Norwegians. Scand, Dahl [/bib_ref] and in other European countries [bib_ref] Dietary iodine intake and urinary iodine excretion in a Danish population: Effect..., Rasmussen [/bib_ref] [bib_ref] Iodine intake and urinary excretion among adults in the Netherlands, Brussaard [/bib_ref] [bib_ref] Prevalence of maternal dietary iodine insufficiency in the north east of England:..., Kibirige [/bib_ref] [bib_ref] Iodine intake in Portuguese pregnant women: Results of a countrywide study, Limbert [/bib_ref] [bib_ref] The Swiss iodized salt program provides adequate iodine for school children and..., Andersson [/bib_ref] [bib_ref] Iodine deficiency in the UK and Ireland, Lazarus [/bib_ref] [bib_ref] Iodine supplementation of pregnant women in Europe: A review and recommendations, Zimmermann [/bib_ref]. Assessment of urinary iodine excretion in Western and Central Europe indicated that more than half of the population is at risk of iodine deficiency [bib_ref] Global iodine status in 2011 and trends over the past decade, Andersson [/bib_ref] [bib_ref] Iodine deficiency disorders in Europe, Vitti [/bib_ref]. This is an issue of public concern, especially in regard to pregnant women [bib_ref] Iodine deficiency in pregnancy: The effect on neurodevelopment in the child, Skeaff [/bib_ref]. A pilot study surveying the prevalence of iodine deficiency in the northeast of England concluded that 3.5% of the pregnant women had evidence of iodine deficiency and that 40% might be borderline deficient, defined by the urinary iodine/creatinine ratio [bib_ref] Prevalence of maternal dietary iodine insufficiency in the north east of England:..., Kibirige [/bib_ref]. Insufficient iodine nutrition in pregnant populations have been reported also in the US, New Zealand and Australia [bib_ref] Are pregnant women in New Zealand iodine deficient? A cross-sectional survey, Pettigrew-Porter [/bib_ref] [bib_ref] Iodine status in pregnant women and their newborns: Are our babies at..., Travers [/bib_ref].
The effects of severe iodine deficiency during critical periods of brain development are well documented, while less is known about the consequences of milder forms of iodine deficiency [bib_ref] Iodine deficiency in Europe and its consequences: An update, Delange [/bib_ref] [bib_ref] Iodine deficiency in pregnancy: The effect on neurodevelopment in the child, Skeaff [/bib_ref] [bib_ref] Iodine deficiency in pregnancy and the effects of maternal iodine supplementation on..., Zimmermann [/bib_ref] [bib_ref] Iodine supplementation of pregnant women in Europe: A review and recommendations, Zimmermann [/bib_ref] [bib_ref] Iodine intake in human nutrition: A systematic literature review, Gunnarsdottir [/bib_ref]. Mild iodine deficiency may influence developmental impairment in children. Although limited, a few studies reported associations between prenatal iodine status or suboptimal maternal iodine intake and cognitive function of infants and children up to 18 months [bib_ref] Subclinical prenatal iodine deficiency negatively affects infant development in Northern China, Choudhury [/bib_ref] [bib_ref] Neonatal thyroxine, maternal thyroid function, and child cognition, Oken [/bib_ref] and with attention deficit hyperactivity disorder (ADHD) symptoms in children [bib_ref] Effect of iodine prophylaxis during pregnancy on neurocognitive development of children during..., Velasco [/bib_ref] [bib_ref] Attention deficit and hyperactivity disorders in the offspring of mothers exposed to..., Vermiglio [/bib_ref]. A prospective study comprising 692 mother-children pairs in Holland reported an inverse association between maternal iodine status (UIC) and executive function in children at four years of age [bib_ref] Low urinary iodine excretion during early pregnancy is associated with alterations in..., Van Mil [/bib_ref]. The median UIC in the Dutch study population (203 μg/L) did not suggest iodine insufficiency at the group level. In the subsample of women having urinary iodine measurements in our study, median UIC was much lower (69 μg/L). The detailed assessment of diet and supplement use in MoBa, along with an on-going ADHD sub-study involving clinical examination of three-year-old children with ADHD symptoms, represents a unique opportunity to investigate whether mild to moderate deficiency of iodine during pregnancy is associated with the risk of developing ADHD symptoms at three years of age. The MoBa study is especially suited to study potential associations between inadequate maternal iodine intake and ADHD in children, because the children are otherwise well nourished.
MoBa is a large pregnancy cohort with participants from both urban and rural regions, representing all age groups and all socioeconomic groups. The study group is not entirely representative of the whole pregnant population of Norway, being somewhat better educated and with a lower percentage of smokers than the overall population of pregnant women [bib_ref] Self-selection and bias in a large prospective pregnancy cohort in Norway, Nilsen [/bib_ref]. The study did not aim to include minority groups, and more than 99% of the participants are of Caucasian ethnicity. Thus, it is all the more remarkable that less than half of the study group had a total iodine intake equal to or above the recommended intake of 175 μg/day, and it is likely that suboptimal iodine intake will be even more prevalent in the total population of pregnant Norwegian women. Data on urinary iodine concentration in the subsample corroborated that inadequate iodine status may be non-trivial even in a privileged pregnancy population in Norway. However, the WHO reference ranges for defining optimal iodine status are based on UIC for population medians [bib_ref] Assessing iodine intakes in pregnancy and strategies for improvement, Skeaff [/bib_ref]. The sample of 119 pregnant women is too small to evaluate iodine status in pregnant Norwegians in general, but the results indicate that suboptimal iodine nutrition is likely. Hence, iodine status should be assessed in a larger sample of pregnant women and in school children, as encouraged by WHO.
In this study, we combined food frequencies with iodine content measured primarily in Norwegian food samples. The highest iodine content was found in foods of marine origin, with lean fish, such as cod, having more than twice the content of fatty fish, such as farmed salmon [bib_ref] Determination of iodine in seafood by inductively coupled plasma/mass spectrometry, Julshamn [/bib_ref]. The consumption of fish, especially lean fish, is declining, and more so in women than in men. The current study showed that milk intake declined over the study period, resulting in a decline in iodine contributed by milk. The iodine content in milk differs with time of year and farming practice [bib_ref] Iodine concentration in Norwegian milk and dairy products, Dahl [/bib_ref]. In Norway, low-fat milk from the summer season had significantly lower median iodine concentration (88 μg/L, range 63-122 μg/L) compared with low-fat milk from the winter season (232 μg/L, range 103-272 μg/L). The median iodine concentration of organic summer milk (60 μg/L) was significantly lower than the iodine concentration of organic winter milk (127 μg/L) [bib_ref] Iodine concentration in Norwegian milk and dairy products, Dahl [/bib_ref]. A limitation of the present study was that a single average iodine value was applied to all milk and yogurt (150 μg/L). Changes in farming practices and legislation may have resulted in lower iodine content in milk over the last decade, but no values of iodine content in Norwegian milk have been published since 2003.
Estimating iodine intake using dietary assessment methods has limitations and uncertainties. The contribution of iodine from iodine fortified household salt was not included in the current study. However, the fortified salt contains very little iodine (5 μg/g), and the use of table salt is low (<2 g/day). Other concerns related to estimating iodine intake are recall bias and changes in appetite and eating patterns due to pregnancy. Misreporting is a serious error in all dietary assessment, and recall of diet over the first trimester is particularly difficult, as many women experience nausea and changes in appetite and eating patterns. Results from the validation study showed that stronger agreement between the FFQ and the test methods was observed for foods perceived as "healthy" than for "unhealthy" foods. Likewise, stronger agreement was seen for total energy intake when we excluded women who reported nausea at the time of answering the FFQ. It should also be noted that in this descriptive study, we used the MoBa FFQ to identify risk groups of low iodine intake, as well as to evaluate dietary sources of iodine, and these data are not independent. It is difficult to predict whether uncertainties in the dietary assessment would most likely lead to over-or under-estimating iodine intake. The results from the subsample analysis indicate that iodine intake of at least 150 μg/day would be needed to get the median UIC up to the optimal range of 150-249 μg/L defined by WHO.
The results of the present study highlight the importance of a balanced diet, including milk and seafood, during pregnancy. The significance of these foods as iodine sources has also been reported in other Nordic countries [bib_ref] Iodine status of pregnant women in a population changing from high to..., Gunnarsdottir [/bib_ref] [bib_ref] Iodine intake before and after mandatory iodization in Denmark: Results from the..., Rasmussen [/bib_ref] [bib_ref] Endemic goitre in Finland and changes during 30 years of iodine prophylaxis, Lamberg [/bib_ref]. Women with low intake of dairy and seafood are particularly vulnerable to low iodine intake and should be encouraged to use iodine-containing dietary supplements. Iodine fortification of table salt is a common strategy for iodine prophylaxis, but this does not ensure sufficient iodine intake in all population groups, except when iodine fortified salt, at concentrations higher than the 5 μg/g NaCl presently permitted in Norway, also is used in bread and other food products [bib_ref] Iodine deficiency in Europe and its consequences: An update, Delange [/bib_ref]. Denmark implemented mandatory iodine fortification of household salt and bread salt at a level of 13 μg/g NaCl in the year 2000. The Danish iodine fortification program was monitored with regard to positive, as well as negative, health effects. Evaluation of urinary iodine excretion in subgroups of the population before and after fortification showed that fortification resulted in increased iodine intake in all investigated groups [bib_ref] Iodine intake before and after mandatory iodization in Denmark: Results from the..., Rasmussen [/bib_ref]. Milk was the strongest dietary determinant of iodine intake before, as well as after, the fortification, and subjects with low milk intake combined with low intake of bread or table salt had iodine intake below the recommended intake, also after fortification.
The importance of iodine-containing supplements has been demonstrated in studies using urinary iodine excretion for assessing sufficient iodine nutrition in pregnancy [bib_ref] Iodine deficiency in Europe and its consequences: An update, Delange [/bib_ref] [bib_ref] Monitoring the adequacy of salt iodization in Switzerland: A national study of..., Hess [/bib_ref]. In Norway, the dietary recommendations to pregnant women do not include use of iodine supplementation. The present study shows that use of iodine-containing supplements is vital to secure optimal iodine intake and is especially important for women who do not include or have low intakes of seafood and/or milk and dairy in their diet.
# Conclusions
In conclusion, this study shows that the current iodine intake in a large proportion of pregnant Norwegian women may give rise to concern. Although the evidence of deleterious effects in terms of subtle cognitive impairment is limited, it is essential to increase the public awareness of dietary iodine nutrition in pregnant women. Our results highlight the significance of a balanced diet, including milk and seafood, during pregnancy. Women with low intake of dairy and seafood are particularly vulnerable to low iodine intake and should be encouraged to use iodine-containing supplements. The dietary sources of iodine in Norway do not secure a sufficient iodine intake for the entire population, and more awareness on iodine nutrition is warranted, especially in women who, for various reasons, limit their consumption of dairy and seafood. It cannot be excluded that a number of children growing up in Norway today have lifelong impairments due to suboptimal iodine nutrition in early life. There is an urgent need for public health strategies to monitor and secure the iodine status in Norway.
[fig] Figure 2: The prevalence of suboptimal (<150 μg/day) and low (<100 μg/day) iodine intake by increasing consumption of milk and/or yogurt in 42,329 non-iodine supplement users. [/fig]
[fig] Figure 3: The prevalence of suboptimal (<150 μg/day) and low (<100 μg/day) iodine intake by increasing consumption of seafood in 42,329 non-iodine supplement users. [/fig]
[fig] Figure 4: Urinary iodine concentration (UIC, μg/L) and urinary iodine excretion (UIE, μg/24 h) in 119 pregnant women in the Norwegian Mother and Child Cohort Study. [/fig]
[table] Table 1: Iodine intake in 61,904 pregnant Norwegian women in the Norwegian Mother and Child Cohort Study, 2002-2008. [/table]
[table] Table 2: Iodine intake, median and range (5th percentile, 95th percentile) by maternal characteristics. [/table]
[table] Table 3: Predictors of iodine intake <100 μg/day in 61,904 pregnant Norwegian women. Additionally adjusted for maternal age, parity, education, marital status, smoking, body mass index, household income and total energy intake. [/table]
[table] Table 4: Intake of iodine and selected iodine sources by year of delivery, n = 61,904 pregnant women. [/table]
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Comprehensive genetic analysis of histological components of combined small cell carcinoma
Background: Combined small-cell lung cancer (cSCLC) is a rare type of small-cell lung cancer (SCLC) that includes both SCLC and non-small-cell lung cancer (NSCLC). The molecular biological mechanisms underlying the heterogeneity of histological types in combined or metachronously transformed SCLC (mtSCLC) remain unclear. This study aimed to investigate the relationship between genetic alterations and each histological component heterogeneously detected in cSCLC and mtSCLC. Methods: This study included four cSCLC cases and one mtSCLC case. Formalinfixed and paraffin-embedded sections of each histological component of these tumors were subjected to next-generation sequencing (NGS) and quantitative reverse transcription-polymerase chain reaction to investigate the genetic mutations and expression levels of neuroendocrine cell-specific transcription factors (achaete-scute homolog-1 [ASCL1], brain-2 [BRN2] also known as POU domain class 3 transcription factor 2, nuclear factor 1 B [NF1B], insulinoma-associated protein 1 [INSM1], and thyroid transcription factor-1 [TTF-1]). Results: NGS analysis revealed that SCLC and NSCLC components share the same somatic mutations detected most frequently in TP53, and also in RB1 and EGFR. Gene expression analysis showed ASCL1 expression was significantly lower in the NSCLC component than in the SCLC component. Conclusion: We conclude that the morphological evolution of heterogeneous histological components in cSCLC may be associated with differences in ASCL1 expression levels, but not in acquired somatic gene mutations.K E Y W O R D Sachaete-scute homolog-1, heterogeneity, next-generation sequencing, small-cell lung cancer, somatic mutations
# Introduction
Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor that accounts for 13% of all lung cancers worldwide.Combined SCLC (cSCLC) is a type of SCLC with an incidence of 28% of all SCLC cases diagnosed by surgical specimens. [bib_ref] Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with..., Nicholson [/bib_ref] The World Health Organization (WHO) Tumor Classification defines it as SCLC combined with additional components of any histological type of non-small-cell lung carcinoma (NSCLC). [bib_ref] Update on small cell carcinoma and its differentiation from squamous cell carcinoma..., Travis [/bib_ref] Cases of NSCLC have been described with epidermal growth factor receptor (EGFR) gene mutation that was metachronously transformed to SCLC after treatment with EGFR-tyrosine kinase inhibitors (TKIs). [bib_ref] Histological transformation after acquired resistance to epidermal growth factor tyrosine kinase inhibitors, Shao [/bib_ref] However, the cell origin of SCLC and NSCLC is usually thought to be different: SCLC generally arises from neuroendocrine cells or neuroendocrine progenitors, whereas adenocarcinoma originates from alveolar type 2 and club cells. [bib_ref] Characterization of the cell of origin for small cell lung cancer, Park [/bib_ref] [bib_ref] Diverse cells at the origin of lung adenocarcinoma, Rowbotham [/bib_ref] Another report indicated that alveolar type II cells might form SCLC, although less frequently than neuroendocrine cells. [bib_ref] Cell of origin of small cell lung cancer: inactivation of Trp53 and..., Sutherland [/bib_ref] The cell origin of cSCLC and metachronously transformed SCLC (mtSCLC) underlying the heterogeneity of different histological components is unknown.
The genetic background of the two histological components of cSCLC is unclear. Only a few reports have investigated the genetic profiles of different histologic components in cSCLC using next-generation sequencing (NGS). [bib_ref] Combined small cell carcinoma of the lung: is it a single entity?, Zhao [/bib_ref] [bib_ref] Clinicopathological and genomic comparisons between different histologic components in combined small cell..., Lin [/bib_ref] Achaete-scute homolog-1 (ASCL1), a basic helix-loophelix transcription factor, is necessary to initiate the development of SCLC in a mouse model and induce neuroendocrine differentiation in SCLC. [bib_ref] Achaete-scute complex homologue 1 regulates tumor-initiating capacity in human small cell lung..., Jiang [/bib_ref] [bib_ref] Class III/IV POU transcription factors expressed in small cell lung cancer cells..., Ishii [/bib_ref] [bib_ref] POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1..., Ishii [/bib_ref] The transcription factor nuclear factor 1 B (NF1B) is targeted by ASCL1 and drives tumor initiation and progression in mouse models of SCLC. [bib_ref] ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroendocrine tumors and regulate distinct..., Borromeo [/bib_ref] [bib_ref] Transcription factor NFIB is a driver of small cell lung cancer progression..., Semenova [/bib_ref] Insulinoma-associated protein 1 (INSM1) is a zinc-finger transcription factor. It is a crucial regulator of ASCL1, brain-2 (BRN2), and neuroendocrine molecules in lung cancer cells and plays a role in the proliferation and apoptosis of SCLC. [bib_ref] Insulinoma-associated Protein 1 is a crucial regulator of neuroendocrine differentiation in lung..., Fujino [/bib_ref] Thyroid transcription factor-1 (TTF-1) expression levels have been associated with neuroendocrine differentiation via the expression of its regulators, such as ASCL1 and NF1B, in SCLC. [bib_ref] Clinicopathological characteristics of thyroid transcription factor 1-negative small cell lung cancers, Iida [/bib_ref] The roles of these transcription factors in the histological differences in cSCLC and mtSCLC have not been elucidated. This study aimed to clarify the status of genetic mutations and gene expressions related to morphological heterogeneity in cSCLC and mtSCLC.
# Methods
## Patients
Patients with cSCLC, mtSCLC, and pure SCLC consecutively diagnosed and treated at the Nihon University Itabashi Hospital (Tokyo, Japan) between 2010 and 2019 were enrolled in this study. The study design was approved by the Institutional Review Board (265-0, 30-14-0) according to the Declaration of Helsinki. The study investigated somatic mutations in targeted cancer panels; germline mutations were excluded.
Patients' clinical information was extracted from the medical records at Nihon University Itabashi Hospital . Diagnoses of cSCLC, mtSCLC, and pure SCLC were based on the 2021 WHO classification of lung tumors 3 by trained histopathologists. Four patients (cases 1-4) had limited cSCLC. One patient (case 5) had advanced mtSCLC that had progressed from adenocarcinoma. The FFPE tissues of primary and/or metastatic lesions of these patients were subjected to immunohistochemical analysis. Three cSCLC samples (cases 1-3) and one mtSCLC sample (case 5) were subjected to NGS analysis, and four cSCLC samples (cases 1-4) and six pure SCLC samples were subjected to quantitative RT-PCR analyses.
## Immunohistochemistry
The expressions of CD56, synaptophysin, chromogranin A, ASCL1, INSM1, TTF-1, and Ki-67 were evaluated using immunohistochemistry. The 4-μm thick sections were mounted on silane-coated glass slides. After deparaffinization, samples were boiled in citrate buffer (pH 6.0) for CD56, synaptophysin, and chromogranin A, and in ethylenediamine tetraacetic acid buffer (pH 9.0) for ASCL1, INSM1, and TTF-1, as antigen retrieval. Subsequently, an automated staining system (Histostainer; Nichirei Bioscience, Tokyo, Japan) was used for immunostaining, which Total RNA extraction and cDNA synthesis
The 8-μm thick sections were mounted on regular glass slides. After deparaffinization, the target tumor cells, identified using HE staining, were dissected and collected in 1.5-mL tubes. Total RNA was extracted using an RNeasy FFPE Kit (Qiagen) according to the manufacturer's instructions. The RNA samples were dissolved in 5 μl of RNase-free water, and the concentration was measured using the Nanodrop (Thermo Fisher Scientific Inc.). Total RNA samples were stored at À80 C until use. Genomic DNA was eliminated and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions.
## Quantitative rt-pcr assay
We investigated the mRNA expression levels of TTF-1, ASCL1, BRN2, NF1B, INSM1, and the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using quantitative RT-PCR. Quantitative RT-PCR was performed using 1 μl of cDNA, TaqMan probes (Thermo Fisher Scientific Inc.), and a Step One Plus quantitative RT-PCR machine (Thermo Fisher Scientific Inc.). The premixed primers and probes were as follows: ASCL1 (Assay ID: Hs04187546_g1), BRN2 (Assay ID: Hs00271595_s1), NF1B (Assay ID: Hs01029175_m1), TTF-1 (Assay ID: Hs00968940_m1), INSM1 (Assay ID: Hs00357871_s1), and GAPDH (Assay ID: Hs99999905_m1). The quantitative RT-PCR thermal cycling profile was as follows: 95 C for 10 min, followed by 50 cycles of 95 C for 15 s and 60 C for 1 min. The expression levels of the target mRNA were calculated using the ΔCt method, with GAPDH mRNA expression as the reference. 21
# Statistical analysis
# Results
## Immunohistochemical findings
Immunohistochemical neuroendocrine and proliferation markers were evaluated in the SCLC and NSCLC components . The SCLC component showed higher positive rates for synaptophysin, CD56, ASCL1, and INSM1, and a higher labeling index for Ki67 compared to the NSCLC component and . The significant difference in the Ki-67 labeling index (%) was higher in the SCLC component (mean 72%) than in the NSCLC component (mean 30%). There were no significant differences in chromogranin A or TTF-1 expression .
## Genetic mutations in sclc and nsclc components
Genetic mutations in three cSCLC cases and one mtSCLC case were analyzed using samples with sufficient levels of DNA quality and volume. The tumor contents comprised 80-90% of the SCLC component and 20-80% of the NSCLC component. lists the somatic mutations that were detected in each case. shows . the somatic mutations validated using Sanger sequencing. In four cases, 17 mutations in nine genes (EGFR, RB1, TP53, MUC16, SMARCA4, KDR, PKHD1, KMT2D, and RBM10) were found. The most common mutation type was the missense mutation (88%, 15/17 mutations). According to the ClinVar database, two pathogenic somatic mutations were clinically significant: p.Ser768Ile in EGFR (case 1) and p.Arg251* in RB1 (case 5), one pathogenic/likely pathogenic somatic mutation, p. Arg158His in TP53 (case 1), and one likely pathogenic somatic mutation, p.Arg175Leu in TP53 (case 5). A drug response variant was also detected (p.Leu858Arg in EGFR [case 5]). The somatic mutations with uncertain significance were p.Pro142Leu in TP53 (case 5), p.Ala159Val in TP53 (case 2), p.Lys1540Arg in SMARCA4 (case 5), p. Val774Met in EGFR (case 1), and p.Glu654Lys in PKHD1 (case 2). In each case, most genetic mutations were shared between SCLC and NSCLC components: 3/3 (100%) in case 1, 5/5 (100%) in case 2, 1/3 (33%) in case 3, and 5/6 (83%) in case 5. The common shared genetic mutations were TP53 (3/4 cases), EGFR (2/4 cases), KDR, PKHD1, KMT2D, MUC16, RB1, and SMARCA4 (1/4 cases).
mRNA expression levels of ASCL1, BRN2, NF1B, TTF-1, and INSM1 in the SCLC and NSCLC components of cSCLC and pure SCLC The mRNA expression levels of ASCL1 were significantly lower in the NSCLC component than in the SCLC component of cSCLCs (p = 0.029; . The ASCL1 expression level tended to be higher in the pure SCLC than in SCLC components of cSCLC, although this was not statistically significant . BRN2, NF1B, TTF-1, and INSM1 expression levels did not differ significantly between the pure SCLC and SCLC components or between the SCLC and NSCLC components of cSCLC .
# Discussion
The present study demonstrated that cSCLC and mtSCLC share the same major gene mutations in the SCLC and NSCLC components. However, in the NSCLC component, the ASCL1 expression was significantly lower than in the SCLC component of the cSCLC. Similar to our result, a previous study also showed that approximately 75% of the identified somatic mutations, such F I G U R E 2 mRNA expression of neuroendocrine-related genes of pure SCLC and SCLC and NSCLC component in cSCLC mRNA expression normalized to GAPDH was compared to that of ASCL1 (a), BRN2 (b), NF1B (c), TTF-1 (d), and INSM1 (e). The results were statistically analyzed using the Mann-Whitney U test. Bars represent mean AE standard deviation. *p < 0.05. NS, not significant as TP53, were present in both components of three cSCLC cases. [bib_ref] Combined small cell carcinoma of the lung: is it a single entity?, Zhao [/bib_ref] These results suggest that SCLC and NSCLC components in cSCLC originate from a common ancestor because they have a similar genetic background. In this study, the most common gene mutation was the TP53 mutation found in all three cases. The TP53 mutations have been reported in approximately 90% of SCLC cases and 46% of NSCLC (adenocarcinoma) cases. [bib_ref] Comprehensive genomic profiles of small cell lung cancer, George [/bib_ref] Furthermore, shared RB1 mutations were observed in one case. RB1 mutations have been reported in approximately 65% of SCLC cases and 4% of NSCLC (adenocarcinoma) cases. [bib_ref] Comprehensive genomic profiles of small cell lung cancer, George [/bib_ref] Bi-allele TP53 and RB1 mutations are early and necessary key events in the development of pure SCLC in humans. [bib_ref] ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroendocrine tumors and regulate distinct..., Borromeo [/bib_ref] [bib_ref] Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer, Peifer [/bib_ref] The same TP53 and RB1 mutations were found in both SCLC and NSCLC histological components in this study. From the results of these gene mutations, the cell origin of each histological component of cSCLC may be closer to that of pure SCLC than that of NSCLC. In addition, pathogenic EGFR mutations were shared by both SCLC and NSCLC. Previous reports have shown that different histological components harbor the same EGFR mutation in transformation into SCLC as a mechanism of resistance to EGFR TKIs. [bib_ref] Histological transformation after acquired resistance to epidermal growth factor tyrosine kinase inhibitors, Shao [/bib_ref] [bib_ref] Epidermal growth factor receptor mutations in small cell lung cancer, Tatematsu [/bib_ref] In this study, one cSCLC case had an EGFR mutation in both histological components, independent of transformation into SCLC as a mechanism of resistance to EGFR TKIs. Since there have been previous reports that cSCLC responds to EGFR-TKI, it may be important to perform an EGFR gene mutation analysis in cSCLC to obtain additional therapeutic options. [bib_ref] Epidermal growth factor receptor mutations in small cell lung cancer, Tatematsu [/bib_ref] ASCL1 regulates neuroendocrine differentiation; in particular, it upregulates synaptophysin and contributes to proliferation and migration by targeting cyclin-dependent kinase 5 in SCLC. [bib_ref] NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine..., Meder [/bib_ref] [bib_ref] Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway, Demelash [/bib_ref] [bib_ref] Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine..., Osada [/bib_ref] [bib_ref] Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin,..., Kashiwagi [/bib_ref] In the present study, the more frequent immunohistochemical positivity for synaptophysin, CD56, ASCL1, and INSM1 and a higher labeling index for Ki67 in the SCLC were correlated with higher ASCL1 expression. Our results support the previous reports and suggest that ASCL1 plays an important role in the development of SCLC. Interestingly, ASCL1 expression tended to be highest in pure SCLC and was lower in the NSCLC component than in the SCLC component. Cases that did not express ASCL1 and NEUROD1 in cSCLC compared to pure SCLC have been described. [bib_ref] SCLC subtypes defined by ASCL1, NEU-ROD1, POU2F3, and YAP1: a comprehensive immunohistochemical..., Baine [/bib_ref] Decreasing ASCL1 expression in the NSCLC component is considered to be associated with the activation of NOTCH signaling regulated by histone modification and differentiation to NSCLC from SCLC. [bib_ref] Correlation between histone acetylation and expression of Notch1 in human lung carcinoma..., Hassan [/bib_ref] Another report hypothesized that cSCLC might originate from pure SCLC, partially decreasing ASCL1 in the NSCLC component. [bib_ref] Notch signaling and Tp53/RB1 pathway in pulmonary neuroendocrine tumorigenesis, Ito [/bib_ref] Our results suggest that morphological transformation into NSCLC from SCLC might occur depending on decreased ASCL1 expression, but not because of genetic mutations, because of the presence of highly similar genetic backgrounds between the SCLC and NSCLC components .
In the present series, there was a peculiar case with lower ASCL1 expression in both SCLC and NSCLC components (case 1). SCLC subtypes are recently defined by differential expression of four key transcription regulators: ASCL1, NEUROD1, yes-associated protein 1 (YAP1) and POU2F3. [bib_ref] Molecular subtypes of small cell lung cancer: a synthesis of human and..., Rudin [/bib_ref] SCLCs with low expression of neuroendocrine markers are classified as YAP1 or POU2F3 types. Whether the cSCLC of case1 represents the YAP1 or POU2F3 type remains to be addressed. SCLC subtyping is more important clinically because the therapeutic potential of inhibitors targeting delta-like protein 3 (DLL3), an actionable target of ASCL1, is currently under further investigation in clinical trials. [bib_ref] AMG 757, a half-life extended, DLL3-targeted bispecific T-cell engager, shows high potency..., Giffin [/bib_ref] In ASCL1-high SCLCs, DLL3-targeting therapy is predicted to be more effective owing to the higher expression of DLL3. [bib_ref] SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting..., Wang [/bib_ref] Our results suggest that targeted therapy may be less effective in cSCLC than in pure SCLC because of lower levels of ASCL1. Further clinical investigations of cSCLC are necessary.
This study has some limitations. First, the sample size was small. The small sample size might make it difficult to clarify the expression levels of NF1B, BRN2, TTF-1, and INSM1. Second, the sensitivity of the methodologies may have influenced the results. The genetic mutations found in NGS were validated using Sanger sequencing, which has lower sensitivity than NGS, therefore gene mutations that could not be verified by Sanger sequencing could not be identified. Third, germline mutations were not analyzed in F I G U R E 3 A hypothetical schematic representation of the carcinogenesis pathway in pure SCLC and cSCLC due to different levels of ASCL1 expression the present study. One mutation (case 3, MUC16) was found in cancerous and noncancerous lung tissues as a secondary finding, which might have resulted from a mixture of cancerous tissues or a germline mutation; however, the ClinVar database has no information regarding this mutation. Thus, this mutation warrants further investigation. Finally, a small number of different histological components might have been admixed. However, each component was collected separately as much as possible via macrodissection.
In conclusion, this study demonstrated that each histological component in SCLC may have morphological evolution depending on the difference in ASCL1 expression, not due to the differences in acquired somatic mutations. |
The use of social media as a tool for stakeholder engagement in health service design and quality improvement: A scoping review
Background: Health-related social media use is common but few health organisations have embraced its potential for engaging stakeholders in service design and quality improvement (QI). Social media may provide new ways to engage more diverse stakeholders and conduct health design and QI activities.Objective: To map how social media is used by health services, providers and consumers to contribute to service design or QI activities.Methods: The scoping review was undertaken using the Joanna Briggs Institute methodology. An advisory committee of stakeholders provided guidance throughout the review. Inclusion criteria were studies of any health service stakeholders, in any health setting, where social media was used as a tool for communications which influenced or advocated for changes to health service design or delivery. A descriptive numerical summary of the communication models, user populations and QI activities was created from the included studies, and the findings were further synthesised using deductive qualitative content analysis.Results: 40 studies were included. User populations included organisations, clinical and non-clinical providers, young people, people with chronic illness/disability and First Nations people. Twitter was the most common platform for design and QI activities. Most activities were conducted using two-way communication models. A typology of social media use is presented, identifying nine major models of use.Conclusion: This review identifies the ways in which social media is being used as a tool to engage stakeholders in health service design and QI, with different models of use appropriate for different activities, user populations and stages of the QI cycle.
# Introduction
Social media is defined as "a group of Internet-based applications that build on the ideological and technological foundations of Web 2.0, and that allow the creation and exchange of User Generated Content" 1 (p.. Any online platform that allows users and audiences to create content and interact with each other can be considered social media.This definition includes platforms such as Facebook, Twitter, Instagram and YouTube, and privately developed platforms with functions that allow user interaction (such as forums and chat rooms). Social media allows for broadcast (one-way) or communicative (two-way) styles of communication 2 and allows for communication to happen in synchronous or asynchronous ways.The use of social media is extremely widespread and growing. It is estimated that 2.77 billion people worldwide use social media sites (equating to 71% of total internet users) with this number predicted to rise to over 3 billion by 2021.The majority of people in the United States of America (USA) use social media, with 68% of all USA adults using Facebook, and 94% using YouTube.In Australia, 88% of internet users have at least one social media profile.Health-related use of social media is very common. An estimated 80% of American internet users have searched for online health information, including information from social media sites,and Fifty-seven percent of Americans with chronic disease have used social media to find information and support for their condition.Health services around the world are also incorporating social media in communication strategies. A study of general and medical hospitals in the USA showed over 99% had a Facebook, Foursquare and/ or Yelp account.A similar study of tertiary hospitals in China showed that 76.2% of hospitals were using the Chinese social media sites Sina Weibo or WeChat.Health-related social media use can increase both consumer to consumer and provider to consumer support,improve self-management of conditions,increase consumer access to information,create more equal relationships between health professionals and patients,and improve health service data collection.Stakeholder engagement in health service design or quality improvement (QI) is an established practice in many health systems. 'Stakeholders' are the individuals and groups who can influence an organisation's success or affect its ability to meet its purpose.Stakeholders in health services come from across all levels of healthfrom the individual to the systems level -and can include providers, consumers, policy makers, health administrators and the general public.Stakeholder engagement activities in health are traditionally conducted face-to-face (e.g., involvement in meetings, deliberative processes, interviews, focus groups) or via surveys.An emerging area of practice and research is the use of social media to facilitate stakeholder engagement in service design and QI. Authors argue for the potential of social media for engaging with a broader range of health stakeholders,leveraging existing online consumer communities for involvement in co-design of services,and using social listening (i.e., the monitoring of online conversations to gather dataas a way to gather patient sentiment and experience data.However, this literature is largely theoretical or opinion-based,and it is unclear whether these potential uses of social media are being realised in real-world stakeholder engagement activities.
Prior to undertaking the scoping review a preliminary search for previous scoping reviews, systematic reviews and qualitative evidence synthesis reviews which aligned with the same topic was conducted. The databases searched were: Campbell Library, JBI Evidence Synthesis, Cochrane Database of Systematic Reviews, PDQ Evidence, and Health Systems Evidence. No previously published reviews were discovered which aligned with the scope of this review.
## Objective
The objective of this scoping review is to map the research on the use of social media by health services, providers and consumers to contribute to service design or QI activities. Research gaps will also be identified. The overarching research question is 'how is social media being used as a tool for health service design and QI activities?' Four research sub-questions (RSQ) are being explored to answer this question: : What are the common features of social media platforms used in health service design and QI activities?
RSQ2: What communication models are used in health service design or QI activities, or to influence changes in health service design? RSQ3: Which populations of people are using social media in health service design or QI activities, or to influence changes in health service design? RSQ4: What types of health service design or QI activities are being undertaken/influenced through social media communications?
# Methods
This scoping review was conducted using Joanna Briggs Institute (JBI) methodologyand has been reported in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR).The objectives, inclusion criteria and methods of analysis for this review were specified in advance in a protocol,and this paper addresses questions concerning features, models, populations and uses of social media. A separate paper will address the additional questions of risks, limitations, barriers and enablers outlined in the protocol.Minor variations in method from the published protocol have been described in Appendix 1 and the the PRISMA-ScR checklist is included in Stakeholder involvement in the co-production of this scoping review This review has been guided by the involvement of an advisory committee of stakeholders, which includes three healthcare consumers and three healthcare providers in clinical and non-clinical roles. Arksey and O'Malley advocate for the use of a consultation exercise with practitioners and consumers within their scoping review framework.In the current review, consultation was expanded to a method of coproduction guided by the INVOLVE Principles. Advisory committee members contributed to the review in the following ways:
- Determining the overarching research question for this review. - Submitting potentially relevant articles for screening. - Reviewing and commenting on the draft data analysis. - Providing feedback on the manuscript.
- Answering specific questions about which findings of the analysis were particularly relevant to their experience of the health system, and these responses were used to shape the content of the discussion section. - Three members of the advisory committee who commented on the draft data analysis, provided feedback on the manuscript, and answered the questions which informed the discussion section are included as co-authors on the review (NJ, CL, SR).
## Eligibility criteria
The eligibility criteria outlined below and the subsequent search strategy and methods for the extraction of the results were guided by the Population-Concept-Context structure recommended in the JBI scoping review methodology.Population. For inclusion in this review, participants in the included studies were:
- Users or potential users of a health service (i.e., patients, consumer representatives, consumers with an acute or chronic condition, carers, family members, consumer organization member, community members, public); and/or - Health service providers (health professionals, health service manager/administrator, health policy makers).
There were no restrictions on the age or gender of study participants considered for inclusion.
Studies only involving participants from non-health service settings (e.g., educational institutions, social care services) were excluded from the review.
Concept. The core concept to be examined in this review is how social media is used as a tool for health service design and QI activities. This includes uses of social media by health services to facilitate user or provider participation in organisational QI or service design activities, and the use of social media by individuals or groups to influence (or attempt to influence) health service or system changes. To capture the full range of potential uses of social media in QI and service design, two broad study types were included. These were:
- Studies where social media was used as a tool within workforce-based health service design or QI activities, and - Studies where social media was used as a tool for communications which influenced or advocated for changes to health service design or delivery.
A date range limitation of '2004 -current' was placed on the search. This limitation was chosen because 2004 is defined as the beginning of Web 2.0, when the internet transitioned from being used primarily as a broadcasting medium to supporting and encouraging user-generated content.This shift paved the way for the rise and dominance of social media platforms and tools.The following types of studies were excluded from the review:
- Social media for disease surveillance only without intention to change health service design/delivery. - Social media for health information dissemination only with no QI-relevant objectives. - Social media for patient treatment/care/peer support without intention to influence health service change or quality improvement. - Social media use in health provider education.
- Consumer/service provider engagement in research only without changes to health service design or delivery. - Social media use for research recruitment.
Context. To be eligible for inclusion, studies needed to be conducted in healthcare or health service settings (hospitals, health services, aged care, community health, primary health, health-specific non-government organisations) or health policy settings (government health departments) and published in English. Studies from any geographic location, regardless of income status, were included.
Studies conducted in non-healthcare settings (e.g., educational institutions, social care services) were excluded from the review.
Study design. Original primary research or evaluation articles (any methods) and secondary research review articles, published in either peer reviewed academic publications or grey literature, were eligible for inclusion in the review. In determining eligibility for inclusion in this scoping review, 'research' was defined as "activities designed to develop or contribute to generalizable knowledge, i.e., theories, principles, relationships, or the information on which these are based, that can be confirmed or refuted by recognized methods of observation, experiment, and inference".To be included in this review, a study had to state the method by which the research was conducted and include some analysis of the data generated through the research method.
Conference abstracts were considered eligible for inclusion if they met the other eligibility criteria. Where it was unclear whether a conference abstract should be included, the author was contacted to determine eligibility.
Sources without original research (e.g., opinion pieces, editorials, commentaries) were excluded from the review. Grey literature was also searched to identify nonindexed researched literature relevant to this study. Grey literature searches were conducted using key search terms (e.g., 'social media', 'consumer engagement') and the search function on the websites of key organisations involved in consumer engagement or health service QI in English-speaking countries. All sections of websites that were likely to house relevant publications (e.g., 'Resources', 'Publications', 'Research' or 'Reports' pages) were also located and searched. The full list of organisational websites and their web addresses searched during the grey literature search is provided in Appendix 4.
In addition to the searches above, the reference lists of included review studies were screened for potentially eligible studies. The members of the research advisory committee were also asked to submit any studies that they thought could be relevant to the authors for assessment for inclusion.
## Screening of studies
Studies were retrieved using the search strategy outlined above. The titles and abstracts of retrieved studies were screened by one review author (LW). The full text of these potentially eligible studies was retrieved and assessed for inclusion by LW, and two other authors (NH, SH) provided additional input where an inclusion decision was unclear.
At the full text screening stage, a forward search was conducted on all conference abstracts to identify whether a journal publication had resulted from the conference presentation. Where a journal publication was associated with the conference presentation, the journal publication was included in the review in place of the conference abstract. Additionally, the authors of all potentially included conference abstracts were contacted to provide additional information to either assist with inclusion/exclusion decisions, or to add further information about the study which could be included in the extraction of results.
## Extraction of the results
Data extraction was performed by one author (LW). A pre-determined data extraction form was used to extract data from the included studies. Extracted information included: article title; year of publication; authors; type of publication (e.g., journal article, conference abstract, book chapter, grey literature); study aims/objectives; methodology; methods; population (using PROGRESS-plus categorisations 37,38 ) setting; description of social media intervention; description of intended or actual health service change; categorisation of communication model (one-way,two-way,or social listening 40,41 ) a priori themes (based on research sub-questions as outlined in the review protocol 33 ) additional emergent themes.
# Synthesis of results
The data were analysed and synthesised in the following ways:
a. An overview of the amount, type and distribution of included studies was constructed in relation features of platforms (RSQ1), communication models (RSQ2), populations of users (RSQ3), and intended health service change (RSQ4) informed by the descriptive numerical summary analysis method recommend by Levac.The numerical overview was constructed by LW, with input from SH and NH. b. A deductive, qualitative content analysis methodwas used to create a new typologydescribing social media communication methods through integrating, interpreting and synthesising the content of extracted data and the findings of the numerical overview.
# Results
# Search results
A total of 2088 titles and abstracts, and 214 full text articles were screened for eligibility. Forty articles from 39 study settings were included in the scoping review. A presentation of the search results using the 'preferred reporting items for systematic reviews and metaanalyses (PRISMA) flow diagram'can be seen in.
## Characteristics of included studies
All studies were published since 2010 and the majority (82.5%) of studies were conducted in high income countries (as classified by The World Bank 45 ) There are 28 primary academic research articles, nine conference abstracts, two systematic reviews (one published in an academic journal, one published as a report) and one dissertation included in this review. The key characteristics of all included studies can be seen in.
Descriptive numerical summary analysis of included studiesand Tursunbayeva (2017)are both systematic reviews with some similarities in scope with this review. Stokes (2015)had a focus on consumer engagement in the design, delivery or evaluation of interventions that targeted individual health outcomes (e.g. healthy eating, physical activity), rather than consumer engagement in a broader range of service design and QI activities which might have other impacts or outcomes on individuals or health organisations (such as improved patient experience). Additionally, the review only included randomised controlled trials. Tursunbayeva (2017)focused on social media for consumer engagement in public health eGovernment, rather than looking at social media for consumer engagement across a range of health settings. Overall, both reviews had narrower inclusion criteria than this review.
Most authors did not identify or describe the specific features of platforms being used for health service design or QI activities. Of those studies which did, forums and blogs were the most commonly used features, with ten studies using forumsand seven using blogs.Privacy settings were a common feature cited in the literature. Twenty-one studiesFor a full numerical summary of the social media platforms in the included studies, see 'RSQ1: Social media platforms' in.
RSQ2: What communication models are used in health service design or QI activities, or to influence changes in health service design? To categorise the communication models described within studies both the direction of the communication and the management of the social media spaces being used were considered.
In the included studies, two-way communication models were used in 22 studies,For a full numerical summary of the communication models used in the included studies, see 'RSQ2: Communication models' on.
RSQ3: Which populations of people are using social media in health service design or QI activities, or to influence changes in health service design? A finding of this review is that, rather than just being a setting where QI and communications activities took place, healthcare organisations were themselves active users of social media, presenting as a population group separate to their providers or consumers. Organisations had social media accounts in their organisational name, they had their own 'voice' and online presence, and the person or people responsible for running the account were generally not identifiable by other users on the platform. Other users interacting with an organisation on social media platforms communicated with the organisation as if it was an individual user, and organisations communicated directly with each other. Therefore, in this review, organisations were identified as a user population.
Social media users were primarily health organisations (n ¼ 25;and hospitals accounted for seven of these organisations. Health service providers (either clinical or non-clinical) were users in 19 studiesand consumers were users in 20 studies.Children or young people (aged <30) (n ¼ 9;and people with a disability or chronic illness (n ¼ 6;were the two most common consumer user populations in the included studies. Only eight studiesindicated one user population, and some of these studies were unclear in their reporting, so it is possible there were other user populations that were not identified.
For a full numerical summary of the user populations in the included studies, see 'RSQ3: Populations of users' on.
RSQ4: What types of health service design or QI activities are being undertaken/influenced through social media communications? Social media was used as a tool in government or international health policy/strategy/guidelines development in 13 included studies.Examples of relevant activities include policy makers gathering public response to drafts of health policy through Twitter,consumers or organisations using social media to organise and advocate for policy changes,and organisations using social media platforms as a virtual space in which to conduct discussions and consultations on guidelines or strategy with stakeholders.Social media was used by health services to gather data from key stakeholders to inform their QI or design activities in 12 included studies.Some of the ways hospitals and health services use social media for planning are by gathering QI-relevant data from posts made by individuals about their care experiences,by using open social media platforms to invite feedback on services,and by using private social media spaces to gather together stakeholders to discuss and provide feedback on service QI activities.Social media was used as a broadcast communication tool to disseminate resources or information which would prompt offline health service QI related actions in seven studies.This was most commonly for providing professional development information to service providers aimed at increasing the number of providers following clinical guidelines.Social media was used as a tool to include stakeholders in the creation of specific interventions, products or resources in seven studies,and as a platform to facilitate improved communication between service providers in six studies.It was used to improve the identification, reporting and response to specific health issues in four included studies,evaluation of activities in two studiesand to monitor health service adherence to national standards in one study.Most of the included studies reported on the experience or process of the QI, service design or change activity. Very few of the included studies demonstrated whether the activities improved the health service or had impact on patient experience or outcomes. Three studies used social media to educate staff about existing guidelines or clinical pathways in an effort to standardise treatment approaches.Two studiesshowed improvements in staff knowledge and awareness of the available resources, while one showed no significant change in knowledge.None of these studies measured patient outcomes or any changes in how treatment was delivered, so it is unknown whether the observed changes in staff knowledge and awareness led to changes at the service-delivery or patient level.
Harris et al.aimed to increase reporting and improve the response to foodborne illness in St Louis, USA, through Twitter interactions between the public and the local health department. This change was demonstrated, with a higher frequency of reporting when the department used their Twitter reporting mechanism alongside their usual reporting mechanisms.The outcomes of the reports (primarily the frequency and severity of food safety violations following inspection) was comparable between Twitter and non-Twitter reporting mechanisms. 52 Gathering data from key stakeholders to inform health service QI or design activities ¼ 12 (30%) Disseminate resources/information to prompt QI actions ¼ 7 (17.5%) Develop specific products, interventions, resources ¼ 7 (17.5%) Develop community of practice/ease communication ¼ 6 (15%) Improve how specific health issues are identified, reported and responded to ¼ 4 (10%) Evaluation of activities ¼ 2 (5%) Monitor organisational adherence to national quality standards ¼ 1 (2.5%) a The total number of studies against each focus area sub-category may not equal the number of included studies (n ¼ 40) because some studies used multiple platforms, had a variety of target user populations, worked across more than one setting, or sub-categories were not described in the paper. b The 'other' category includes two systematic reviews that only included studies from high income countries; one study which included participants from Africa, Latin America, Brazil, Asia Pacific, Eastern Europe, Central Asia, Caribbean, Central Europe and China; one study with participants from Africa, North and South America and Europe; one study from Spain; and one from Romania and Moldova. c All platforms in the 'other' category were only used in one study. These included the platforms WhatsApp, Bebo, Tumblr, Choicebook, RenRen, High5, Vkontake, ZorgkaartNederland and an email moderated discussion forum.
Finally, Hoxwoth et al.examined a virtual community of practice of healthcare providers and organisations in Colorado working together to share data and find collaborative approaches to reducing the rates of healthcare onset and healthcare acquiredcommunity onset clostridium difficile infections (CDI). The goal was to reduce these CDI rates by at least 15% from baseline.This was achieved, with the rates of the infections reducing by 17% over the study period.For a more detailed description of the health service design or QI activity within each included study, see 'Description of QI activity' on. For a full numerical summary of the health service design or QI activities in the included studies, see 'RSQ4: Intended health service change/QI activity' on.
## Typology of social media use in qi and health service design
The importance of generating new theories and knowledge from the analysis of data from included studiesrather than just mapping the literature -is a feature of the qualitative content analysis method used in this scoping review.By analysing the models of social media communication identified in the included studies alongside the platform features and user populations, several recurring methods or types of social media use were identified.presents the results of this analysis, summarising how different user populations use social media platforms (and their associated features) to communicate for a variety of aims and within a range of different activity types. In the 'method of social media use' column, each method in the typology is named based on the direction of communication, whether communication occurs in public or privately, and who hosts or manages the online space.
# Discussion
In this review we examined the various ways in which social media is used by health services, providers and consumers to contribute to service design or QI activities.
## Main findings
Platforms and user populations. Commercially available platforms were used more often than purpose-built platforms, and these were most often used in public, openly accessible ways. Social media channels were most commonly managed by health services or occurred in largely unmanaged and unmoderated public spaces (such as Twitter), rather than being managed by provider or consumer groups. The most common user populations were organisations, consumers and service providers.
The findings in this review about who is using social media and how it is being used supports ideas proposed by previous authors that social media could be used to reach different audiences and engage diverse consumer communities in health service design and quality improvement.In this review social media engagement methods were used to engage children and young people,First Nations communitiesand culturally and linguistically diverse communities in English-speaking countries.However, within the included studies, users often identified as belonging to more than one population, or studies involved multiple user populations. In total, only 14 studies in the review included user populations who have been identified as at higher risk of experiencing health inequalities 37 or representative of groups that health programs often fail to reach.Half of the included studies (n ¼ 20) did not include health service consumers. These findings demonstrate that while social media use can be a successful strategy to engage communities in health service design and QI activities, the theoretical potential for using social media to reach groups of people which health services often fail to reach -or even health service consumers in general -may not yet be fully realised.
Only six studies included users who identified as having chronic illness or disability,and in only four of these studies was the social media consumer community pre-existing.Tapping into chronic illness and disability communities that are already well established online was viewed as an important potential benefit of social media use in QI and service design in some of the background literature which prompted this review.Our results demonstrate that while some organisations and providers are working with existing online groups, this number is still small, and engaging with pre-existing groups of people who discuss their chronic illness or disability online may still be a largely untapped source of knowledge and experience that can be integrated into -or lead -health service design and QI activities in the future.
## Models of communication.
Past research of health service use has shown that social media is primarily used as a one-way broadcast medium.In this review, most studies used two-way models of social media communication. Two-way communication is seen as one of the advantages of social media over other forms of online communication both generally 2 and specifically within health when engaging stakeholders in service design and QI. 22 Public communication aimed at a known audience
Purposeful attempt at sharing information with a group, organisation or individual in a public forum to address a specific interest, issue or concern.
One-way, directed, attempt to get a reply or prompt an action. forums; responses to consultative activities, initiating advocacy activities.
Studies:An online breast cancer community of consumers and providers hosted an ongoing public conversation on Twitter using the #BCSM hashtag to inform QI and research.(continued) Reporting who was responsible for management of the social media spaces in the included studies emerged through the analysis as an important feature of the social media communication models being used. Responsibility for management reflects who has 'control' of the space. This is important in a context of stakeholder engagement in health services because being aware of who has 'control' enables us to consider issues of power and participation in social media spaces, can inform the development of new ways to categorise of social media use in QI and health service design, and may also help to identify models that support more meaningful consumer engagement in QI activities.In half of the included studies the social media spaces were managed by a health organisation. This creates an 'invited space' where an organisation (often with greater power than the stakeholders) owns and structures how people participate.Participation within an invited space can result in tokenistic participation,due to constraints on engagement that are either intentionally created by the managing organisation, or through unintentionally creating power imbalances between the manager of the space and the people participating.In the typology we have developedSocial media could also provide opportunities for expanding beyond existing models of stakeholder participation in closely curated and moderated 'invited spaces'. In our typology, 'covert social listening', 'non-targeted broadcast', 'public communication aimed at a known audience' and 'public conversation' methods all happen in public, largely unmoderated or unmanaged social media spaces (e.g., Twitter). Additionally, social media allows stakeholder groups with common interest or experience to establish their own communities, and in this review there are two studies where spaces were managed by consumers,and one study where the space was managed by providers.Groups that sit outside of invited spaces are typically less marked by differences in power and control between members, give members the opportunity to develop their own approaches to the issues they face, and can include people that are either not invited into, or are unwilling to participate in, spaces created by organisations.The spaces that sit outside organisational control have the potential to uncover insights which could inform QI and service design which would not necessarily be shared within organisationcontrolled spaces. Additionally, within these types of groups there is more likelihood of achieving a 'citizen power' model of participation.Types of QI and design activities. The most common QI or design activities undertaken through social media were the development of health policy, strategy or guidelines (n ¼ 13;and gathering data from key stakeholders to inform QI or design activities at the hospital or health service level (n ¼ 12;.
Social media was generally used either as a way for organisations to gather data to inform QI and design activities, or as a virtual meeting space for people to collaborate on projects or advocate for change. When used as a data gathering tool, communication methods such as covert social listening, and public and private consultation were common (see. When used as a meeting space, public conversation, organisation hosted collaborative spaces and consumer-or provider-initiated spaces communication methods were favoured. Cycles of data gathering and meetings of stakeholders to discuss and decide on actions based on the available data are a common feature of QI modelsand based on our review social media can be used as a place for these activities to be undertaken.
However, the use of social media as a source of data is an area of ongoing debate. Some researchers believe that social media posts made on public sites can be used without consent, and consent is only necessary where posts are made in private, password protected or invitation-only, groups.Other researchers believe that consent should be gained for the use of any social media posts,or that historical social media content is not an appropriate source of data at all.A study of user views about the ethics of social media researchdemonstrated that what was seen as 'ethical' or 'unethical' by study participants was determined by a large range of different factors -including, but not limited to, the mode of the posts (e.g., written, photos), the subject matter (e.g., sensitive, 'mundane'), the type of platform being used (e.g., social, professional), the original audience for the post (e.g., private, public), and the nature of the research (e.g., research purpose, research affiliation) and the participant's individual views about social media. In general, participants in this study felt that the passive use of data without the user's consent or knowledge (e.g., social listening, data mining) raised ethical concerns even when posts were made on public sites, whereas actively participating in research activities through forums or groups raised fewer ethical concerns.These ethical questions may be even more complex in the area of health service QI, where the requirements for ethical oversight are not always clear.This complexity highlights the need for people conducting service design and QI activities to involve target audiences in the design of social media-based engagement activities, and include discussions of ethical concerns as part of this design process.
## Gaps in the literature and opportunities for future research
This review identified several areas where there are potential gaps in the existing literature.
Service and practitioner rating sites (e.g, CareOpinion, 98 PatientOpinion 99 ) are considered social media and a number of articles on service rating sites were found through the literature search but did not meet the inclusion criteria because they examined the validity/reliability of patient ratings compared to other measures rather than the use of rating sites to inform QI or design activities. Only one article about rating sites met the inclusion criteria of this review.The experience of using rating sites and the use of rating site data to inform QI and service design activities could be an emerging area of research, particularly if rating sites expand their coverage across more institutions and more locations.
There were few included studies from low-and middle-income countries (LMICs). However, we know from the digital disruption literature that it is likely that people in these countries are adopting digital technologies to overcome issues relating to access to health care, data collection and the economics of health.It is possible that consumer and community input into health service design and QI via social media is already happening in LMICs and is published in languages other than English or is yet to be published. As a result, there may be a need for specific research into how health services and stakeholders in LMICs use social media to engage in health service QI, design and change activities, particularly in the context of rapid uptake of digital technologies.
Finally, while most of the studies examined the process of QI or service design, and the experience of participants using social media (to be presented in a future publication), very few included studies measured either patient or health service outcomes arising from the QI, service design or change activities. There is an opportunity for future research into the impacts of QI and design activities on patient and service outcomes, and to compare activities conducted either solely or partially on social media platforms with those undertaken through more 'traditional' methods of engagement.
# Limitations
As this is a scoping review, no assessment of quality was conducted on included studies. This means that no conclusions can be drawn about how robust or generalisable the findings of individual studies are, and no weighted evidence around the various approaches to social media use can be produced.Additionally, non-English publications were excluded from the review. As a result, some studies -particularly those which addressed the gap of evidence from LMICsmay have been excluded during the search stage. Finally, it must be noted that only one author (LW) was responsible for the bulk of the screening and selection of included studies. This approach differs from the JBI methods which state that "source selection . . . is performed by two or more reviewers, independently".As this scoping review forms the literature review component of a PhD project, a decision was made that the PhD candidate (LW) would do the bulk of the screening and selection of studies, with NH and SH providing input only when inclusion was unclear. This approach may have introduced bias into the selection of included studies which may impact the quality of the scoping review findings.
# Conclusion
This review addressed the overarching question 'how is social media being used as a tool for health service design and QI activities' and demonstrated that social media is used in a range of ways in health service design, QI and change activities. Engagement through open-access platforms was more common than restricting access through passwords or registration. Social media has been most commonly used as tool for engagement in national or international health policy or strategy design, and in design/QI projects in individual health services. Communication was most often two-way, but social media spaces are often managed by organisations, which may have implications for the quality of stakeholder engagement.
This review demonstrates that social media platforms are suitable for engaging health stakeholders in the cycles of data gathering and planning/implementation meetings that characterise most QI models. More importantly, it shows the diversity of participation and engagement approaches that are possible through social media, including using the different platforms and communication models in strategic ways to engage a range of social media users in QI and service design activities. The diversity of potential communication approaches available through using social media creates new opportunities for innovation in designing and trialling new ways of engaging stakeholders in QI and health service design. DIGITAL HEALTH Contributorship: LW conceived the study, developed the protocol, conducted the search, data extraction, screening, and data analysis, wrote the first draft of the manuscript, and integrated feedback from co-authors into subsequent drafts. NH and SH provided input into the study design, coauthored the protocol, assisted with screening decisions and data analysis, and were major contributors to the manuscript. NJ, CL and SR, provided input into the study design, reviewed and edited multiple versions of the manuscript and approved the final version of the manuscript.
Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical approval: As this is a scoping review of existing literature no ethics committee approval was required. Supplemental material: Supplemental material for this article is available online. |
Newborn pulse oximetry screening for critical congenital heart defects
We looked at existing recommendations and supporting evidence addressing the effectiveness of pulse oximetry effective for detecting critical congenital heart defects (CCHDs) in newborns. We also looked at the impact of timing of oximetry and the site of testing in the accuracy of screening, and at the potential harms and limitations of pulse oximetry screening, We conducted a literature search up to the 13 th of August 2019 by using key terms and manual search in selected sources. We summarized the recommendations and the strength of the recommendation when and as reported by the authors. We summarized the main findings of systematic reviews with the certainty of the evidence as reported. Current evidence supports consistent accuracy for detection of CCHDs in newborns by pulse oximetry screening in addition to antenatal ultrasonography and clinical examination. Overall, early diagnosis of CCHD with pulse oximetry is judged to be beneficial and cost-effective, and potential harms associated with false-positive tests are not serious, while missing CCHDs and other serious diseases detected by hypoxaemia in absence of pulse oximetry screening can lead to serious consequences. The site of testing (post-ductal versus pre-and post-ductal) had no significant effect on sensitivity nor specificity for detection of CCHDs.
# Introduction
The World Health Organization (WHO) European Region is developing a new pocket book for primary health care for children and adolescents in Europe. This article is part of a series of reviews, which aim to summarize the existing recommendations and the most recent evidence on preventive interventions applied to children under five years of age to inform the WHO editorial group to make recommendations for health promotion in primary health care. In this article, we looked at existing recommendations and supporting evidence addressing the effectiveness of pulse oximetry effective for detecting critical congenital heart defects (CCHDs) in newborns. We also looked at the impact of timing of oximetry and the site of testing (post-ductal versus pre-and post-ductal) in the accuracy of screening, and at the potential harms and limitations of pulse oximetry screening.
Why is screening for critical congenital heart defects important?
CCHD is defined as any cardiac lesion from which infants die or require surgery or cardiac catheterization within the first 28 days of life to prevent death or severe end-organ damage. Early detection of CCHD before acute cardiovascular collapse leads to improved cardiopulmonary and neurological outcomes. However, most newborns are asymptomatic at birth. Newborn screening for CCHD can help identifying some cases to allow prompt diagnosis and treatment, and may prevent disability or fatal outcome.
## Context
Congenital heart defects (CHDs) constitute the most common group of birth defects, with a prevalence of around 6 to 11 per 1000 live births for moderate and severe cases. They account for up to 10% of all infant deaths, and 46% of deaths related to congenital malformations. About 25% of CHDs are lifethreatening CCHDs. Antenatal ultrasound screening and newborn clinical examination are already established methods to detect malformations such as CCHDs. However, it was estimated that antenatal ultrasound can detect around two thirds of CCHDs (sensitivity of 68.1%; 95% confidence interval [CI] 59.6 to 75.5%), and that the newborn examination also has a low detection rate. Indeed, detection of hypoxaemia by visual assessment of the newborn colour has limitations, and cardiac murmurs are not always present in cases of CCHDs and accidental murmurs can be heard in up to 60% of healthy newborns. The combination of antenatal ultrasonography and clinical examination of the newborn lead to sending home up to 30% of cases of CCHDs before diagnosis, with mortality rates up to 50%. Several studies have reported that adding routine pulse oximetry (PO) to the antenatal ultrasound screening and clinical examination of the newborn can potentially improve the detection of CCHDs. PO is a simple, non-invasive and painless tool that measures oxygen saturation, and therefore could detect CCHDs with ductal-dependent systemic or pulmonary blood flow that usually present with hypoxemia. Seven severe lesions have been identified as primary targets for screening by PO; those are hypoplastic left heart syndrome, pulmonary atresia, tetralogy of Fallot, total anomalous pulmonary venous return, transposition of the great arteries, tricuspid atresia, and truncus arteriosus.
## Key questions
1. Is PO effective for detection of CCHDs in newborns? 2. Does timing of oximetry have an impact in the accuracy of screening? 3. Is pre-and post-ductal more effective than postductal measurement for detection of CCHDs? 4. What are the harms and limitations of PO screening?
Cost-effectiveness of the implementation of PO screening and acceptability to parents and health workers are beyond the scope of this summary but were assessed by others.
## Search methods and selected manuscripts
We described the search methods, data collection and data synthesis in the second paper of this supplement (Jullien S, Huss G, Weige R. Supporting recommendations for childhood preventive interventions for primary health care: elaboration of evidence synthesis and lessons learnt. BMC Pediatr. 2021 https://doi.org/10.1186/s12887-021-02638-8).
The search was conducted on the 13 th of August 2019, by manual search and by using the search term "congenital heart" OR "congenital cardi*". We did not find any recommendations from the WHO. No document was identified from the US Preventive Services Task Force (USPSTF) (published recommendations or recommendations in progress) or the PrevInfad workgroup (Spanish Association of Primary Care Pediatrics), but we did include the consensus document with recommendations from the Spanish National Neonatal Society, Spanish Association of Paediatrics (AEP, from acronym in Spanish). We did not find recommendations from the National Institute for Health and Care Excellence (NICE) other than the physical examination of the newborn that should include 'heart; check position, heart rate, rhythm and sounds, murmurs and femoral pulse volume'. From the Centers of Disease Control and Prevention (CDC) website, we included a fact sheet and recommendations addressing this topic. We also included the 2011 recommendations from the American Academy of Pediatrics (AAP) and their comprehensive website within the "Program to Enhance the Health & Development of Infants and Childre"'. The Royal College of Paediatrics and Child Health (RCPCH) included this topic within their chapter on "Physical examination." Finally, we also included the recommendations stated by the UK National Screening Committee (NSC) in their recommendations report from 2014-15, and their current update on this matter.
The search in the Cochrane library by using the search strategy 'congenital heart OR congenital cardi*' in titles, abstracts or keywords returned 41 reviews and three protocols. By screening the titles and abstracts, we included one systematic review and two protocols. However, one protocol entitled 'Routine screening by echocardiography to reduce morbidity and mortality from congenital heart disease in neonates with Down syndrome' dated from 2005, and the other one, entitled 'Clinical assessment for diagnosing congenital heart disease in newborn infants with Down syndrome' was out of date and consequently withdrawn from the authors and editors of Cochrane Neonatal. Therefore, we did not contact authors from any of these two protocols and only included one All the included manuscripts for revision in this article are displayed in.
## Existing recommendations
We summarized the existing recommendations and the strength of recommendations as per their authors in.
## Existing evidence
## Short history of po used for newborns screening
In 2009, the AAP and the American Heart Association (AHA) published conjointly a statement of evidence on the routine use of PO in newborns for detection of CCHDs. The evidence supporting this document was mainly based on two studies (de Wahl 2009 and Riede 2010), both included in the Cochrane systematic review by Plana et al. Although the document showed benefits of PO screening, it was concluded that "future studies in larger populations and across a broad range of newborn delivery systems are needed to determine whether this practice should become standard of care in the routine assessment of the neonate". Based on this document, the Secretary's Advisory Committee on Heritable Disorders in Newborns and Children (SACHDNC) recommended that CCHDs to be added to the recommended uniform screening panel (RUSP). After this, the SACHDNC in collaboration with the AAP, the AHA, and the American College of Cardiology Foundation convened a work group to elucidate how to implement this screening safely and with efficiency. The working group evaluated the new findings of large population-based screening activities in Sweden and England, and published their updated recommendations in 2011, which are the basis for the AAP and CDC current recommendations. Previously called "cyanotic congenital heart diseases", this group recommended renaming the target conditions of the PO screening as "critical congenital heart disease" (CCHD), as "many newborns with the targeted congenital heart defects do not develop clinically appreciable cyanosis until after nursery discharge, and some lesions (e.g., hypoplastic left heart syndrome) may present with significant cardiovascular compromise without apparent cyanosis." As a conclusion, "the work-group members found sufficient evidence to begin screening for low blood oxygen saturation through the use of pulse-oximetry monitoring to detect CCHD in well-infant and intermediate care nurseries", however they identified research gaps, mainly regarding some specifics populations and delivery strategies. Therefore, "the Secretary of the US Health and Human Services has directed an interagency work group to develop a plan to address these critical gaps before recommending that CCHD be a part of the recommended uniform screening panel". The CCHD newborn screening was added to the RUSP in 2011, and two years later, another document was published, addressing some of the implementation challenges. While routine PO screening for detection of CCHDs in all newborns has been introduced in the US, other countries have been conducting similar assessments to evaluate the potential harms and benefits of introducing the screening at a national level. In the UK, a pilot study was conducted in 2015 in order to elucidate effectiveness and feasibility of the implementation of the CCHD newborn screening (findings below). In addition, "Public Health England undertook a review of the extent to which PO met the UK NSC criteria for screening, particularly focussing on the harms and benefits of potential for over-diagnosis, over-treatment, falsepositives, false reassurance, uncertain findings, and complications". Based on the pilot study and the review conclusions, the UK NSC recently recommended against the introduction of routine PO screening, which is disputed by others. In May 2019, the UK NSC "announced a public consultation on its decision not to introduce routine PO for CCHD in all newborn babies". By the time this 2018 "There is sufficient evidence to recommend neonatal screening by pulse oximetry in the first hours post birth, in addition to prenatal ultrasound and the physical examination." (Level of evidence A) "The timing of screening affects its sensitivity, with a higher sensitivity the earlier it is performed." (Level of evidence A) "Early screening, within 24h of birth, reduces the risk of onset with severe or very severe symptoms in CCHD at the expense of a greater number of false positives, although most of the latter are indicative of other disorders that may also require observation, diagnosis and treatment, so early screening is preferable to late screening (>24 h). Very early screening (<12 h) may result in an excessive number of false positives, an issue that needs to be weighed at the local level. In case of very early discharge, screening should be performed before discharge, regardless of timing. It is recommended that the screen be performed between 6 and 24 h post birth." (Level of evidence B)
[formula] CDC [4] 2018 [/formula]
The CDC recommends screening of all newborns in well-baby nursery at ≥24 hours of age or shortly before discharge if <24 hours of age, with a subsequent algorithm according to findings (reported by Kemper et al.. In addition, it is recommended that 'Pulse oximetry screening should not replace taking a complete family health history and pregnancy history or completing a physical examination, which sometimes can detect a critical CHD before the development of low levels of oxygen (hypoxemia) in the blood.' NICE 2015 "heart; check position, heart rate, rhythm and sounds, murmurs and femoral pulse volume"
[formula] AAP [3, 10] 2019 [/formula]
- "All newborns at risk for undetected CCHD should be screened. In other words, the only babies who do not need to be screened are those who are already known to have CCHD, such as those identified by prenatal ultrasound or who have already had an echocardiogram." - "Screening should begin after 24 hours of age or shortly before discharge if the baby is less than 24 hours of age. Waiting until 24 hours of life will decrease the false-positive results." - "The screening should occur in the right hand and either foot. If using only one pulse oximeter, test one right after the other." - "CCHD screening should be conducted by individuals who have pulse-oximetry testing within their scope of practice, who are trained in the use of pulse oximetry and the CCHD algorithm, and who regularly use pulse oximetry for other purposes." - "In the event of a positive screening result, CCHD needs to be excluded with a diagnostic echocardiogram. Infectious and pulmonary causes of hypoxemia should also be excluded.". Of them, 75 (78%) neonatal units felt that screening did not increase unnecessary investigations, and 10 (10%) felt "that any small increase was justified and offset by the benefits of identifying considerable cardiac and non-cardiac pathology". Findings of this survey call for unified national recommendation.
## Rcpch
## Accuracy of po for detection of cchds
Threshold for considering PO result pass or failed
As mentioned by Kemper et al, "selecting the threshold for a positive pulse-oximetry monitoring result is challenging, because it must trade-off the harm of missing CCHD against the harm of false-positive screen results". Several institutions currently agree on the following criteria to consider a screen failed: any oxygen saturation measure less than 90% in the initial screen or in repeated screens; oxygen saturation less than 95% in the right hand and foot on three measures, each separated by one hour; or an absolute difference in oxygen saturation of three percentage points between the right hand and foot on three measures, each separated by one hour.
## Studies reporting accuracy of po for detection of cchds
Accuracy of PO for detection of CCHDs was assessed by one Cochrane review, and by one project conducted in the UK. The Cochrane review conducted by Plana et al assessed the diagnostic accuracy of screening with PO compared to echocardiography or clinical follow-up in the first 28 days of life for detection of CCHD in asymptomatic newborn infants. The literature search was conducted up to March 2017. Twentyone studies were included (16 prospective cohorts and five retrospective cohorts), published between 2002 and 2017, mainly from Europe (UK, 5 studies; Italy, 2 studies; Germany, Norway, Poland, Sweden, Switzerland, and Turkey, 1 study each), but also from other settings (US, 3 studies; Australia, China, Mexico, Saudi Arabia, and South Africa 1 study each). Across the included studies, data was provided for 457,202 newborns and several pulse oximeters models were used. Different thresholds were used to establish a pass or failed screening: postductal saturation <95% (8 studies), post-ductal saturation ≤95% (3 studies), pre-and post-ductal saturations <95% (6 studies), pre-and post-ductal saturations ≤94% (1 study), post-ductal saturation <96% (1 study). The applicability concerns were judged as low for all included studies per the review authors.
In 2013, the UK NSC decided to assess the feasibility and impact of PO screening in a wide context, and started a multicentric pilot study across England. This took place from July to December 2015, and it consisted in taking pre-and post-ductal measurements between 4 and 8 hours of life.
## Main findings
Plana et al considered <95% or ≤95% as a threshold to include studies for primary analysis. From 19 studies (n = 436,758), they found that PO for detection of CCHDs had a sensitivity of 76.3% (95% confidence interval [CI] 69.5 to 82.0%) and a specificity of 99.9% (95% CI 99.7 to 99.9%) with a false-positive rate of 0.14% (95% CI 0.07 to 0.22). Summary positive and negative likelihood ratios were estimated at 535.6 (95% CI 280.3 to 1023.4) and 0.24 (95% CI 0.18 to 0.31), respectively. As detailed by the review authors and considering a median prevalence of 0.6 per 1000 newborns, 'these results showed that out of 10,000 apparently healthy late preterm or full-term newborn infants, six will have CCHD'; 'screening by PO will detect five of these infants as having CCHD and will miss one case' and 'screening by PO will falsely identify another 14 infants out of the 10,000 as having suspected CCHD when they do not have it'. The certainty of the evidence was graded as high for specificity and low for sensitivity, downgraded due to serious imprecision (due to small number of cases with CCHD included) and serious risk of differential verification bias ('diagnosis was established by echocardiography in test positive cases however test negatives were usually confirmed by clinical follow-up or by accessing congenital malformation registries and mortality databases').
Main findings from the English pilot study showed that among the 32,836 newborns screened, there were 239 cases (0.73%) with a positive screen, out of which eight were CCHD cases. The screening was performed within the target time of 4 to 8 hours for half (52%) of the participants, within 12 hours for 78%, and after 24 hours for 8.5%. More in detail, around half the newborns with a positive screen were admitted to the neonatal unit. Among those not admitted, 97% had transitional circulation and 2 cases had culture negative sepsis. Among the 114 newborns admitted to the neonatal unit, eight (7%) had a CCHD, 86 (75%) had a 'significant illness which required medical intervention', and 22 (9% of newborns with positive screen, 0.07% of all newborns screened) were healthy babies. From these findings, investigators interpreted that 'earlier screening (within 24 hours) results in a higher proportion of babies detected with a clinical condition but at the expense of a slightly higher screen positive rate'. Two CCHD cases were missed by a negative screen result. These two newborns also passed the antenatal ultrasonography screening and the clinical examination screening without detecting anomalies. One of them had a fatal outcome, and the other one presented with cardiovascular collapse. Overall, authors concluded that 'the pilot has demonstrated that in general, it is feasible to introduce PO screening in an NHS environment, however there are important clinical considerations' and that 'the routine introduction of PO screening could be considered once these issues have been satisfactorily resolved.'
Accuracy considering different thresholds Different thresholds were considered by two studies included in the Cochrane review. According to the review authors, one study used a threshold of ≤94% and found a sensitivity of 100% (95% CI 29 to 100) and specificity of 100% (95% CI 100 to 100); and another study used a threshold of <96%, and found a sensitivity of 100% (95% CI 3 to 100) and specificity of 100% (95% CI 100 to 100), respectively. While it is difficult to understand 100% for both sensitivity and specificity, we noted some inconsistencies between the findings of this systematic review on this aspect and what is reported in the original included papers. We have contacted the review authors for clarification but have not received any reply.
## Accuracy considering antenatal diagnosis
The review authors looked whether antenatal diagnosis could have an impact in the findings and found that both sensitivity and specificity did not change significantly when newborns with suspicion of CHD by antenatal ultrasound screening were included versus excluded.
## Accuracy considering risk of bias
The Cochrane review authors also looked at the impact of risk of bias in the findings. For the 'flow and timing domain', nine studies were judged at unclear risk of bias and 10 studies at low risk of bias. It was found that risk of bias for this domain had no significant effect on sensitivity, but that studies classified at unclear risk of bias had higher specificity than those judged at low risk of bias (100% [95% CI 99.9 to 100%] versus 99.7% [95% CI 99.3 to 99.8%]; P = 0.016).
## Accuracy considering other factors
Other factors that could impact the findings of pulseoximetry in the detection of CCHDs have been reported. Algorithm and cut-offs for pass or failed PO screening were based on studies conducted at low altitude. As oxygen saturations are lower at higher altitudes, using the same algorithm in newborns at high altitudes might lead to higher false-positive rate. While there is global awareness around this question, there is a lack of data to support modification of the current algorithm on PO screening. The altitude where screening was performed was not reported in the studies included in the Cochrane review.
Performing the screening in alert newborns was associated with lower false-positive rates, 'possibly by reducing the likelihood of low oxygen saturations caused by hypoventilation in deep sleep', although this was reported from anecdotal reports. Performing PO screening around the time of the hearing screening was also associated with an improved efficiency, 'assuming that the hearing screening is conducted after 24 hours or immediately before discharge'.
## Conclusions on accuracy of pf for detection of cchds
Current evidence supports consistent accuracy for detection of CCHDs in newborns by pulse oximetry screening in addition to antenatal ultrasonography and clinical examination.
## Timing of po screening
With the closure of the arterial ductus and other physiologic changes after birth, timing might have an impact in the findings of PO screening. A recent study estimated the median ductal closure time to be 27 hours in boys and 45 hours in girls. However, the cut-off considered in the studies is usually 24 hours, which coincides with timing discharge of apparently healthy neonates without maternal complications leading to performing neonatal examination and screening within the first 24 hours of life. In the review conducted by Plana et al (primary analysis), PO screening was performed within the first 24 hours of life in eight studies, with an overall sensitivity of 79.5% (95% CI 70.0 to 86.6) and specificity of 99.6% (95% CI 99.1 to 99.8). Among the 11 studies that performed the screening test after 24 hours of life, the overall sensitivity was 73.6% (95% CI 62.8 to 82.1) and specificity 99.9% (95% CI 99.9 to 100). There were no significant differences on sensitivity between performing the screening before versus after 24 hours of life, but the false-positive rate for detection of CCHDs was significantly lower among the group of newborns screened after 24 hours of life (0.06% [95% CI 0.03 to 0.13] versus 0.42% [95% CI 0.20 to 0.89]; P = 0.027).
The Spanish National Neonatal Society affirmed the opposite: "The timing of screening affects its sensitivity, with a higher sensitivity the earlier it is performed" with a level of evidence judged as A. From the supporting evidence provided by the consensus document, authors argued that "an analysis of late screens (>24 hours) demonstrated that half of CCHDs manifest in the first 24 hours and 20% do so with cardiovascular compromise", but no data was given on direct comparison between screening before versus after 24 hours of life. However, there is a general agreement in the literature that early screening leads to an increased rate of false-positive cases, although it is highly valuable to detect most of these cases, as most of them are indicative of other severe disorders that may also require prompt diagnosis and treatment. Indeed, in the UK, early screening of around 26,000 newborns led to a screening positive rate of 0.8%, with the detection of nine CCHDs cases and within the false-positive cases, 79% had a significant medical condition. Conversely in the US, late screening of around 73,000 newborns led to a very low false-positive rate of 0.04%, at the expenses of a low number of CCHDs cases detected (three cases). As reflected by Ewers et al, "the likelihood is that in the US cohort, many infants with CCHD presented before screening took place". These considerations are the main rationale why early screening is finally recommended by several associations, such as the Spanish National Neonatal Society, as it is judged that benefits outweigh risks.
## Conclusions on timing of oximetry
The timing of pulse oximetry screening had no significant difference on sensitivity, but the false positivity rate was significantly lower among newborns screened after 24 hours of life, when compared with newborns screened within the first 24 hours of life. There is some evidenceand wide acceptance among physiciansthat early screening leading to early detection of CCHDs allows timeliness of appropriate medical intervention and therefore reduces the risk of onset with severe or very severe symptoms, with subsequent better outcome. Newborns with false-positive screening results might undergo unnecessary additional tests such as echocardiogram. However, early screening allows detection of hypoxemia due to other clinically severe conditions that benefit from prompt intervention. Overall, benefits of early screening are judged to be greater than the potential negative impact of the increased rate of positive rate by some societies, but this is not the position of other institutions such as the United Kingdom National Screening Committee and the Royal College of Paediatrics and Child Health.
Post-ductal versus pre-and post-ductal screening A pulse oximeter device can measure pre-ductal oxygen saturation when the probe is placed on the right hand, and post-ductal oxygen saturation when used on either foot. Eleven studies included in the Plana et al (primary analysis) review measured post-ductal oxygen saturation only for the detection of CCHDs, with a sensitivity of 81.2% (95% CI 70.9 to 88.4), a specificity of 99.9% (95% CI 99.7 to 100), and a false-positive rate of 0.13% (95% CI: 0.05 to 0.31%). Pre-ductal and post-ductal screening was performed in 8 studies, with a sensitivity of 71.2% (95% CI 58.5 to 81.3), a specificity of 99.8% (95% CI 99.5 to 99.9), and a false-positive rate of 0.17% (95% CI 0.06 to 0.46). It was found that the site of testing (post-ductal versus pre-and post-ductal) had no significant effect on sensitivity nor specificity.
## Conclusions on site of testing
The site of testing (post-ductal versus pre-and postductal) had no significant effect on sensitivity nor specificity for detection of CCHDs.
Potential harms of PO screening PO is a safe and harmless tool. Newborns with a false-positive screen will however receive additional testing such as an echocardiography or a chest radiography, and might be referred or admitted to a neonatal unit. This has the potential to cause discomfort to the newborn, although additional tests are very unlikely to be invasive. Authors from the pilot study conducted in the UK concluded that "there was little evidence of additional significant harm to the majority of babies who had a screen positive outcome. It is possible however, that some babies underwent unnecessary admission and investigation as a result of testing screen positive, particularly some of those with culture-negative sepsis, these are likely to be in a minority". In settings where echocardiography is not available within a short period, the delay in performing the test for confirmation or exclusion of the CCHDs diagnosis might increase parental anxiety, in addition to increase the workload for health workers performing echocardiography and additional potential admissions until the test is performed. Indeed, like any screening of a potential severe disease, CCHDs screening might raise unnecessary parental anxiety. However, clinical practice and available literature suggests good acceptability to mothers and no increased anxiety among mothers given falsepositive results compared to mothers given true-negative results.
However, it was found that the number of falsepositive results generated by PO screening was lower than those generated by clinical examination alone, which is worth to consider when balancing benefits and harms of PO screening.
In addition, it is important to note that detection of hypoxemia that is not due to CCHDs allows the identification of many cases of clinically significant conditions that cause hypoxemia such as pneumonia or sepsis, that benefit from early recognition and management. As neonates with a clear non-cardiac diagnosis are unlikely to require an echocardiography, the number of false-positive cases lead to a lower number of echocardiography needed. In the UK, health staff were not aware of any increase in the number of echocardiograms during the period of the pilot study introducing PO screening.
## Conclusions on harms and limitations of po
Overall, early diagnosis of CCHD with pulse oximetry is judged to be beneficial and cost-effective, and potential harms associated with false-positive tests are not serious, while missing CCHDs and other serious diseases detected by hypoxaemia in absence of pulse oximetry screening can lead to serious consequences.
## Summary of findings
Current evidence supports consistent accuracy for detection of CCHDs in newborns by pulse oximetry screening in addition to antenatal ultrasonography and clinical examination. Overall, early diagnosis of CCHD with pulse oximetry is judged to be beneficial and costeffective, and potential harms associated with falsepositive tests are not serious, while missing CCHDs and other serious diseases detected by hypoxaemia in absence of pulse oximetry screening can lead to serious consequences. Timing of oximetry:
○ The timing of pulse oximetry screening had no significant difference on sensitivity, but the false positivity rate was significantly lower among newborns screened after 24 hours of life, when compared with newborns screened within the first 24 hours of life. ○ There is some evidenceand wide acceptance among physiciansthat early screening leading to early detection of CCHDs allows timeliness of appropriate medical intervention and therefore reduces the risk of onset with severe or very severe symptoms, with subsequent better outcome. ○ Newborns with false-positive screening results might undergo unnecessary additional tests such as echocardiogram. However, early screening allows detection of hypoxemia due to other clinically severe conditions that benefit from prompt intervention. ○ Overall, benefits of early screening are judged to be greater than the potential negative impact of the increased rate of positive rate by some societies, but this is not the position of other institutions such as the United Kingdom National Screening Committee and the Royal College of Paediatrics and Child Health.
The site of testing (post-ductal versus pre-and postductal) had no significant effect on sensitivity nor specificity for detection of CCHDs.
Acknowledgments I am very grateful to María Jesús Esparza, Laura Reali, and Gottfried Huss for carefully reviewing and providing valuable feedback for each article. I am also grateful to Ralf Weigel and Gottfried Huss for proofreading the final version of this document.
## About this supplement
This article has been published as part of BMC Pediatrics Volume 21, Supplement 1 2021: Defined preventive interventions for children under five years of age: evidence summaries for primary health care in the WHO European region. The full contents of the supplement are available at https://bmcpediatrics.biomedcentral.com/articles/supplements/volume-21supplement-1.
Author's contributions SJ was identified as the researcher in the development of the synthesis of evidence and writing the report. For each selected topic on preventive interventions, SJ defined the key questions, established and run the literature search, screened the returned manuscripts for eligibility, extracted data and summarized the existing recommendations and supporting evidence. The principal advisors of this project were Dr. Gottfried Huss, MPH General Secretary of ECPCP, Project-Coordinator and Prof. Ralf Weigel, Friede Springer endowed professorship of Global Child Health, Witten/Herdecke University (scientific advice). The author(s) read and approved the final manuscript.
# Funding
Publication charges for this article have been funded by the Friede Springer endowed professorship for Global Child Health at the Witten Herdecke University, Germany.
Availability of data and materials Not applicable.
Ethics approval and consent to participate Not applicable.
## Consent for publication
Not applicable.
Competing interests SJ had a contract and was paid as an independent consultant by the WHO via Witten/ Herdecke University, ECPCP and EPA/UNEPSA for developing the different articles of this supplement. |
Migrant health in French Guiana: Are undocumented immigrants more vulnerable?
Background: Few data exist on the health status of the immigrant population in French Guiana. The main objective of this article was to identify differences in its health status in relation to that of the native-born population.Methods: A representative, population-based, cross-sectional survey was conducted in 2009 among 1027 adults living in Cayenne and St-Laurent du Maroni. Health status was assessed in terms of self-perceived health, chronic diseases and functional limitations. The migration variables were immigration status, the duration of residence in French Guiana and the country of birth. Logistic regression models were conducted.Results: Immigrants account for 40.5% and 57.8% of the adult population of Cayenne and St-Laurent du Maroni, respectively. Most of them (60.7% and 77.5%, respectively) had been living in French Guiana for more than 10 years. A large proportion were still undocumented or had a precarious legal status. The undocumented immigrants reported the worst health status ] for self-perceived health, OR = 2.79 [1.22-6.34] for a chronic disease, and OR = 2.17 [1.00-4.70] for a functional limitation). These differences are partially explained by socioeconomic status and psychosocial factors. The country of birth and the duration of residence also had an impact on health indicators.
# Background
A growing body of studies suggests that there are health disparities between immigrants and local populations [bib_ref] A framework for analysing migrant health policies in Europe, Mladovsky [/bib_ref] [bib_ref] Migration: A social determinant of migrants' health, Davies [/bib_ref]. Despite growing knowledge, the relationship between migration and health remains complex and dynamic, for many migration-related determinants can have an impact on health. French studies, too, have reported that disparities in health outcomes exist between immigrants and native-born individuals [bib_ref] An overview of the health status of migrants in France, in relation..., Darmon [/bib_ref] [bib_ref] Time since the last HIV test and migration origin in the Paris..., Lapostolle [/bib_ref] , although such studies are rare in France, where categorizing people as immigrants is viewed as a sensitive issue and is governed by strict legal rules.
French Guiana is located in a humid equatorial zone of South America, between Brazil to the southeast and Surinam to the northwest. A former French colony, French Guiana became, in 1946, a French overseas territory, with the same legislation as in mainland France. French Guiana has a multiethnic population, the result of successive migration waves. Up until the early 1960s, the history of French Guiana was characterized by problematic and insufficient human settlement (in 1954, the population was still only 27,000, over an area of 83,350 km 2 , essentially Creoles, Amerindians and Bushinenge). It subsequently attracted a great deal of foreign labour enticed by a job market that had become attractive with the creation of the Guiana Space Centre and the launch of large infrastructure projects. In the 1970s and 1980s, it took in a large number of migrants fleeing from the political instability and economic hardships in their countries: political turmoil in Haiti, a civil war in , and social and economic problems in Guyana. French Guiana has been going through a major economic crisis since the 1990s, with a high unemployment rate (20.6% of the active population in 2006), a huge trade deficit and heavy economic dependence on public transfers . Despite this economic crisis and increasingly restrictive immigration policies, there is still significant migratory pressure. In 2009, this department had 229,000 inhabitants, [bib_ref] Integrating Ethnicity and Migration As Determinants of Canadian Women's Health, Vissandjee [/bib_ref].5% of whom were immigrants . There are few data on the health of this immigrant population.
The objective of this article is to analyze health disparities between immigrants and native-born people in light of several migratory characteristics (the immigrants' legal status, their duration of residence in French Guiana, and their country of origin).
# Methods
## Study design
A representative, population-based, cross-sectional survey was conducted in French Guiana's two largest cities: Cayenne and Saint-Laurent du Maroni, which had 58,004 and 33,707 inhabitants, respectively, as at . The target population consisted of the resident adult population (≥ 18 years), "resident" meaning having lived or intending to live in either of these two cities for at least 6 months.
A four-stage random sample was constituted. The objective was to conduct 600 interviews in Cayenne and 400 in Saint-Laurent du Maroni (in order to respect the population ratio between the two cities) and to interview 60 people per neighbourhood. These neighbourhoods constitute an intermediate aggregated geographical level between residential IRIS [10] (IRIS, a French acronym for "blocks for incorporating statistical information", are aggregated census blocks) and census blocks. First, 10 neighbourhoods were selected from the 34 neighbourhoods in Cayenne (which has 25 IRIS) and 7 were selected from the 17 in Saint-Laurent du Maroni (10 IRIS) in proportion to the number of households (according to the 2009 census), and they were stratified according to whether or not they are designated as "underserved neighbourhoods" by French urban public policies [fig_ref] Figure 1: Map of the randomly selected neighbourhoods in Cayenne and Saint-Laurent du Maroni [/fig_ref]. Second, in each neighbourhood, census blocks were selected proportionally to the number of households. In all, 40 census blocks were randomly selected from the 474 eligible census blocks in Cayenne, and 25 were randomly selected from the 160 in Saint-Laurent du Maroni. Subsequently, households were randomly selected using a sampling interval calculated for each block in proportion to the number of households in that block (the sampling interval varied between 1 and ¼). Lastly, one adult within each household was randomly selected by the interviewer. The questionnaire was administered face-to-face at the individuals' residences by local, multilingual interviewers from February to April 2009. This survey did not fall into the category of biomedical research (as defined by French law) and did not collect any personal identification data. Therefore it did not need ethical approval in France. On the other hand, it has been approved by the Department of research of the Agence française de développement (AFD).
## Data collection health status
We used the three health-related questions from the Mini European Health Module (MEHM) that concern self-assessed health, chronic diseases and functional limitations [bib_ref] Creating a coherent set of indicators to monitor health across Europe: the..., Robine [/bib_ref] [bib_ref] The reliability of the Minimum European Health Module, Cox [/bib_ref] :
- Self-assessed health was based on the question, "How would you describe your general health?", to which the possible answers were "very good", "good", "fair", "poor" and "very poor". This indicator was dichotomised between the individuals who assessed their overall health as very poor, poor or fair and those who assessed it as good or very good.
- Chronic disease status was assessed by the question, "Do you have any longstanding illness or longstanding health problem?", "longstanding" referring to illnesses or health problems that had lasted or were expected to last for 6 months or longer.
- Functional limitations were assessed by the question, "For at least the past six months, have you been limited because of a health problem in activities people usually do?"
## Migration variables
Three variables were examined: 1) Migration status was defined on the basis of four variables: the country of birth, nationality at birth, nationality on the day of the interview and, for those of foreign nationality, their legal status on the day of the interview. Six migration statuses were thus defined:
-Native-born French. Applies to people of French nationality born in French Guiana. They were chosen as the reference category.
-Born French outside French Guiana (in mainland France, another French overseas territory or abroad). Such individuals were differentiated from the previous group in that they constitute a special subgroup (migration is often temporary, and they often enjoy a privileged socioeconomic status).
-Naturalized immigrant. Applies to people who had acquired French citizenship.
-Long-term documented immigrant. Applies to migrants of foreign nationality who had a 10-year French territory residence card. The few citizens of the European Union were included in this subgroup.
-Temporary documented immigrant. Applies to migrants of foreign nationality with a 1-year temporary stay document, authorization for a temporary stay (usually 6 months) or, more rarely, a refugee claim in progress.
-Undocumented immigrants. Applies to migrants of foreign nationality who had no valid stay document on the day of the interview.
Immigrants (born non-French abroad) are therefore represented by the last four categories.
2) Duration of residence. In addition, immigrants were classified into two groups according to their duration of residence in French Guiana: ≤ 5 years (recent immigrant) or > 5 years (established immigrant).
3) Country of birth. In light of the sample size limitations, the analyses concerned only the two main groups of immigrants: those born in Haiti and those born in Surinam.
## Covariables
The demographic variables included gender and age. Median age and the interquartiles were calculated for the description of the population, and four categories , , and > 50 years) were used in logistic regression models.
Socioeconomic status was characterized by three variables: education level, of which there were three categories (none or primary, secondary and tertiary); occupational status, which was categorized as civil servant, upper whitecollar, lower white-collar, blue-collar (including farmer), unemployed, housewife, student, retired, and inactive; and perceived financial situation. The latter was assessed by a question put to the head of the household ("Presently, for this household, would you say that financially..."), for which there were five possible answers ("We don't have enough to live on; we can't get by.", "We have just enough to live on, but we go without a lot of things", "We have enough to live on as long as we're careful.", "We aren't lacking for anything important.", and "We don't go without anything at all; we're very well off."). This variable was divided into three categories: good (the last two answers), fair and poor (the first two answers). Lastly, two binary psychosocial variables were taken into account. One was fluency in French (fluent in French, with no difficulty or with some difficulty, versus not fluent in French at all, with a great deal of difficulty). This variable provided an indication of acculturation to French society. The other one was feeling of loneliness, which was assessed by the question, "In general, would you say that you...?" ("have a very good circle of people around you" or "have a fairly good circle of people around you" versus "feel fairly alone" or "very alone").
## Statistical analyses
All of the following analyses were weighted in order to account for the sample design and the poststratification adjustment for age, gender and citizenship status (French or foreigner) according to the general population census performed in 2006 by the Institut National de la Statistique et des Études Économiques (the French Bureau of Statistics).
First, we described and compared the demographic characteristics, socioeconomic conditions and health status of the migration status subgroups using a chisquare test. The comparisons of the median durations of residence and age used the nonparametric test of Kruskal-Wallis. Second, we performed logistic regression models, which were systematically adjusted for age and gender, to estimate the associations between the abovementioned covariables and each of the three health status variables. Third, we compared the odds ratio (OR) estimating the strength of the association between each of the three migration variables and each of the three health status variables separately when successively adding the covariables to the respective models. Fourth, we constructed a new variable -migration status and origin -that combined the undocumented immigrants' migration status and country of birth, and, in the same manner as in step 3, we determined whether the covariables contributed to the associations observed between this migration status-and-origin variable and each of the three health status variables. All the analyses were performed with Stata ® software, version 10.0.
# Results
In all, 1027 people were interviewed (607 in Cayenne and 420 in Saint-Laurent du Maroni). The participation rate was 81.2%. Of the study population, 52.9% were women, and the median age was 36 years [fig_ref] Table 1: Description of the population by migration status [/fig_ref]. The distribution of this population by migration status was as follows: 37.8% were born in French Guiana and were of French nationality; 16.1% were born French outside French Guiana (70.9% of them were born in mainland France); 6.9% were naturalized immigrants (more than half were from the Caribbean, and the median duration of residence was 25 years); 14.2% were long-term documented immigrants and 11.0% were temporary documented immigrants (the median duration of residence was 21 years and 9 years, respectively. These two subgroups consisted mostly of people from Haiti, Surinam and Brazil); and lastly, 14.0% were undocumented immigrants (half of this subgroup were from Surinam, and the median duration of residence was 9 years). It is also worth noting that the age and gender distributions of the native-born French and the undocumented immigrants were quite similar to each other and to the overall distribution, as compared to other migration status groups. A comparison of the socioeconomic conditions according to these six migration profiles showed strong disparities. For the people who perceived their financial situation as having enough to live on, the civil servants and the people who had a higher education, we observed a socioeconomic gradient based on the following six migration profiles: those born French outside French Guiana were always in a more favourable situation, followed by the native-born, naturalized immigrants, longterm documented immigrants and temporary documented immigrants, in that order, with, at the very bottom of this gradient, undocumented immigrants, who were in the most unfavourable socioeconomic circumstances. People who were fluent in French followed an exactly identical gradient.
All comparisons used Chi2 Test, except comparisons of ages and durations of residence, which used the Kruskal-Wallis test
Since advanced age and female sex were associated with poorer health status indicators, the rest of the analyses were systematically adjusted for these two demographic variables. [fig_ref] Table 2: Logistic regression models analyzing the health variables according to the demographic, socioeconomic... [/fig_ref] shows that, after such an adjustment, the characteristics significantly associated with poorer health were (regardless of the health variable) being an unemployed, retired or some other inactive individual, being in a poorly perceived financial situation, feeling socially isolated, and having poor fluency in French. Being a homemaker and having a low education level were associated with poorer perceived health and a reported functional limitation, but not of a chronic disease. The bluecollars and lower white-collars were more likely to report poor perceived health.
The analysis of the associations between the migration variables and the health variables [fig_ref] Table 3: Logistic regression models explaining the health variables according to the migration variables [/fig_ref] shows that, after adjustment for age and gender, the temporary documented immigrants and undocumented immigrants reported poor perceived health more often than the native-born
# Discussion
To the best of our knowledge, this study is the first one carried out in French Guiana that describes and analyzes social and health disparities in specific populations on the basis of their origins and migration status. This study shows that these two cities have large immigrant populations (40.5% of the adult population in Cayenne and 57.8% in Saint-Laurent du Maroni) and that many of these individuals' had been there for long while (60.7% of the immigrants living in Cayenne and 77.5% in Saint-Laurent du Maroni had been living there for more than 10 years). Despite this long duration of residence in French Guiana, a substantial portion of the immigrant population had no stay documents or had a precarious status. An analysis of the population's social and economic conditions shows strong inequalities that follow a gradient according to the individual's legal status with regard to his or her stay. The analyses showed that the health of these populations depends on several migration-related factors, but also on how health is measured. Three key findings are noted. First, in general, of all the subgroups of migrants that were studied, those most vulnerable and with the worst health status were those who were undocumented, regardless of which social and health indicators were considered. Second, when health was measured as perceived health, the analyses showed that the undocumented immigrants and the documented immigrants with a precarious status (with a stay document valid for one year or less) reported poorer perceived health than the native-born. The country of origin and the duration of residence did not change these results very much. These observed associations are only partially explained by the individuals' socioeconomic status. Third, with regard to functional limitations, certain groups of immigrants (recent immigrants and those born in Haiti) reported a more favourable situation than the native-born for a comparable socioeconomic status.
Although the literature on this topic is sparse, several studies and reports suggest it is undocumented immigrants who are the most vulnerable with regard to health [bib_ref] Undocumented migrants in Canada: a scope literature review on health, access to..., Magalhaes [/bib_ref] [bib_ref] Illegality as risk factor: a survey of unauthorized migrant patients in a..., Castaneda [/bib_ref] [bib_ref] Access to health care for undocumented migrants in Italy, Ravinetto [/bib_ref] [bib_ref] Irion O: Undocumented migrants lack access to pregnancy care and prevention, Wolff [/bib_ref]. They suffer from a combination of socioeconomic conditions and working conditions that are precarious or even harmful to their health [bib_ref] Migration and "low-skilled" workers in destination countries, Benach [/bib_ref] , and they have difficulty accessing health care. In our study, it was mainly the undocumented immigrants who seemed to be the worst off socioeconomically. The socioeconomic indicators used in this study explain only some the observed differences in health. The remaining differences could be explained by socioeconomic factors that were not taken into account in this study (such as income, working conditions or housing conditions) and by difficulty accessing health care. In French Guiana, as in mainland France, undocumented immigrants can theoretically access health care free of charge through a specific health insurance system called "Aide Médicale État" (government medical assistance, which is government-run, unlike the usual health insurance system, which is run by Social Security). If, as several reports have shown, there is, in France, a gap between theoretical rights and actual rights to health care (due to the complexity of the system, the difficulty people have in presenting the required administrative documents, the lack of information on the part of administrative personnel, differences in their practices, and so on, then these difficulties are surely much worse in French Guiana.
Our results for perceived health are consistent with those of several international and French studies. A systematic review with the objective of examining and comparing self-perceived health among migrants and ethnic minority groups in EU countries showed that most migrants and ethnic minority groups appeared to be disadvantaged in relation to the majority population, even after controlling for age, gender and socioeconomic factors [bib_ref] Poorer self-perceived health among migrants and ethnic minorities versus the majority population..., Nielsen [/bib_ref]. A study carried out in mainland France among a sample of more than 20,000 people that was representative of the general population (Enquête décennale santé [Decennial Health Survey]) found that people of foreign origin living in France reported poorer health than the French born in France. It did not find any differences in health between foreign immigrants and those who had been naturalized. As in our study, these populations' poor socioeconomic conditions only partially explained their poorer perceived health. A study carried out on Mayotte Island, a French overseas territory in the Comoros Archipelago, found that the health of foreigners was less good there as well (and they were found to have more difficulty accessing health care) than that of the French [bib_ref] Migration, health and access to care in Mayotte Island in 2007: lessons..., Florence [/bib_ref]. The recent immigrants to French Guiana (≤ 5 years) reported fewer functional limitations than the nativeborn French. This finding supports the "healthy immigrant effect" hypothesis, according to which migrants represent a selectively healthy group that is not representative of all potential migrants from origin societies [bib_ref] Inequalities in death-specific explanations of a general pattern, Marmot [/bib_ref] [bib_ref] Test of the 'healthy migrant hypothesis': a longitudinal analysis of health selectivity..., Lu [/bib_ref] [bib_ref] The healthy migrant effect: new findings from the Mexican Family Life Survey, Rubalcava [/bib_ref]. This hypothesis is also supported by additional analyses in this study suggesting that the migration of sick people (or health care migration) accounts for only a minority of migration movements [bib_ref] Migration, health, and care in French overseas territories, Jolivet [/bib_ref]. This is not observed for perceived health, which may be due to the cut-off that was chosen. Indeed, other studies suggest that the decline in self-perceived health occurs over a very short period after migration [bib_ref] Integrating Ethnicity and Migration As Determinants of Canadian Women's Health, Vissandjee [/bib_ref] [bib_ref] Self-rated health within the Canadian immigrant population: risk and the healthy immigrant..., Newbold [/bib_ref] [bib_ref] Deteriorating health satisfaction among immigrants from Eastern Europe to Germany, Ronellenfitsch [/bib_ref]. In addition, several studies have found relatively better health outcomes for immigrants for indicators such as mortality, chronic conditions and impaired activity than for selfassessed health [bib_ref] Test of the 'healthy migrant hypothesis': a longitudinal analysis of health selectivity..., Lu [/bib_ref] [bib_ref] Integrating Ethnicity and Migration As Determinants of Canadian Women's Health, Vissandjee [/bib_ref] [bib_ref] Comparing three measures of health status (perceived health with Likert-type scale, EQ-5D,..., Leung [/bib_ref] [bib_ref] Migration-related health inequalities: showing the complex interactions between gender, social class and..., Malmusi [/bib_ref] , which suggests that health selection is stronger for chronic and severe conditions.
After adjustment for the socioeconomic conditions, the people born in Haiti reported fewer functional limitations than the native-born French. This subgroup of immigrants had the worst socioeconomic indicators (47.2% of the people born in Haiti had no or only a primary education, 60.7% reported that they did not have enough to live on, and only 34.5% were working). Moreover the proportion of recent immigrants (≤5 years) among immigrants born in Haiti (14.2%) was not different from the one among immigrants from other countries (15.5%). Therefore, three hypotheses could explain this paradoxical finding. One is that of cultural differences in reporting functional limitations, although it hardly seems plausible (this hypothesis will be detailed below). Another is that of selection bias due, in this case, to the return of migrants in poor health to their country of origin, which seems even more unlikely, given the overall situation in Haiti. A third hypothesis seems the most probable: that of greater migration selection among migrants from Haiti, the poorest country in the Americas. A recent study carried out in Spain found that "[f]oreign immigrants from poor countries reported the worst socio-economic conditions, but relatively good health" [bib_ref] Migration-related health inequalities: showing the complex interactions between gender, social class and..., Malmusi [/bib_ref]. Other studies suggest that long distance migration may be associated with a stronger selection effect [bib_ref] Test of the 'healthy migrant hypothesis': a longitudinal analysis of health selectivity..., Lu [/bib_ref]. It may be that Haitians in better physical health are the ones more likely to move to French Guiana because they are able to manage the difficulties and stress associated with immigrating. The undocumented immigrants from Surinam had poor health indicators, regardless of which health indicator was used or which adjustments were made. These people have special attributes: all of them were living in Saint-Laurent du Maroni (a town on the border with Surinam), and their median duration of residence in French Guiana was 16 years (as opposed to 6 years for the other undocumented immigrants). Thus, a number of hypotheses can be proposed to explain their particularly poor health status: the circumstances of their immigration to French Guiana (fleeing from the civil war between 1986 and 1992 and economic hardships in Surinam), the geographical proximity of Saint-Laurent du Maroni (which limits the possibility of positive immigrationselection bias), and the many years spent underground.
## Limitations and strengths of this study
This study has a certain number of strengths: a sampling method ensuring that the final sample would be representative, a high participation rate, and the inclusion of several migration variables.
Several limitations should be discussed. First, this was a cross-sectional study, and no definite conclusions can be drawn regarding causality. Second, this survey was conducted among people over the age of 18 years who had been living or were intending to live in French Guiana for at least 6 months and who were residing in single-family dwellings. It therefore excluded people living collectively, people with no fixed address, and transient migrants. Third, we did not have a means of measuring the representativeness of the subgroup consisting of undocumented immigrants, since they are, by definition, undocumented in the national statistics. On the other hand, the sampling procedure (the stratification and sampling intervals used) and the large proportion of this population in the two survey cities make it unlikely that we under-or overrepresented the neighborhoods inhabited by undocumented immigrants. Lastly, a few words need to be said about the choice of indicators. The three health indicators of the MEHM had the advantage of being widely used in epidemiological surveys, and their reliability had been evaluated in a European population [bib_ref] The reliability of the Minimum European Health Module, Cox [/bib_ref]. However, they have not been validated in the populations of French overseas departments (especially in French Guiana). Moreover, questions remained about their interindividual comparability, since health perceptions vary according to health norms and people's aspirations, who are influenced by their social and cultural environment [bib_ref] Self-rated health and mortality: a review of twentyseven community studies, Idler [/bib_ref] [bib_ref] Socio-economic and demographic variation in health and in its measures: the issue..., Shmueli [/bib_ref]. Of the three health indicators used, a self-reported chronic disease is the most prone to differential reporting bias between social groups. In this study, homemakers and individuals with little schooling reported poor perceived health and functional limitations more often, but these associations were not found for the indicator 'chronic disease'. Several analyses have reported a trend toward chronic diseases in population groups in the lowest education and income brackets being underreported [bib_ref] Differences in the misreporting of chronic conditions, by level of education: the..., Mackenbach [/bib_ref] [bib_ref] Inequalities in health related to women's marital, parental, and employment status-a comparison..., Elstad [/bib_ref]. This can be explained by less medical information, which is due to less use of the health-care system. In addition, it is questionable whether the concept of chronic disease is clearly understood by all sociocultural groups. Perceived health is the mostly widely used indicator, and numerous studies have shown associations with mortality [bib_ref] Self-rated health and mortality: a review of twentyseven community studies, Idler [/bib_ref] [bib_ref] Perceived health and mortality: a nine-year follow-up of the human population laboratory..., Kaplan [/bib_ref] , morbidity and the use of the health-care system [bib_ref] Equity in the delivery of health care in Europe and the US, Van Doorslaer [/bib_ref] [bib_ref] Predicting mortality and healthcare utilization with a single question, Desalvo [/bib_ref] , regardless of the ethnic group [bib_ref] Validating self-rated health in different ethnic groups, Chandola [/bib_ref]. However, a few studies found that this indicator tended, once again, to underestimate social health inequalities [bib_ref] Using selfrated health for analysing social inequalities in health: a risk for..., Delpierre [/bib_ref]. As for the indicator 'functional limitations', its transcultural validity has not been investigated, but several studies that have examined this indicator between different ethnic groups suggest that information biases are weak [bib_ref] Limiting long-term illness among black Caribbeans, black Africans, Indians, Pakistanis, Bangladeshis and..., Harding [/bib_ref] [bib_ref] Migration status and limiting long standing illness: a longitudinal study of women..., Robertson [/bib_ref] [bib_ref] Social mobility and self-reported limiting long-term illness among West Indian and South..., Harding [/bib_ref] [bib_ref] Long-term illness among indigenous and foreign-born people in Sweden, Sundquist [/bib_ref].
The choice of migration variable has its limitations, too. The main one is that the groupings that were made (to construct the six subgroups based on migration status) mask very different sociocultural situations and migration paths. For instance, the subgroup consisting of people born in French Guiana was actually quite heterogeneous (among the main ethnic groups that make up the population in French Guiana are the Creoles, the Bushinenge and Amerindians). Furthermore, when constructing these six groups, we took into account the individual's status on the day of the survey. This categorization did not take into account how long the person had had that status, for some statuses are not stable. Immigrants with a temporary stay document can have their renewal request turned down and quickly become undocumented. In contrast, some of the interviewees may have very recently regularized their status.
# Conclusion
Overall, the results of this study suggest that, although the determinants of migrant health in French Guiana mainly have to do with the multiple dimensions of the social determinants of health and social health inequalities, other parameters specific to immigration (the country of origin, the duration of residence, the reason for immigrating, and the conditions of residence in French Guiana) play a role of their own. Data on migrant health are scarce in France, and more generally, migrant health problems have been largely ignored in public health policies. Indeed, the notion of a "specific approach" to health issues is creating a debate. Any differential treatment, in particular, according to nationality or ethnic group, is generally perceived as wrong, since it is contrary to the principle of equal treatment for all citizens guaranteed by the Constitution and that is part of a long republican tradition. In reality, such a view shows a lack of understanding, all the while contributing to the denial of the problems specific to immigrants. We recommend that the specific determinants associated with migration be taken into account in different epidemiological surveys and the current local information systems to improve knowledge of the health of specific populations in French Guiana. The health status of migrants is of crucial interest to health policy planners, and it is especially relevant, considering the size of the foreign-born population in that region.
[fig] Figure 1: Map of the randomly selected neighbourhoods in Cayenne and Saint-Laurent du Maroni. [/fig]
[table] Table 1: Description of the population by migration status [/table]
[table] Table 2: Logistic regression models analyzing the health variables according to the demographic, socioeconomic and psychosocial variables (OR and 95% CI) * The models for each socioeconomic and psychosocial variable are adjusted for age and gender [/table]
[table] Table 3: Logistic regression models explaining the health variables according to the migration variables (OR and 95% CI) On the other hand, undocumented immigrants born in Haiti reported fewer functional limitations than the native-born French, regardless of which adjustments were made (Model 3: OR = 0.13; 95% CI = [0.05-0.36]). In addition, this table suggests that the undocumented immigrants born in Surinam had poor health indicators, regardless of which health indicator was used or which adjustments were made: all models combined, the ORs associated with poorer health varied, for this subgroup, from 2.40 (95% CI = [0.91-6.34]) to 4.29 (95% CI = [1.55-11.84]). [/table]
[table] Table 4: Logistic regression models explaining the health variables according to migration status and country of birth (OR and 95% CI) [/table]
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Splicing imbalances in basal-like breast cancer underpin perturbation of cell surface and oncogenic pathways and are associated with patients’ survival
Despite advancements in the use of transcriptional information to understand and classify breast cancers, the contribution of splicing to the establishment and progression of these tumours has only recently starting to emerge. Our work explores this lesser known landscape, with special focus on the basal-like breast cancer subtype where limited therapeutic opportunities and no prognostic biomarkers are currently available. Using ExonArray analysis of 176 breast cancers and 9 normal breast tissues we demonstrate that splicing levels significantly contribute to the diversity of breast cancer molecular subtypes and explain much of the differences compared with normal tissues. We identified pathways specifically affected by splicing imbalances whose perturbation would be hidden from a conventional gene-centric analysis of gene expression. We found that a large fraction of them involve cell-to-cell communication, extracellular matrix and transport, as well as oncogenic and immune-related pathways transduced by plasma membrane receptors. We identified 247 genes in which splicing imbalances are associated with clinical patients' outcome, whilst no association was detectable at the gene expression level. These include the signaling gene TGFBR1, the proto-oncogene MYB as well as many immunerelated genes such as CCR7 and FCRL3, reinforcing evidence for a role of immune components in influencing breast cancer patients' prognosis.Breast cancer is a heterogeneous disease that comprises tumour subgroups with substantial differences in biology, clinical outcomes and responses to treatment. Whilst the debate on the most appropriate definition of breast cancer subtypes is still open, it is now accepted that breast cancer consists of at least five different molecular subtypes which include -according to the PAM50 classification scheme 1 -basal-like, HER2, Luminal A, Luminal B and the additional category of Normal-like, made of tumours which transcriptionally resemble normal breast tissue samples 2 . These molecular subtypes have clear, although not complete, correlation with clinically defined tumour classes, based on the histological assessment of the oestrogen (ER) and progesterone (PR) receptors and human epidermal growth factor receptor 2 (HER2). Basal-like breast cancers overlap to a large degree with clinically defined triple-negative tumours (ER-negative, PR-negative and HER2-negative), whilst Luminal A/B and HER2
correspond respectively to ER negative and ER-negative/HER2-positive tumours. What makes the discovery and exploration of these subtypes relevant is the evidence of their association with different clinical outcomes, ranging from the best-prognosis Luminal A tumors to poor prognosis HER-2 and basal-like tumors, as well as underlying differences in biology reflected in different patterns of response to therapeutic agents [bib_ref] Basal-like breast cancer: a critical review, Rakha [/bib_ref].
In the last decade, genomics analyses have significantly improved our knowledge of breast cancer. Extensive and integrated molecular studies of increasing size and resolution are revealing the existence of additional tumour subgroups with distinct molecular properties [bib_ref] The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups, Curtis [/bib_ref] [bib_ref] Comprehensive molecular portraits of human breast tumours, Cancer Genome Atlas [/bib_ref] [bib_ref] The landscape of cancer genes and mutational processes in breast cancer, Stephens [/bib_ref] [bib_ref] The clonal and mutational evolution spectrum of primary triple-negative breast cancers, Shah [/bib_ref]. However, only limited information is currently available on the role of alternative splicing in the establishment and progression of these tumours, and on the contribution of splicing to breast cancer heterogeneity and its potential for biomarker development Alternative splicing is a key post-transcriptional mechanism affecting more than 90% of human genes and is responsible for the generation of protein isoforms with very different biological properties and functions [bib_ref] Alternative isoform regulation in human tissue transcriptomes, Wang [/bib_ref] [bib_ref] Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput..., Pan [/bib_ref]. Antagonistic splice variants of genes involved in differentiation, apoptosis, invasion and metastasis often exist in a delicate equilibrium that is found to be perturbed in tumours. Indeed, a number of studies have demonstrated that changes in splicing during cancer development alter hallmarks of cancer metastases such as cell morphology, adhesion, migration, apoptosis and proliferation processes, and that oncogenes are inactivated by alternative splicing in normal differentiation [bib_ref] Unbalanced alternative splicing and its significance in cancer, Venables [/bib_ref].
To have an insight into the molecular perturbations induced by splicing imbalances in breast cancer we have used the Affymetrix GeneChip Exon 1.0 ST platform to analyse a well characterised patient cohort encompassing 176 samples composed primarily of tumours classified as basal-like according to PAM50 [bib_ref] Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with..., De Rinaldis [/bib_ref] [bib_ref] Comparison of basal-like triple-negative breast cancer defined by morphology, immunohistochemistry and transcriptional..., Gazinska [/bib_ref]. This technology allows for expression profiling of individual exons and has already been applied in several cancer studies to assess transcriptional splicing variants [bib_ref] Exon-level transcriptome profiling in murine breast cancer reveals splicing changes specific to..., Bemmo [/bib_ref] [bib_ref] Alternative splicing in colon, bladder, and prostate cancer identified by exon array..., Thorsen [/bib_ref] [bib_ref] Exon arrays provide accurate assessments of gene expression, Kapur [/bib_ref] [bib_ref] Alternative splicing and differential gene expression in colon cancer detected by a..., Gardina [/bib_ref].
The exon-level resolution allowed for the measurement of the relative abundances of the exons -and therefore indirectly of the underlying isoforms -transcribed from each gene, a concept we referred to as gene's splicing balance. Results reveal that an additional layer of transcriptional diversity between tumours and normal breast tissues and between different tumour molecular subtypes exists based on genes' splicing imbalances, which goes beyond what has been observed so far in breast cancer by measuring overall gene expression levels 2 . We have attempted to quantify and to qualify this layer, investigating on the pathways affected by splicing imbalances and on the use of this information to identify therapeutic targets and clinical prognostic biomarkers.
# Results
## Samples data and clinical and molecular classification. the study is based on the analysis of
Affymetrix GeneChip Exon 1.0 ST data from a set of 176 invasive breast carcinomas extracted from an equivalent number of patients, and an additional group of 9 normal breast tissues (hereby referred to as NBT samples) extracted from mammary reductions of unrelated individuals. The same cohort was analyzed in previous studies by our group [bib_ref] Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with..., De Rinaldis [/bib_ref] [bib_ref] Comparison of basal-like triple-negative breast cancer defined by morphology, immunohistochemistry and transcriptional..., Gazinska [/bib_ref]. Of the 176 tumours analysed, 148 were immunohistochemically ER-negative, 93 being also triple-negative. Molecular characteristics of tumour samples were analysed in association with clinical and pathological information (Additional File 1). In addition to the assignment to clinical subgroups based on ER, PR and HER2 status, tumour samples were classified according to the five intrinsic molecular subtypes (basal-like, luminal A, luminal B, HER2 and normal-like). For this we used the expression of predefined intrinsic gene lists according to the PAM50 centroid-based classification method 1 . In line with our previous analyses on the same data set [bib_ref] Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with..., De Rinaldis [/bib_ref] [bib_ref] Comparison of basal-like triple-negative breast cancer defined by morphology, immunohistochemistry and transcriptional..., Gazinska [/bib_ref] , triple-negative breast cancers were found to correspond mostly with the basal-like tumours (84%) while ER-positive lesions corresponded to luminal A and B subtypes (79%) (Additional File 2). Differential expression and differential splicing across normal tissues and breast cancer molecular subtypes. We compared different breast cancer subtypes between themselves and with respect to NBT samples, on the basis of three different measurements: (i) the overall expression of genes (GE), in which multiple probes on different exons are summarised into a cumulative expression value for all transcripts of the same gene; (ii) the expression of individual exons (EE), inferred from exon-specific probes; (iii) the exon splicing levels, as measured by the splicing index (SI) metric (see methods) [bib_ref] Detection and measurement of alternative splicing using splicing-sensitive microarrays, Srinivasan [/bib_ref]. This metric captures the contribution of each exon to the overall expression of a gene. Differences in an exon's SI between two sample groups reflect indeed different inclusion or exclusion rates of that exon with respect to the overall gene expression, and thus different splicing balances between the two groups [fig_ref] Figure 1: Exon expression, gene expression and splicing balance values of an exemplary three-exons... [/fig_ref].
First we computed coefficients of determination using the GE/EE/SI values to assess the overall degree of similarity among tumours from the same and from different subtypes. As expected, we observed pairwise correlation levels to be significantly higher when calculated from within-then from between-subtypes. These differences are very high when GE and EE values are used, and lower when using SI values, in keeping with the fact that subtypes are defined based on overall gene expression and not splicing information (Additional File 3).
Then we compared tumour subtypes between themselves and with normal breast tissues using GE/EE/SI metrics. In this way it was possible to reveal different splicing balances, in the presence or absence of whole-gene differential expression, thus adding a layer of resolution to standard gene-centric transcriptional analyses.
By analysing the deviation of the obtained distribution of p-values for each comparison from the distribution generated under the null hypothesis of no average differences we could then infer the overall diversity between groups, due to each of these measures respectively, along with their statistical significance (see Materials and Methods).
The observed pairwise differences are beyond what would be observed by random fluctuations under the null hypothesis, thus pointing to their internal statistical significance (Additional File 4).
Having established the principle that splicing imbalances contribute to overall breast cancer diversity and to the differences between tumour and NBT samples, we then tried to quantify and to define the borders between gene expression and splicing imbalance effects.
In all comparisons we could identify, along with genes showing both differential expression and splicing balance (GE and SI overlaps), also genes having differential splicing balances but not overall differential expression (GE and SI disjunctions) [fig_ref] Figure 2: Pairwise transcriptional differences between tumour subtypes and NBT [/fig_ref]. Perturbation of these genes would not have been detected by looking at GE values alone. By quantifying GE/SI overlaps and disjunctions we could therefore assess in each comparison the GE and SI relative contributions to the overall transcriptional diversity across different sample groups: on one extreme is the Luminal A/Luminal B pair, whose differences are mainly explained by GE levels; on the other is the basal-like tumours and NBT pair, showing marked differences both in GE and SI levels. Noticeably, whilst the absolute number of genes differentially expressed and spliced might differ from pair to pair due to different samples sizes, their percentage contribution to the overall set of perturbed genes is stable and independent of both sample sizes and the q-value thresholds used for statistical significance.
These results indicate the distinct value of looking at differential splicing in addition to differential expression and demonstrate that splicing mechanisms significantly contribute to the diversity across tumour subtypes and to their differences with respect to normal breast tissue counterpart.
On this basis we also investigated the value of splicing data for potential application to molecular diagnostics and tumour subtype classification. We adopted a decision-tree algorithm of classification, seeking to identify basal-like tumours, from a pool of basal-like tumour and NBT samples -either by using GE, EE, SI, or using SI after having filtered out genes differentially expressed between basal-like tumours and NBT samples (see Materials and Methods). Results show that not only GE data but also SI information, used as the sole input data, is capable of correctly classifying tumour samples, with a performance of over 90% specificity and 70% sensitivity using 1000 randomly selected genes (Additional File 5, see also Methods). The ability of SI information to distinguish between sample types, was also confirmed by unsupervised clustering, based on principal component analysis (PCA) (Additional File 6). Comparative assessment of splicing-level results. The validity of our results was assessed through comparison with three independent studies, respectively on a panel of breast cancer cell lines using exon array-based method [bib_ref] Exon-level microarray analyses identify alternative splicing programs in breast cancer, Lapuk [/bib_ref] , on a small group of triple-negative primary breast cancers using RNA sequencing-based technology [bib_ref] RNA sequencing of cancer reveals novel splicing alterations, Eswaran [/bib_ref] and on a larger RNA sequencing data set of basal-like breast tumours and NBT from The Cancer Genome Atlas (TCGA: http://cancergenome.nih.gov/). In all three cases we compared the list of genes found to have differential splicing balances in our data (based on differential SI) with the equivalent list in the external data set.
Comparison with the array-based cell line study analysis 8 showed a significant overlap, with 21 out of 58 genes differentially spliced between basal-like vs luminal cell lines confirmed in our study (45% of overlap, Fisher test p-value < 10 −4 ) (Additional File 7). The second check against the triple-negative (n = 6) vs. normal breast tissues (n = 3) analysis carried out using RNA sequencing technology 9 produced also a very significant overlap, with 121 out the 371 genes identified in this study to be differentially spliced confirmed by our results (32% of overlap, Fisher test p-values respectively < 10 −6 ) (Additional File 7).
The third data encompassed 92 basal-like tumours and 133 NBT samples and was used to run a more thorough comparison, where genes were selected for being differentially spliced but not differentially expressed in basal-like tumours vs NBT in both data sets. Out of 1.822 identified in the external data set to fulfill these criteria, 408 were confirmed by our study (22% of overlap, Fisher test p-value < 10 −12 ) (Additional File 7).
We also looked for experimental evidences in support of the differential splicing observed in our data set between basal-like tumours and NBT samples. The 100 genes with the lowest p-values for differential splicing balance between basal-like tumours and NBT samples in our data were selected and used for automated literature searches to explore experimental evidences in support of our findings.
Several of them had previously been reported to have breast cancer specific splicing events or differential isoform expression. Examples are FANCD2, RAD54, BIRC5 (survivin) and ASF1B [bib_ref] Polymorphic variations in the FANCA gene in high-risk non-BRCA1/2 breast cancer individuals..., Litim [/bib_ref] [bib_ref] Mutations in the RAD54 recombination gene in primary cancers, Matsuda [/bib_ref] [bib_ref] Transcriptional expression analysis of survivin splice variants reveals differential expression of survivin-3alpha..., Moniri Javadhesari [/bib_ref] [bib_ref] − )-Epigallocatechin-3-gallate Modulates the Differential Expression of Survivin Splice Variants and Protects..., Al-Ajmi [/bib_ref] [bib_ref] Predictive value of survivin alternative transcript expression in locally advanced breast cancer..., Boidot [/bib_ref] [bib_ref] Association of p53 gene alterations with the expression of antiapoptotic survivin splice..., Vegran [/bib_ref] [bib_ref] Distinct expression of Survivin splice variants in breast carcinomas, Vegran [/bib_ref] [bib_ref] Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in..., Corpet [/bib_ref]. Others amongst our list were detected to be differentially spliced in other forms of cancer, or cancer cell lines: FoxM1 CDKN3, ZBTB16, AURKB, CHEK1, SGOL1, SULF1, CDC45 and UBE2C [bib_ref] Dysregulated expression of FOXM1 isoforms drives progression of pancreatic cancer, Kong [/bib_ref] [bib_ref] FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of..., Liu [/bib_ref] [bib_ref] Proliferation marker pKi-67 occurs in different isoforms with various cellular effects, Schmidt [/bib_ref] [bib_ref] FOXM1b, which is present at elevated levels in cancer cells, has a..., Lam [/bib_ref] [bib_ref] Aberrant splicing of cyclin-dependent kinase-associated protein phosphatase KAP increases proliferation and migration..., Yu [/bib_ref] [bib_ref] Identification of a novel promyelocytic leukemia zinc-finger isoform required for colorectal cancer..., Jones [/bib_ref] [bib_ref] Expression of Aurora B and alternative variant forms in hepatocellular carcinoma and..., Yasen [/bib_ref] [bib_ref] Tumor-specific usage of alternative transcription start sites in colorectal cancer identified by..., Thorsen [/bib_ref] [bib_ref] A novel tumor-derived SGOL1 variant causes abnormal mitosis and unstable chromatid cohesion, Kahyo [/bib_ref] [bib_ref] Mammalian Sulf1 RNA alternative splicing and its significance to tumour growth regulation, Gill [/bib_ref] [bib_ref] Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70..., Kukimoto [/bib_ref] [bib_ref] Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumors, Jiang [/bib_ref]. Other cases had been shown to have cell cycle dependent isoform expression. These are: KIF18A, NEK2, MKI67 and CCNA2 [bib_ref] Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B, Lee [/bib_ref] [bib_ref] Alternative splice variants of the human centrosome kinase Nek2 exhibit distinct patterns..., Hames [/bib_ref] [bib_ref] Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A..., Fletcher [/bib_ref] [bib_ref] The Ki-67 protein: from the known and the unknown, Scholzen [/bib_ref] [bib_ref] An intron-retaining splice variant of human cyclin A2, expressed in adult differentiated..., Honda [/bib_ref]. By taking a complementary approach and looking at genes previously shown to be differentially spliced in breast cancer we also observed a high level of concordance, for example Tenascin C, CD44, CD47, RELA, PTK2, ESR1, SYK, BRCA1, LARP1 and ADD3 9,45-52 .
The convergence of our results with these independent genomic studies and experimental evidences pointed to the overall reliability of our results and provided the basis for further downstream analyses. Experimental validation of differential splicing results. To provide experimental support to our findings, a selection of genes showing differential splicing was assayed on a Bioanalyzer DNA7500 after RT-PCR based amplification (Additional File 8). We have selected 11 genes to be either differentially spliced between basal-like tumours and normal samples or differentially spliced in basal-like tumours, between patients with respectively better and worse outcome. Amplified full length cDNAs from each gene were size separated on a Bioanalyzer DNA7500 (see Materials and Methods), allowing comparison between the patterns of transcriptional isoforms expressed in two groups of tumours. Among the 11 genes selected we could successfully amplify 9 of them, of which 8 differentially spliced between basal-like tumours and normal samples (AURKA, AURKB, BCL2-a, NEK2, RRM2, TGFBR1, UBE2C, ZBTB16) and 2 differentially spliced in basal-like tumours, between patients with respectively better and worse outcome (CCR7, TGFBR1). Results obtained for these genes confirm differential splicing, with one of more isoforms of each gene showing differential expression between the two compared groups (Additional File 8).
The analyses on this small gene panel served as a proof of concept to validate our methodological framework for identification of genes undergoing differential splicing, based on the Affymetrix Exon Array 1.0 ST arrays, a platform which has also been extensively validated elsewhere 18,53-55 .
## Cell functions and pathways affected at splicing level in basal-like tumours.
We aimed to identify the cellular functions and pathways altered as a consequence of differential splicing balances in basal-like tumours, as compared to NBT samples. We started from the list of genes showing differential splicing balances but not differential expression between these two groups and evaluated the affected pathways using gene set enrichment and Ingenuity-based analyses [bib_ref] Identification of novel therapeutics for complex diseases from genome-wide association data, Grover [/bib_ref]. We observed that in basal-like tumours splicing imbalances determine, or contribute to the deregulation of many key cancer "hallmarks" 57 . These include known oncogenes (BCL2, BRAF), caspases (CASP6/7), transcription factors (E2F3), cell cycle genes (CDC42, CDK2, CDKN2A), cancer related kinases (JAK2/3, MAPK4/6/14) and DNA repair genes (PARP1, RAD50 and BRCA1). Moreover, we found a clear enrichment for cell surface and extracellular matrix genes, controlling cellular adhesion and cellular motility. Equally striking is the enrichment for oncogenic signaling pathways, mediated by cell surface receptors (complete results are listed in Additional File 9). In these cases surface receptors as well as downstream intracellular signaling proteins showed splicing level imbalances. Examples are the integrin and paxillin signalling pathways which emerged with highest ranking. These membrane mediated pathways are involved in cellular spreading, cell motility and cancer development 58 and exert their function by transducing the extracellular signal to key oncogenic pathways such as MAPK/ERK, Wnt, Rho, mTOR, PTEN and PI3K/AKT signaling pathways [fig_ref] Figure 3: The integrin signalling pathway [/fig_ref] and Additional File 10).
We also checked whether these pathways would have emerged from standard whole-gene expression levels. To this aim we carried out a parallel gene set enrichment analysis of the two gene lists respectively based on differential gene expression (GE), and differential splicing index (SI) between basal-like breast cancer and NBT samples. We found that many of the described perturbations were specifically affected by splicing imbalances and would have been missed or largely underestimated had the same samples been analyzed at a whole-gene expression perspective. Examples include the integrin signalling pathway mentioned above, the VEGFR1 pathway and the oncogenic MAPK/ERK, mTOR and RAS signaling pathways, which also appear to be perturbed exclusively at the splicing level [fig_ref] Figure 4: Heatmap of gene sets and pathways specifically perturbed by splicing imbalances [/fig_ref] and Additional File 11, Additional File 9 for complete results).
We also observed basal-like splicing specific enrichments in other sets of genes related to breast cancer. These include a cancer mesenchymal transition signature ("ANASTASSIOU CANCER MESENCHYMAL TRANSITION SIGNATURE"), genes up-regulated in metastatic breast cancer ("RAMASWAMY METASTASIS UP") as well as genes found mutated and amplified ("NIKOLSKY MUTATED AND AMPLIFIED IN BREAST CANCER") [fig_ref] Figure 4: Heatmap of gene sets and pathways specifically perturbed by splicing imbalances [/fig_ref]. Other interesting enrichments relate to the perturbation of immune-related pathways, such as those mediated by CD8, TCR, CDC42 and JNK, as well as sets of genes previously found to be perturbed in different types of immune cells (e.g. "CD4 T-CELL VS B-CELL UP") (purple group in [fig_ref] Figure 4: Heatmap of gene sets and pathways specifically perturbed by splicing imbalances [/fig_ref].
The same approach was used to explore the differences between tumour molecular subtypes. Despite the limited sample size of the non-basal-like groups (resulting into diminished statistical power) we could identify splicing-specific differences between basal-like and Luminal and HER2 subtypes, with plasma membrane Splicing and association with breast cancer survival. As the next step we explored the possible associations between gene splicing balances -as measured by SI -and disease outcome, using patients' breast cancer specific survival as the clinical end point (see Materials and Methods). Keeping the same analytical framework described, we ran parallel and independent analyses using GE, EE and SI data as the predictor variables in Cox-regression univariate model, followed by Wald test. We observed that the distributions of the p-values obtained from the three analyses significantly deviate from uniform distributions, indicating that -at a general level -all these three measures, GE, EE and SI, hold statistically significant prognostic information (Additional File 12). External validation of our gene-level survival analysis results came from comparison with a large public database of Affymetrix-based tumour gene expression data, herewith referred to as the KMP database 59 . Out of the 204 genes associated with basal-like prognosis in our dataset (q-val < 0.1), 168 (83%) had q-val < 0.1 in the KMP database (Fisher-test p-value of the overlap < 10 −20 ). As a negative control, when we took the 204 genes with lowest association with prognosis from our dataset, only 25% had a q-value < 0.1 in the KMP database (a more extensive description of the comparative validation of our survival results can be found in Additional File 13). Interesting patterns emerged from the gene-by-gene comparative analysis of the results obtained by using the GE and SI metrics. We found a total number of 344 genes whose respective splicing index values are associated with survival. Of these, 97 genes were found to have both GE and SI levels associated with survival [fig_ref] Figure 5: Association with breast cancer specific survival [/fig_ref] and Additional File 14 for complete results). Examples are CYFIP2, WIPF1 and SLAMF1 (Additional Files [bib_ref] Exon-level transcriptome profiling in murine breast cancer reveals splicing changes specific to..., Bemmo [/bib_ref] [bib_ref] Alternative splicing in colon, bladder, and prostate cancer identified by exon array..., Thorsen [/bib_ref] [bib_ref] Exon arrays provide accurate assessments of gene expression, Kapur [/bib_ref]. For these genes the overall gene expression levels -comprising the sum of all transcriptional isoforms -is associated with survival, and at the same time the splicing balance relative to one exon -that is, the contribution to the overall gene expression of one particular exon and its related transcript isoforms -is also associated with survival.
A more intriguing pattern is represented by the 247 genes whose overall expression does not show association with survival, whilst the splicing balance (as determined by the SI) of one of its exons is. In these cases what drives the association with survival is not the expression of a gene as a whole, but instead the relative abundance of the transcriptional isoforms containing a given exon. We describe two examples of genes following this pattern: CCR7 and TGFBR1 [fig_ref] Figure 6: Gene models and Kaplan-Meier curves [/fig_ref]. Others include proto-oncogenes such as MYB and immune-related genes such as FCRL3.
We, as well as others, have shown in previous studies that the percentage of lymphocytic infiltration represents an important prognostic factor in basal-like and triple-negative breast cancer [bib_ref] Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with..., De Rinaldis [/bib_ref] [bib_ref] Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized..., Loi [/bib_ref] [bib_ref] Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive..., Loi [/bib_ref]. In order to assess whether the observed associations of genes splicing balance with clinical outcome are prognostic factors independent of lymphocytic infiltration we moved to a multivariate Cox-regression model, which included this as an additional predictor variable (see Materials and Methods). Our results indicate that if lymphocytic abundance is taken into account, the significance for association with survival obtained using GE, EE, and SI gene levels is lost or significantly reduced for most of the genes (Additional File 18). In other words, transcriptional information does not contribute much to the prediction of clinical outcome when lymphocytic abundance is also available.
Taken together these data suggest that genes associated with survival in univariate analysis act in the model as a surrogate for the abundance of lymphocytic infiltration, implying that these genes are expressed in lymphocytic cells.
To investigate on this hypothesis, we examined in more details genes whose total expression levels or splicing balances were associated with patients' survival in univariate analysis (respectively 204 and 344 genes). We observed that a significant proportion -respectively 28 and 37 -were indeed genes specifically expressed in lymphocytes -as determined by using an external transcriptional data set -or annotated to play a role in the interaction between epithelial and immune tumour compartments (see Materials and Methods and Additional File 18). Notwithstanding, we also identified two (SCGB2A1, SCGB1D1) and seven genes (TGFBR1, CD3E, UHRF1BP1L, SBF2, CCDC121, SETD8, NUCB2) whose respectively gene expression (GE) and splicing balance (SI) retain prognostic value independently of the level of lymphocytic infiltration (see Additional File 14 for complete results). Of note, the transforming growth factor TGFBR1 is a membrane protein receptor involved in many cancers and whose polymorphisms were previously observed to be associated with risk for several forms of cancer, in particular breast cancer 62 .
# Discussion
In this work we have analyzed exon level expression data of 176 breast cancer tissue and 9 non-tumour breast samples, with the aim of detecting splicing imbalances occurring in breast cancer subtypes and inferring their functional and prognostic significance.
First, we characterized splicing-driven differences across breast cancer molecular subtypes and in comparison with normal breast tissues (NBT).
Parallel analyses of whole-gene and exon-level measurements run across different group pairs showed that in addition to largely known differences at gene expression levels, a great proportion of transcriptional perturbations occurring in breast cancer can be ascribed to differences in splicing balance. These perturbations do not necessarily affect the overall expression of genes -that is the sum of all their expressed isoforms -but relate to the balance between the different splicing variants of each gene. Large proportions attributable to splicing differences are observed in basal-like tumours when compared to all other subtypes and to NBT samples [fig_ref] Figure 2: Pairwise transcriptional differences between tumour subtypes and NBT [/fig_ref]. By focusing our analyses on the basal-like subtype, we identified genes and pathways that with respect to normal tissues are significantly affected by differential splicing balance. These include cancer hallmarks of various types: oncogenes (BCL2, BRAF), caspases (CASP6/7), transcription factors (E2F3), cell cycle genes (CDC42, CDK2, CDKN2A), cancer related kinases (JAK2/3, MAPK4/6/14) and DNA repair genes (BRCA1, PARP1 and RAD50).
We could also infer information on the pathways that were exclusively affected by splicing imbalances. We found a clear enrichment for pathways involving cell surface and extracellular matrix genes, controlling cellular adhesion, cellular motility and spreading. Also perturbed specifically at splicing levels are a number of key oncogenic signaling pathways mediated by cell surface receptors such as the MAPK/ERK, mTOR and RAS signaling pathways, as well as pathways related to the immune response. Of note, these pathways would have been completely overlooked from a gene-centric perspective (using for example Affymetrix 3′ microarrays), as they are not On the bottom are Kaplan-Meier breast cancer specific survival curves for Gene Expression, Exon Expression, and Splicing Index. In each plot, the three lines represent the top tertile (red), middle tertile (blue), and lower tertile (green) for the value of the variable. q-values for association with survival, and the hazard ratio with 95% confidence intervals are reported.
Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 altered at the overall gene expression level. Relating exon level information extracted from Exon Array data to individual splicing isoforms is not straightforward and is complicated by the fact that many times the same exon can be shared across several isoforms. Despite this limitation, the general quantification of the volume of splicing imbalance events in basal-like cancers, along with the general observation that many of them involve surface proteins and oncogenic pathways, has important consequences and might open considerable translational perspectives. An example is the development of blood-based molecular assays for the detection of specific isoforms to diagnose this specific breast cancer subtype. Surface-specific cell protein isoforms are also attractive candidate therapeutic targets for development of monoclonal antibody-based therapies. This point is particularly relevant as for this subtype of breast tumours only limited therapeutic options other than systemic chemotherapy are currently available.
In the second part of our work we aimed at evaluating the potential of exon-level splicing information as biomarkers to predict clinical outcome, which represents yet another challenge posed by these tumours.
Previous demonstrations of how clinical outcome can depend on the expression of alternatively spliced isoforms in cancer suggest potential for the use of splicing information to predict patients' prognosis. For example, RHAMM and HAS1 genes in bone marrow and TKS5 in lung have isoform imbalances that have been shown to be prognostic indicators for multiple myeloma and lung adenocarcinoma, respectively [bib_ref] RHAMM expression and isoform balance predict aggressive disease and poor survival in..., Maxwell [/bib_ref] [bib_ref] Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of..., Adamia [/bib_ref] [bib_ref] Differential Tks5 isoform expression contributes to metastatic invasion of lung adenocarcinoma, Li [/bib_ref].
Through parallel exon and gene level survival analyses we could disentangle associations between gene expression and exon splicing levels with clinical outcomes.
We identified 247 genes whose splicing levels were significantly associated with basal-like tumour patients' survival, whilst the same association did not emerge from whole-gene expression analysis. Interestingly, what appears to drive the association of these genes with patients' prognosis is the balance of different transcriptional gene isoforms rather than their overall expression levels. Among them are cancer-related genes such as MYB and TGFBR1 as well as many immune-related genes such as CCR7 and FCRL3, whose prognostic association is likely to reflect the inflammatory process and the presence of lymphocytic cells in the tumour. Indeed, when we included the percentage of lymphocytic infiltration in the model we found that the prognostic association of many of these genes is lost. Through expression analyses of lymphocytic specific genes we showed that many of these splicing variants are in fact expressed in immune cells.
These findings confirm the relevance of immune system related genes in determining tumour control or progression and extend this notion by showing that the splicing levels of many immune-related genes also hold prognostic information. Whether this is a reflection of the engagement of different T-and B-cell types in tumour inflammation, each expressing a specific isoform and with different effects on tumour progression and clinical outcome, will require further investigation.
We were also able to identify 7 genes whose splicing levels have statistically significant prognostic association, independently of the abundance of lymphocytic cells in the tumour. Among these is the TGF beta receptor TGFBR1, a membrane protein receptor involved in many cancers and whose non-synonymous single-nucleotide polymorphisms were previously observed to be associated with risk for several forms of cancer, including breast [bib_ref] Exon-level microarray analyses identify alternative splicing programs in breast cancer, Lapuk [/bib_ref]. We showed that in addition to the identified SNPs TGFBR1 splicing balances also hold prognostic information.
# Conclusion
This work reveals the role of splicing mechanisms in altering key processes in basal-like breast tumours, involving cell surface proteins, immune-related and oncogenic pathways, and provides the basis for the identification of novel isoform-specific membrane therapeutic targets. Our findings disclose aspects of breast cancer transcriptional biology that have so far been largely unexplored. We investigated the use of splicing information in relation to prognosis and have identified genes whose internal splicing balances are associated with patient clinical outcome, in absence of an association at the overall gene-level of expression.
These results highlight the relevance of splicing information for translational applications as potential prognostic biomarkers and in revealing cancer specific targets for therapy. Whilst conclusive assessment of the prognostic value of each of these spliced gene exons will have to await confirmation in larger data sets, our study demonstrates the potential of splicing information as a prognostic biomarker and for the discovery of isoform-specific therapeutic targets in basal-like breast cancer.
# Materials and methods
All methods were carried out in accordance with the approved guidelines.
## Patient characteristics and sample preparation.
This study was based on the same patients' cohort and tumour samples analyzed in previous studies by our group [bib_ref] Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with..., De Rinaldis [/bib_ref] [bib_ref] Comparison of basal-like triple-negative breast cancer defined by morphology, immunohistochemistry and transcriptional..., Gazinska [/bib_ref]. The clinical endpoint considered here was breast cancer specific survival (BCSS), therefore events of death due to other reasons were ignored. In addition, 9 samples of Normal breast tissue (NBT) were obtained from patients undergoing mammoplasty for aesthetic reasons, under protocols approved by the Institutional Review Board and by Guy's Research Ethics Committee, in compliance with the Human Tissue Act. Informed consent was obtained from all subjects from where the tissue samples were taken. The tissues were processed as described in ref. [bib_ref] ALDH1 is a marker of normal and malignant human mammary stem cells..., Ginestier [/bib_ref]. Exon-level transcriptional profiles were obtained by using the Affymetrix Exon 1.0 ST array platform. Tumour molecular subtypes were assigned as described in ref. Exon-Array data pre-processing. An overview of the workflow used for Exon-Array data pre-processing is given in Additional File 19 following the analytical strategy proposed by Lockstone et al. [bib_ref] Exon array data analysis using Affymetrix power tools and R statistical software, Lockstone [/bib_ref]. ExonArray data pre-processing was performed on the R platform using the "aroma.affymetrix" R package (www.aroma-project.
Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 org). RMA was used to remove the array signal background, followed by quantile normalisation to correct for inter-arrays global differences and by gene level summarisation. For this latter step probe sets were mapped to ENSEMBL genes using the mapping file (HuEx-1_0-st-v2, U-Ensembl49, G-Affy.cdf) generated by the aroma. affymetrix team [bib_ref] FIRMA: a method for detection of alternative splicing from exon array data, Purdom [/bib_ref]. Quality of individual arrays was assessed by visual evaluation of RLE (relative log expression), NUSE (normalised unscaled standard error) and hierarchical clustering plots (Additional .
Once expression levels were obtained for each gene and probe set, they were tested for differential expression between different sample groups. Tumour samples were annotated according to the PAM50 molecular classification and on the basis of the ER, PR and HER2 status 1 . Gene level expression measures were tested for differential expression using the moderated t-test implemented in the Limma package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) as part of the R/Bioconductor platform [bib_ref] Bioconductor: open software development for computational biology and bioinformatics, Gentleman [/bib_ref]. Likewise, for the exon analysis, the expression recorded for each probe set was evaluated and compared in the same way. With the genomic mapping of probe sets coordinates of the hg19 genome assembly, they can be mapped on to specific gene and exon locations. The obtained p values were corrected for multiple hypotheses testing using the Benjamini and Hochberg method 70 and the resulting corrected values are hereafter referred to as q values. Except where otherwise noted, a gene was considered to be differentially expressed when its q value for the test is lower than 0.001.
Splicing Index (SI) values were calculated by dividing expression values captured for each probe by the sum of the expression values of all the probes of that gene, as reported elsewhere [bib_ref] Detection and measurement of alternative splicing using splicing-sensitive microarrays, Srinivasan [/bib_ref]. Differential splicing index between samples was then tested with the identical procedure: i.e. using the SI as input values to the Limma package to calculate p values of as described above. By using the SI metric it is possible that when comparing two groups of samples, the average SI of one exon is higher (e.g. due to exon inclusion or to higher expression of an isoform containing that exon), and the average SIs for the other exons of the same gene are lower. In this case our analysis would detect an overall splicing imbalance for that gene, due to different SI values of the exons in the two samples. Splicing imbalances are reported at the gene level; therefore results are not affected if the imbalance is detected from one or more exons within the same gene. The p-values were adjusted for multiple hypotheses testing using the Benjamini Hochberg method 70 and the resulting corrected values are referred to as q values. A gene was deemed to have a splicing imbalance between two groups when the q value in one or more of its probes was lower than 0.001.
The comparison of multiple pairwise combinations of subtypes needs also to be taken into account as a further element for multiple testing correction. However, standard multiple testing procedures assume independence of individual tests and our pairwise comparisons violate the assumption of independence (i.e. subtypes of the same cancer type cannot be considered independent). We have addressed this problem by using a very stringent threshold (q-val < 0.001), which accounts also for the multiple pairwise subtypes comparisons (n. of tests = 10).
## Analysis of p-value distributions from pairwise comparisons. distribution of p-values obtained for
each pairwise comparison were compared against the theoretical distribution under the null hypothesis of differential expression as the result of random noise. The latter was modeled in two ways: (i) as the uniform distribution (ii) as the Montecarlo distribution obtained upon permutation of sample labels. In all cases distribution of p-values obtained for pairwise comparisons showed clear deviation from the null-hypothesis distribution(s), indicating the presence of statistically significant signals in the data.
Overlaps with external data sets. Lists of genes with differential splicing balances were extracted from our data upon pairwise comparisons between TNBC or basal-like tumour samples with luminal tumours or NBT samples. These lists were compared with equivalent lists published in refs 8, 9 as described in the results section. A third comparison was done against RNA-Seq data downloaded from The Cancer Genome Atlas project (TCGA, http://cancergenome.nih.gov/). The data was in the form of "Level 3 data" according to the TCGA nomenclature, which represents gene and isoform level read counts. All samples for which the status of ER, PR and HER2 receptors was available, annotated as "basal-like" according to the PAM50 molecular classification 1 were used in the subsequent analysis. Differential gene expression and differential isoform expression between basal-like tumour (n = 92) and NBT samples (n = 133) was calculated using edgeR [bib_ref] Count-based differential expression analysis of RNA sequencing data using R and Bioconductor, Anders [/bib_ref]. Genes and isoforms with q-value lower than 0.001 were considered to be differentially expressed. From that data we compiled a list of genes having at least an isoform differentially expressed, but not found to be differentially expressed when analyzed at overall gene-level. Similarly, from our data, we selected genes which had one probe indicating differential splicing index (q-value < 0.001) but not found to be differentially expressed when analyzed at overall gene-level. Considering as background population of genes all the gene symbols that could be mapped to isoform names and Ensemble gene Ids, we calculated the statistical significance of the overlap of these two gene lists using the hypergeometric test. Experimental validation of differential splicing results. We have selected 11 genes in total, to be either differentially spliced between basal-like tumours and normal samples (8 genes: AURKA, AURKB, BCL2-a, NEK2, RRM2, TGFBR1, UBE2C, ZBTB16) or differentially spliced in basal-like tumours, between patients with respectively better and worse outcome (4 genes: CCR7, RASSF5, PARP12, TGFBR1). Full length cDNAs were prepared from intact RNA using Primescript Reverse transcriptase (Clontech), oligo dT and a custom transcript switching Oligo (TSO). cDNAs were amplified using semi-nested PCR using the Advantage PCR kit (Clontech) with gene specific primers located near the poly adenylation signal and TSO. Amplified full length cDNAs were analysed using Bioanalyzer DNA7500 kit to size separate all full length isoforms arising from each gene (Additional File 8).
## Analysis of cellular functions and pathways.
We evaluated what cellular functions and pathways were affected at the gene splicing balance or gene expression levels. We analyzed separately a number of gene lists derived from comparative analyses of differential splicing index (splicing imbalance) and differential gene Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 expression, using gene set enrichment analyses based on Fisher-test. The gene sets we used were extracted from the Ingenuity (www.ingenuity.com) as well as the MSigDB data base [bib_ref] Molecular signatures database (MSigDB) 3.0, Liberzon [/bib_ref].
Ingenuity gene sets were used for the analysis of the list of genes differentially spliced (therefore had differential splicing index values) but not differentially expressed, between basal-like tumours and breast normal tissues. MSigDB was used for all comparative lists. For any gene list the Fisher-test assessed the probability that the number of overlapping genes between our gene list and the pre compiled gene set would happen by chance. The background population for the test consisted of all genes represented on the Affymetrix GeneChip Exon 1.0 ST platform. Classification models. Classification models were built to assess the diagnostic potential of three different levels of information that can be extracted from Exon Array: gene expression, exon expression, and splicing index. An additional data type was used consisting of all splicing indexes of the exons of genes not differentially expressed between the categories to be classified (i.e. the q value for difference in the expression of that gene was greater than 0.01). The classification model was, in all cases, based on decision trees as implemented in the R package "tree" (http://cran.r-project.org/web/packages/tree/index.html). For each data type the procedure used was the same: (1) n number of variables were selected randomly from the data matrix (for example gene expression values) (2) we randomly assigned two thirds of biological samples to be the training samples. Those are used to calibrate the model using the n variables. (3) the model is then used to classify the remaining third of samples (the test samples). For every number n of variables this procedure (steps 1-3) is repeated 1000 iterations always randomly selecting the test and training samples, as well as the specific n variables to use. From those classifications we calculated sensitivity and selectivity associated to each model.
## Survival analysis. kaplan-meier analysis was used for calculation and visualization of survival curves, and
Cox-regression models followed by Wald test were used to determine the statistical association between the expression of each GE, EE, SI value and breast cancer specific survival (BCSS). Two different Cox-regression models were used, with or without consideration of the percentage of lymphocytic infiltration as an additional covariate. To adjust for multiple testing, false discovery rate (FDR) q-values were calculated from the Wald test p-values, using Benjamin-Hochberg method. We considered GE, EE, SI to be associated with BCSS using FDR q-value < 0.1. Distributions of the resulting p-values were compared with random uniform distribution (from 0 to1), representing p-values that would be obtained by chance. With all models the obtained p-values were clearly deviating from uniform distribution with an overall bias towards low p-values, and therefore deviating from the results that would be obtained by chance (Additional File 12). Percentage of lymphocytic infiltration covariate was used as a categorical variable, as follows: < 15% = "low", > = 15% = "high". All analyses were run using R software and the 'survival' R package (http://cran.r-project.org/web/packages/survival/index.html).
Annotation of Lymphocytic associated Genes. Genes were annotated as lymphocytic according to two criteria: i) expression in lymph node tissues, based on the arbitrary threshold of at least 10 read counts from the normalized RNA-Seq data set deposited in the Array Express database, data set E-MTAB-513 (http:// www.ebi.ac.uk/gxa/experiments/E-MTAB-513) ii) the gene was present in any of the following data sets from the MSigDb 72 : "BIOCARTA_BLYMPHOCYTE_PATHWAY","REACTOME_IMMUNOREGULATORY_ INTERACTIONS_BETWEEN_A_LYMPHOID_AND_A_NON_LYMPHOID_CELL","SIG_PIP3_ SIGNALING_IN_B_LYMPHOCYTES"."PID_LYMPHANGIOGENESIS_PATHWAY","LYMPHOCYTE_ DIFFERENTIATION","POSITIVE_REGULATION_OF_LYMPHOCYTE_ACTIVATION","REGULATION_ OF_LYMPHOCYTE_ACTIVATION", "LYMPHOCYTE_ACTIVATION".
Availability of supporting data. Patient clinical and pathological information used for the analyses, are reported in and include the patients' survival data, age at diagnosis, tumour grade, percentage of lymphocytic infiltration, estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER2) status.
Microarray data have been deposited in GEO public repository with ID GSE40267 (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc= GSE40267).
[fig] Figure 1: Exon expression, gene expression and splicing balance values of an exemplary three-exons gene in two samples. Panel (A): a multi exon gene model. Panel (B): illustration of a case of two samples expressing the gene with different balances of exons. Panel (C): Gene and exon level measures of expression. In the example, the two samples have equal gene expression (GE), but different splicing balances, as detected by different exon-level contributions to the overall gene expression (D): The Splicing Index (SI) metric used to quantify splicing imbalances is explained, showing the different contribution of each exon to the total gene expression. Note that SI, unlike exon expression, is invariant to total gene expression. This exemplifies how SI captures the splicing balance level of information, different than gene or exon expression. Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 [/fig]
[fig] Figure 2: Pairwise transcriptional differences between tumour subtypes and NBT. Each bar represents the results of comparison between two groups of samples. Key: B = basal-like, H2 = HER2, La = LuminalA, Lb = LuminalB, N = Normal like, O = Others (non-basal-like rumour), NBT = normal breast tissue. The relative fractions of genes differentially expressed, with differential splicing balances or both are reported in different colours. The absolute numbers of genes falling into each of these categories is also reported. Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 [/fig]
[fig] Figure 3: The integrin signalling pathway (Ingenuity ® Systems). In purple are genes affected by splicing imbalances between basal-like tumours vs NBT, with no evidence of whole-gene differential expression.Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 receptors showing up again as specifically deregulated at splicing levels in basal-like tumours (complete results are listed in Additional File 9). [/fig]
[fig] Figure 4: Heatmap of gene sets and pathways specifically perturbed by splicing imbalances. Each column represents a pairwise comparison (either at GE or SI level), each row is a gene set or a pathway, and color-coded is the significance level of the enrichment. On the left are represented the complete results of the analysis. On the right, a selection of specific gene sets and pathways is highlighted showing specific perturbations related to cancer, immune system, and transport and membrane. All reported gene sets and pathways are significantly enriched in at least one of the three pairwise comparisons illustrated. Gene sets are grouped in five different classes as indicated by the side colour bar.Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 [/fig]
[fig] Figure 5: Association with breast cancer specific survival. Number of genes where GE or SI are associated with breast cancer specific survival in basal-like tumour patients. Scientific RepoRts | 7:40177 | DOI: 10.1038/srep40177 [/fig]
[fig] Figure 6: Gene models and Kaplan-Meier curves. CCR7 and TGFBR1 genes and their association with breast cancer specific survival. For each panel, on the top is a schematic representation of the gene model (from the UCSC Genome Browser). Highlighted in green are probes where the SI could be associated with survival. [/fig]
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PGAM1, regulated by miR-3614-5p, functions as an oncogene by activating transforming growth factor-β (TGF-β) signaling in the progression of non-small cell lung carcinoma
# Background
Globally, lung cancer is the most prevalent malignant tumor and accounts for approximately 27% of all cancerrelated deaths [bib_ref] Cancer treatment and survivorship statistics, Miller [/bib_ref]. Lung cancer is generally divided into two histological subtypes: small cell lung cancer and nonsmall-cell lung cancer (NSCLC), which accounts for approximately 80% of all lung cancers 2 . Despite great effort has been made, NSCLC patients with advancedstage disease exhibit high mortality after resection due to metastatic lesions or local recurrence [bib_ref] Lung cancer: current therapies and new targeted treatments, Hirsch [/bib_ref]. Thus, elucidating the molecular mechanisms underlying NSCLC progression and metastasis is crucial for the treatment of the disease.
Recently, phosphoglycerate mutase 1 (PGAM1), a key aerobic glycolysis enzyme, has been identified to be frequently upregulated and involved in the tumorigenesis of different types of human cancers, such as hepatocellular carcinoma, oral squamous cell carcinoma, glioma, urothelial bladder cancer, renal clear cell carcinoma and colorectal cancer [bib_ref] Correlation of expression levels of ANXA2, PGAM1, and CALR with glioma grade..., Gao [/bib_ref] [bib_ref] Expression of PGAM1 in renal clear cell carcinoma and its clinical significance, Li [/bib_ref] [bib_ref] Identification of potential genes/proteins regulated by Tiam1 in colorectal cancer by microarray..., Liu [/bib_ref] [bib_ref] Proteomics identification of PGAM1 as a potential therapeutic target for urothelial bladder..., Peng [/bib_ref] [bib_ref] Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target..., Ren [/bib_ref] [bib_ref] Phosphoglycerate mutase 1 predicts the poor prognosis of oral squamous cell carcinoma..., Zhang [/bib_ref]. In addition, proteomic analysis results indicated that PGAM1 was highly expressed in NSCLC [bib_ref] Proteome analysis of human lung squamous carcinoma, Li [/bib_ref]. Moreover, Sun et al. revealed that high PGAM1 expression was associated with reduced patient survival, and PGAM1 silencing suppressed aggressive tumor phenotypes and mTOR-dependent glycolysis of NSCLC cell [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref]. Interestingly another novel PGAM1 allosteric inhibitor, HKB99, has been reported to suppress tumor growth and metastasis and overcome drug resistance in NSCLC [bib_ref] A novel allosteric inhibitor of phosphoglycerate mutase 1 suppresses growth and metastasis..., Huang [/bib_ref]. Together, these work provides a preclinical proof of concept for PGAM1 as a novel candidate target in NSCLC. However, the relationship between PGAM1 expression and prognosis in NSCLC and the functional role and regulatory network of PGAM1 remain largely unknown.
MicroRNAs (miRs, miRNAs) are small noncoding single-stranded RNAs that lack protein-encoding functions. MiRNAs can bind to the 3′ untranslated regions (3′-UTRs) of their target mRNAs to suppress protein expression [bib_ref] MicroRNA biogenesis and cancer, Gregory [/bib_ref]. Mounting evidence has indicated that miRNAs play crucial roles in the progression of most human cancers by acting as oncogenic RNAs or tumor suppressors [bib_ref] MicroRNA expression and function in cancer, Garzon [/bib_ref]. MiR-3614-5p, with a length of 24 nt, is located on chromosome 17q22. It has been reported that miR-3614-5p can combat dengue virus by regulating the action of adenosine deaminase acting on RNA 1 (ADAR1) in human macrophages [bib_ref] MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p..., Diosa-Toro [/bib_ref]. Another report indicated a close relationship between dysregulated miR-3614-5p expression and autoimmune disease risk [bib_ref] Evidence for a potential role of miR-1908-5p and miR-3614-5p in autoimmune disease..., Wohlers [/bib_ref]. Additionally, overexpression of miR-3614-5p dramatically inhibited the proliferation capacity of breast cancer cells [bib_ref] Blockade of miR-3614 maturation by IGF2BP3 increases TRIM25 expression and promotes breast..., Wang [/bib_ref]. Bioinformatics analysis indicated that miR-3614-5p may suppress WNT signal pathway through targeting NFATC2 in NSCLC cell [bib_ref] Pseudogene CHIAP2 inhibits proliferation and invasion of lung adenocarcinoma cells by means..., Shang [/bib_ref]. Nevertheless, whether miR-3614-5p plays a role in the development and progression of NSCLC remains largely unclear.
In the present study, we demonstrated that PGAM1 was significantly upregulated in tumor tissues and associated with poor clinical prognosis of NSCLC. Functional experiments revealed that PGAM1 could promote the proliferation and invasion of NSCLC cell lines. Moreover, we discovered that PGAM1 was inhibited by miR-3614-5p and could reverse miR-3614-5p's inhibitory effects on NSCLC tumorigenesis. An investigation of the mechanism found that the transforming growth factor-β (TGF-β) signaling pathway was regulated by the miR-3614-5p/PGAM1 axis. Finally, in vivo experiments further validated that miR-3614-5p inhibited NSCLC tumor growth by targeting PGAM1. Taken together, our findings reveal a critical role of the miR-3614-5p/PGAM1 axis in mediating TGF-β signaling pathway activation in NSCLC progression.
# Results
PGAM1 mRNA is significantly upregulated and correlated with the prognosis of NSCLC patients First, we determined that PGAM1 mRNA expression were markedly high in most solid cancer tissues through TCGA data analysis [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref]. We also confirmed that the other isoform PGAM2 was negatively expressed in NSCLC tissues [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref]. The PGAM1 mRNA levels in the NSCLC tissues lines were significantly higher than those in normal tissues, as confirmed by RT-PCR analyses in 30 paired NSCLC specimens [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref]. In addition, a similar result was also observed in the TCGA [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref] and several GEO NSCLC cohorts [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref]. In addition, we found that there was a significant positive correlation between the PGAM1 and advanced TNM stage [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref] and tumor proliferation marker (Ki-67 and PCNA) expression levels (Supplementary [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref]. To explore the prognostic value of PGAM1, the overall survival (OS) and disease-free survival (DFS) rates were analyzed in TCGA cohorts and three additional independent GEO cohorts. The Kaplan-Meier analysis results showed that both OS and DFS time were significantly shorter in patients with high-PGAM1-expression than in patients with low-PGAM1expression in almost all NSCLC cohorts [fig_ref] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis [/fig_ref]. Additionally, a ROC curve was used to evaluate the diagnostic value of PGAM1, and we found that the proportion of PGAM1 under the ROC curve (AUC) was 0.79 (TCGA), 0.879 (GSE19188), 0.746 (GSE7670) and 0.774 (GSE10072), respectively (Supplementary . Collectively, these results revealed that PGAM1 was significantly upregulated, and potentially represents a novel prognostic and diagnostic biomarker for NSCLC patients.
## Upregulation of pgam1 protein levels predicts unfavorable prognosis in nsclc
We further confirmed the dysregulated PGAM1 expression in NSCLC at the protein level. As shown in [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref] , PGAM1 expression was dramatically increased in NSCLC tissues when compared to their normal counterparts. In addition, the relationship between PGAM1 expression and clinical prognosis in two independent NSCLC tissue microarray (TMA) cohorts (ZZU cohort and Outdo cohort) was evaluated through IHC assay. PGAM1 expression was scored from 1+ to 5+ according to the staining intensity of each specimen [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref]. The results confirmed that PGAM1 displayed markedly higher expression in NSCLC tissues than in in noncancerous tissues, and the immunoreactivity of PGAM1 was mostly distributed in both the cytoplasm and cell membrane [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref]. Moreover, we found that high PGAM1 expression was significantly correlated with advanced TNM stage and lymph node metastasis [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref]. Furthermore, consistent with the results observed in the public genome cohort, strong PGAM1 IHC staining was significantly associated with poor survival in both the ZZU and Outdo cohorts [fig_ref] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in... [/fig_ref].
Furthermore, univariate and multivariate analysis suggested that PGAM1 expression, lymph node metastasis and TNM stage were independent predictors of OS in patients with NSCLC [fig_ref] Table 1: Clinicopathological characteristics and expression of PGAM1 in ZZU NSCLC cohort [/fig_ref]. Taken together, these findings strongly suggested that high PGAM1 expression status was positively associated with poor prognosis and might serve as a prognostic biomarker in NSCLC patients.
## Pgam1 promotes nsclc cell growth, migration and invasion in vitro
The above findings prompted us to elucidate the potential biological function of PGAM1 in NSCLC cells. Initially, we detected PGAM1 expression in NSCLC cell lines. The results showed that PGAM1 was highly expressed in NSCLC cell lines compared with normal lung cells at both the mRNA and protein levels . To evaluate the effects of PGAM1 on aggressive tumor phenotypes in NSCLC cells, loss-and gain-of-function experiments were conducted. First, the transfection efficacy of PGAM1 siRNA was confirmed . CCK-8 and EdU assays indicated that PGAM1 silencing significantly inhibited the proliferation capacity and DNA synthesis rate of NCI-H226 and SK-MES-1 cells , e). Similarly, colony-formation ability was reduced after PGAM1 silencing . In addition, transwell and b Representative expression pattern of PGAM1 with different IHC staining score. c Distribution of PGAM1 IHC staining score in NSCLC tissue samples or non-tumor tissues. d, e Representative expression pattern of PGAM1 in NSCLC tissue samples from patients with different TNM stages or with absent/present lymph node metastasis. Kaplan-Meier analysis of the OS rate in NSCLC patients in ZZU cohort (f) and Outdo cohort (g). *p < 0.05, **p < 0.01.
wound healing assays indicated that the invasion and migration capacities of NSCLC cells in the PGAM1 silencing groups were markedly reduced compared to those in the control group .
Furthermore, to further confirm whether PGAM1 silencing stimulated cell apoptosis, we performed a TUNEL assay, and the findings suggested that knockdown of PGAM1 potently augmented the apoptosis of NCI-H226 and SK-MES-1 cells . Consistently, PGAM1 knockdown largely reduced the expression of invasion-related proteins MMP2/7/9 , increased the expression of the proapoptotic protein BAX, cleaved caspase 3 and cytochrome C and suppressed the expression of the anti-apoptotic protein Bcl-2 (Supplementary . Conversely, PGAM1 overexpression promoted the proliferation and invasion of NSCLC cells [fig_ref] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway [/fig_ref]. Collectively, these results indicated the crucial role of upregulated PGAM1 expression in facilitating NSCLC cell growth and metastasis.
## Unregulated pgam1 enhances tumorigenicity in vivo
To further elucidate the oncogenic role of PGAM1 in vivo, stable PGAM1 knockdown or overexpression cells and control A549 cells were injected subcutaneously into BALB/c nude mice model (6 mice per group). The tumor volume and luciferase signal were measured on a weekly basis. After 5 weeks, we observed that the mice injected with PGAM1-knockdown A549 cells had reduced tumor weight and volume compared with those in mice injected with control cells . Consistently, the bioluminescence signal of tumors formed by the sh-PGAM1 cells was significantly decreased . Furthermore, PGAM1 and Ki-67 were significantly weaker in the PGAM1-silenced xenograft tumors than in control xenograft tumors . Conversely, as shown in -j, tumors formed by PGAM1-overexpressing A549 cells were heavier and larger than tumors formed by control cells . Furthermore, IHC analysis revealed that PGAM1-overexpressing tumors showed higher percentages of Ki-67-positive cells . Collectively, our findings emphasize the role of oncogenic PGAM1 in NSCLC progression in vivo.
## Pgam1 overexpression promotes the tgf-β signaling pathway
The underlying mechanism by which PGAM1 contributes to NSCLC progression remains unclear. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set variation analysis (GSVA) to explore PGAM1-related downstream pathways in the TCGA NSCLC dataset. We found that the TGF-β signaling pathway was closely correlated with PGAM1 overexpression [fig_ref] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway [/fig_ref]. We further confirmed the significant activation of the TGF-β signaling pathway, cell adhesion molecules and focal adhesion pathways in PGAM1-high-expression NSCLC tissues by gene set enrichment analysis (GSEA) analysis, indicating that the TGF-β signaling pathway may be involved in the oncogenic role of PGAM1 in NSCLC [fig_ref] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway [/fig_ref]. To test this hypothesis, the expression of TGF-β signaling pathway related molecules (TGF-β, BMP4, ICAM1 and VCAM1) was determined through qRT-PCR and western blotting and the results showed that the expression levels of these molecules were significantly downregulated in PGAM1 silencing NSCLC cells [fig_ref] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway [/fig_ref]. Consistently, IHC staining of xenograft tumor tissues using TGF-β, BMP4, ICAM1 and VCAM1 antibodies indicated suppression of the TGF-β signaling pathway status in PGAM1 silencing cancer cells compared to that in the control group [fig_ref] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway [/fig_ref]. Moreover, it has been suggested that the TGF-β signaling pathway can induce the epithelial-mesenchymal transition (EMT) process in NSCLC cells [bib_ref] Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial-mesenchymal transition in non-small-cell lung..., Wang [/bib_ref]. Thus, we further investigated whether PGAM1 affects EMT in NSCLC cells. As expected, EMTrelated gene sets were significantly enriched in the PGAM1-high expression phenotype, suggesting that PGAM1 may contribute to TGF-β-induced EMT of NSCLC cells . Furthermore, PGAM1 silencing led to an increased E-cadherin expression and reduced N-cadherin, Snail and Slug expression in NCI-H226 and SK-MES-1 cells (Supplementary . Collectively, our results reveal that PGAM1 activates the TGF-β signaling pathway and induces EMT during NSCLC tumorigenesis.
## Pgam1 is a direct target of mir-3614-5p
We next identified the molecular mechanisms responsible for PGAM1 overexpression in NSCLC. Numerous studies have identified that miRNAs exert critical roles in cancer progression by regulating target genes [bib_ref] MicroRNA expression and function in cancer, Garzon [/bib_ref]. Seven candidate miRNAs targeting PGAM1 were selected based on an online prediction tool (StarBase 3.0) and expression status . Subsequently, we further performed validation experiments in vitro and found that miR-3614-5p expression significantly reduced PGAM1 mRNA levels . It has been reported that miR-3614-5p functions as a tumor suppressor in breast cancer [bib_ref] Blockade of miR-3614 maturation by IGF2BP3 increases TRIM25 expression and promotes breast..., Wang [/bib_ref]. We first confirmed the interaction between miR-3614-5p and PGAM1 with the sites predicted . We also found that miR-3614-5p expression was significantly decreased in NSCLC tissues . Consistently, miR-3614-5p expression was inversely associated with mRNA levels and strong IHC staining intensive of PGAM1 in NSCLC tissues . Moreover, we found that knockdown of miR-3614-5p significantly increased PGAM1 expression, whereas enforced expression of miR-3614-5p significantly decreased PGAM1 expression in NSCLC cells . Additionally, dual reporter luciferase assays indicated that the miR-3614-5p mimic significantly decreased the luciferase activity of PGAM1-wt but not PGAM1-mut . RIP assay results showed that miR-3614-5p and PGAM1 were both enriched in Ago2-coated beads relative to the IgG control group, further indicating the direct binding between miR-3614-5p and PGAM1 in NCI-H226 and SK-MES-1 cell lines . Additionally, given the previous reports that HIF1α could promote PGAM1 expression [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref] , we further determined the potential effects of hypoxic condition on miR-3614-5p/PGAM1 axis. The results indicated that miR-3614-5p regulated the PGAM1 expression in a hypoxia-independent manner (Supplementary .
Moreover, although there was no significant relationship between miR-3614-5p expression level and NSCLC clinical outcome , patients with a profile of miR-3614-5p high /PGAM1 low profiles had a better survival rate that patients with miR-3614-5p low / PGAM1 high profiles . Overall, these results indicated that PGAM1 is a direct functional target of miR-3614-5p in NSCLC.
The miR-3614-5p/PGAM1 axis promotes NSCLC progression via the TGF-β signaling pathway Given the potential tumor suppressive role of miR-3614-5p in cancer, we further explored whether PGAM1 mediated the tumor suppressive role of miR-3614-5p in (see figure on previous page) PGAM1 knockdown inhibit NSCLC cell proliferation and invasion. a, b The mRNA and protein expression levels of PGAM1 in bronchial epithelial cell 16HBE and NSCLC cell lines (A549, H1299, NCI-H226 and SK-MES-1) were analyzed by RT-qPCR (up panel) and western blot (lower panel). c NCI-H226 and SK-MES-1 cells were untreated (Blank), transfected with negative control (shCtrl), or transfected with shRNAs targeting PGAM1 (sh-PGAM1-1/2/3). The expression of PGAM1 protein was analyzed by western blot 48 h later. The representative result of three independent experiments was shown. Cell proliferation capability of NCI-H226 and SK-MES-1 cells transfected with shCtrl or shPGAM1-1/2 was determined by CCK-8 assay (d), EdU assay (e) and colony formation assay (f). Transwell experiment (g) and wounding healing assay (h) was performed to analyze the cell invasion and migration capability of NCI-H226 and SK-MES-1 cells transfected with shCtrl or shPGAM1-1/2. The results are presented as the mean ± SD. *p < 0.05, **p < 0.01 by Mann-Whitney U-test. PGAM1 promotes the proliferation of NSCLC cells in vivo. A549 cells infected by lentiviral to achieve PGAM1 stable knockdown (sh-PGAM1) or PGAM1 overexpression (PGAM1) or infected by negative control (shCtrl or MOCK, respectively) were implanted into the nude mice and tumor growth was recorded. Tumor weight in nude mice was assessed at week 5 (a, b) and tumor volume (c) was determined based on tumor size measured every week in nude mice from shCtrl or shPGAM1 group. Representative bioluminescent images and quantification of bioluminescent imaging signal intensities in nude mice from shCtrl or shPGAM1 group (d). Representative images of HE staining, Ki-67 and PGAM1 IHC staining of tumor tissues obtained from nude mice from shCtrl or shPGAM1 group. Scale bar = 100 μm (e). Tumor weight in nude mice was assessed at week 5 (f, g) and tumor volume (h) was determined based on tumor size measured every week in nude mice from MOCK or PGAM1 group. Representative bioluminescent images and quantification of bioluminescent imaging signal intensities in nude mice from MOCK or PGAM1 group (i). Representative images of HE staining, Ki-67 and PGAM1 IHC staining of tumor tissues obtained from nude mice from MOCK or PGAM1 group. Scale bar = 100 μm (j). The results are presented as the mean ± SD for each group (n = 6). *p < 0.05, **p < 0.01 by Mann-Whitney U-test. NSCLC. Ectopic expression of miR-3614-5p markedly suppressed PGAM1 expression, while co-transfection of miR-3614-5p with the PGAM1 overexpression plasmid mildly rescued the PGAM1 expression in NSCLC cell lines . Functional experiments showed that reintroduction of PGAM1 could partially reverse the inhibition of NSCLC cell proliferation ability caused by the miR-3614-5p overexpression . In addition, PGAM1 counteracted the decrease in cancer cell metastasis potential induced by ectopic expression of miR-3614-5p, as revealed by the transwell and migration assay . Furthermore, the expression of TGF-β signaling pathway related molecules was decreased after enforced expression of miR-3614-5p in NSCLC cells, while reintroduction of PGAM1 abolished the suppressive effect of miR-3614-5p mimics on the TGF-β signaling pathway .
Additionally, as PGAM1 is an important enzyme in the aerobic glycolysis process, we further confirmed the effect of miR-3614-5p/PGAM1 axis on NSCLC cell glucose metabolism [bib_ref] A novel allosteric inhibitor of phosphoglycerate mutase 1 suppresses growth and metastasis..., Huang [/bib_ref] [bib_ref] Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing..., Hitosugi [/bib_ref]. Taken together, these findings revealed that the miR-3614-5p/PGAM1 axis promotes NSCLC progression, at least in part, via the TGF-β signaling pathway.
## Mir-3614-5p inhibits tumor growth in an nsclc xenograft model
We further confirmed the suppressive role of miR-3614-5p in an NSCLC xenograft model. We subcutaneously inoculated A549 cells with overexpression of miR-3614-5p subcutaneously into nude mice. The results showed that mice injected with miR-3614-5p-overexpressing A549 cells (lenti-miR-3614-5p group) exhibited markedly reduced transplanted tumor weight and volume of transplanted tumors compared to that in the control group (lenti-MOCK) . Consistently, in vivo live imaging indicated that ectopic expression of miR-3614-5p significantly reduced photon flux . Additionally, IHC analysis demonstrated that PGAM1 and Ki-67 expression was significantly weaker in the lenti-miR-3614-5p group than in the lenti-MOCK group . Moreover, consistent with the results observed after PGAM1 knockdown, the TGF-β signaling pathway was significantly suppressed in mouse tumors formed by miR-3614-5p-overexpression cells . These results further demonstrate that miR-3614-5p expression suppresses NSCLC tumor growth by inhibiting PGAM1 through attenuating the TGFβ signaling pathway .
# Discussion
PGAM1 is a critical enzyme in coordinating glycolysis and biosynthesis [bib_ref] Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing..., Hitosugi [/bib_ref]. Emerging evidence indicates that PGAM1 is frequently upregulated and promotes the progression of several cancers [bib_ref] Expression of PGAM1 in renal clear cell carcinoma and its clinical significance, Li [/bib_ref] [bib_ref] Proteomics identification of PGAM1 as a potential therapeutic target for urothelial bladder..., Peng [/bib_ref] [bib_ref] Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target..., Ren [/bib_ref] [bib_ref] Phosphoglycerate mutase 1 predicts the poor prognosis of oral squamous cell carcinoma..., Zhang [/bib_ref] [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref] [bib_ref] Phosphoglycerate mutase 1 (PGAM1) promotes pancreatic ductal adenocarcinoma (PDAC) metastasis by acting..., Liu [/bib_ref] [bib_ref] S1P/S1PR3 axis promotes aerobic glycolysis by YAP/c-MYC/ PGAM1 axis in osteosarcoma, Shen [/bib_ref]. In NSCLC, Sun et al. revealed that PGAM1 was significantly upregulated and that elevated PGAM1 was positively correlated with poor survival [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref]. Consistently, we determined the elevated expression of PGAM1 both at the mRNA and protein levels by combined analysis of publicly available datasets and our cohort, which was consistent with the results observed by proteomic analysis [bib_ref] Proteomic analysis of secreted proteins of non-small cell lung cancer, Huang [/bib_ref]. Additionally, PGAM1 in NSCLC tissues had a relatively high diagnostic efficacy. We also provide solid clinical evidence that PGAM1 overexpression in NSCLC correlated with poor prognosis in several independent NSCLC cohorts. Together, these findings and ours suggest that PGAM1 might serve as a potential diagnostic and prognostic biomarker for NSCLC patients.
Evidence has shown that repression of PGAM1 expression could inhibits proliferation, metastasis and chemotherapy resistance in multiple cancers [bib_ref] Proteomics identification of PGAM1 as a potential therapeutic target for urothelial bladder..., Peng [/bib_ref] [bib_ref] Phosphoglycerate mutase 1 predicts the poor prognosis of oral squamous cell carcinoma..., Zhang [/bib_ref] [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref] [bib_ref] Phosphoglycerate mutase 1 (PGAM1) promotes pancreatic ductal adenocarcinoma (PDAC) metastasis by acting..., Liu [/bib_ref]. For instance, PGAM1 promotes pancreatic ductal adenocarcinoma proliferation and metastasis [bib_ref] Phosphoglycerate mutase 1 (PGAM1) promotes pancreatic ductal adenocarcinoma (PDAC) metastasis by acting..., Liu [/bib_ref]. In lung cancer, Sun et al. found that PGAM1 knockdown inhibited NSCLC xenografting tumorigenesis [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref]. In agreement with these findings, we demonstrated that the PGAM1 promoted the malignant biological behavior of NSCLC cells. Additionally, decreased DNA synthesis and activation of the apoptosis pathway were observed after PGAM1 silencing. Based on these findings, we propose a (see figure on previous page) PGAM1 expression is downregulated by miR-3614-5p directly targeting of the 3'-UTR of PGAM1. a The expression status of candidate miRNAs targeting PGAM1 were analyzed based on TCGA dataset (left panel). Pearson analysis of the correlation between PGAM1 and candidate miRNAs based on TCGA database (right panel). b Expression of PGAM1 was detected under treatment of candidate miRNA mimics in NCI-H226 cells. c PGAM1 was found for the potential regulatory targets of miR-3614-5p using prediction tool (Starbase 3.0). The PGAM1 mRNA levels were determined by real-time PCR analysis in 30 paired NSCLC tissues (d), and in GSE53882 (e) and GSE56036 (f) datasets. g Pearson analysis of the correlation between PGAM1 and miR-3614-5p expression in 30 paired NSCLC specimens. h Representative IHC staining of PGAM1 and miR-3614-5p in NSCLC tissues (left panel) and quantification of PGAM1 staining scores in NSCLC patients from ZZU cohort with high or low miR-3614-5p expression (right panel). Scale bars, 100μm. The PGAM1 expression levels were determined by real-time PCR analysis (i) western blot (j) after transfection with the miR-3614-5p mimics or negative control or after transfection with the miR-3614-5p inhibitor or negative control in NCI-H226 and SK-MES-1 cells. GAPDH served as an internal control. k Luciferase activity assays for luciferase reporters with wild-type or mutant PGAM1 3'-UTR were performed after co-transfected with miR-3614-5p mimics or miR-control in 293 T cells. The luciferase activity of each sample was normalized to Renilla luciferase activity. l RIP assays confirmed the binding status between miR-3614-5p and PGAM1 in untreated and treated NSCLC cell lines, respectively. The representative result of at least three independent experiments was shown. *p < 0.05; **p < 0.01. potential therapeutic strategy for NSCLC that involves targeting PGAM1. KH3, an allosteric PGAM1 inhibitor, has shown desirable drug-like properties with satisfactory efficacy and limited toxicity in pancreatic ductal adenocarcinoma [bib_ref] A novel allosteric inhibitor of phosphoglycerate mutase 1 suppresses growth and metastasis..., Huang [/bib_ref]. Interestingly another novel PGAM1 allosteric inhibitor, HKB99, has been reported to could suppress tumor growth and metastasis and overcome drug resistance in NSCLC [bib_ref] MicroRNA-421 confers paclitaxel resistance by binding to the KEAP1 3'UTR and predicts..., Duan [/bib_ref]. Taken together, these findings strongly suggest that targeting PGAM1 is a potential strategy for treating NSCLC.
A previous study demonstrated that PGAM1, as a critical metabolic enzyme involved in glycolysis and biosynthesis, contributes to cancer progression by regulating cancer metabolism [bib_ref] Phosphoglyceric acid mutase-1 contributes to oncogenic mTORmediated tumor growth and confers non-small..., Sun [/bib_ref] [bib_ref] Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing..., Hitosugi [/bib_ref]. Consistently, we also confirmed that PGAM1 knockdown potently inhibited glucose metabolism. However, whether and how PGAM1 facilitates NSCLC progression through other mechanisms remains a subject for further study. To elucidate this issue, comprehensive bioinformatics analysis was performed, and the results indicated a significant positive correlation between elevated PGAM1 and activation of the TGF-β signaling pathway. Moreover, functional experiments confirmed that PGAM1 knockdown significantly suppressed the activation of the TGF-β signaling pathway. Emerging evidences has shown that TGF-β signaling participates in a diverse set of cellular processes, including cell proliferation, apoptosis and metastasis [bib_ref] TGF-beta signaling in tumor suppression and cancer progression, Derynck [/bib_ref]. Previous publications also reported that TGF-β signaling plays a crucial carcinogenic role in NSCLC development by inducing EMT [bib_ref] Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial-mesenchymal transition in non-small-cell lung..., Wang [/bib_ref]. Consistently, our current study shows that PGAM1 augments EMT, which consequently strengthens the role of TGF-β signaling in the EMT process. These findings imply that PGAM1 could be involved in lung cancer tumorigenesis and progression, at least in part, by activating the TGF-β signaling pathway.
A large amount of evidence suggests that certain miR-NAs participate in the cancer progression by targeting distinct mRNAs [bib_ref] MicroRNA biogenesis and cancer, Gregory [/bib_ref]. miRNAs, such as miR-421, miR-125b, miR-195, miR-374a and miR-216a, can function as either tumor suppressors or oncogenes in NSCLC progression [bib_ref] Ferritin heavy subunit enhances apoptosis of non-small cell lung cancer cells through..., Biamonte [/bib_ref] [bib_ref] MicroRNA-421 confers paclitaxel resistance by binding to the KEAP1 3'UTR and predicts..., Duan [/bib_ref] [bib_ref] Long non-coding RNA HOTTIP promotes BCL-2 expression and induces chemoresistance in small..., Sun [/bib_ref] [bib_ref] miR-195 targets cyclin D3 and survivin to modulate the tumorigenesis of non-small..., Yu [/bib_ref] [bib_ref] Dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal..., Zhao [/bib_ref]. In this study, we identified PGAM1 as a functional target of miR-3614-5p, and confirmed miR-3614-5p regulated PGAM1 in a hypoxia-independent manner. To date, there has few reports addressing miR-3614-5p in human cancers. Wang, Z. et al. reported that miR-3614-5p functions as a tumor suppressive role through targeting TRIM25 in breast cancer [bib_ref] Blockade of miR-3614 maturation by IGF2BP3 increases TRIM25 expression and promotes breast..., Wang [/bib_ref] showed that that miR-3614-5p may suppress WNT signal pathway through targeting NFATC2 in NSCLC cell [bib_ref] Pseudogene CHIAP2 inhibits proliferation and invasion of lung adenocarcinoma cells by means..., Shang [/bib_ref]. Consistently, our functional experiments revealed that the tumor inhibition effect of miR-3614-5p is in part mediated by the downregulation of PGAM1 in vitro and in vivo. Furthermore, the suppressive effect of miR-3614-5p on the glucose metabolism and TGF-β signaling pathway was attenuated by ectopic expression of PGAM1. In addition, we found that NSCLC patients who with miR-3614-5p low /PGAM1 high profiles always had a poorer prognosis than those with miR-3614-5p high /PGAM1 low profiles. Together, these data identify miR-3614-5p as a novel determinant of PGAM1 expression and establish a novel miR-3614-5p/PGAM1/TGF-β signaling pathway for NSCLC progression.
# Conclusions
In conclusion, our findings further strengthened the conclusion that PGAM1 plays a critical oncogenic role in the progression of NSCLC. Mechanistic investigations suggested that PGAM1 was a functional target for miR-3614-5p. Furthermore, miR-3614-5p/PGAM1 axis regulated the malignant phenotype, at least in part, through activating the TGF-β signaling pathway in NSCLC. Overall, our data further emphasize the potential of miR-3614-5p/PGAM1 axis as a therapeutic target for NSCLC patients.
# Methods
## Expression data sets
Gene expression data for eight human lung cancer cohorts and corresponding clinical information were obtained from the Gene Expression Omnibus (GEO; GSE10072, GSE19188, GSE7670, GSE40791, GSE31320, GSE37745 and GSE42127) and The Cancer Genome Atlas (TCGA) database. For Kaplan-Meier analysis, patients were stratified into 'low' and 'high' expression based on auto-select best cutoff using R language (version 3.51), and the detailed codes were shown in Supplementary materials.
## Patient samples and tma
Two independent cohorts containing paraffin embedding lung cancer specimens were used in present study: (1) TMA cohort containing a total of 94 NSCLC and (see figure on previous page) MiR-3614-5p inhibits NSCLC cell proliferation and invasion in vitro by inhibiting PGAM1. NSCLC cells NCI-H226 or SK-MES-1 were transfected with negative control (Ctrl), miR-3614-5p mimics, or miR-3614-5p mimics & PGAM1 overexpression plasmid. a The protein expression levels of PGAM1 in different groups were analyzed by western blot 48 h. b Cell viability was assessed at indicated time points by CCK-8 assay, c EDU incorporation, d transwell assays and e wound healing assays were performed to evaluate the NSCLC cell proliferation and invasion in vitro. f The protein expression of TGF-β, BMP4, ICAM1 and VCAM1 were analyzed by western blot. The representative result of at least three independent experiments was shown. Results were shown as mean ± SD. *p < 0.05, **p < 0.01. adjacent normal tissues were purchased from Outdo Biotech (Shanghai, China) (Outdo cohort); (2) 85 pairs of NSCLC and adjacent normal tissues were collected from NSCLC patients who underwent surgical resection at the First Affiliated Hospital of Zhengzhou University from 2011 to December 2013 (ZZU cohort). The study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University. All patients provided written informed consent and the project was in accordance with the Helsinki Declaration of 1975.
## Cell lines and cell culture
Human bronchial epithelial cell 16HBE and NSCLC cell lines A549, H1299, NCI-H226, SK-MES-1 were purchased from Cell Bank of Chinese Academy of Sciences and maintained in RPMI 1640 (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) at 37°C in a humidified incubator with 5% CO 2 .
# Western blotting analysis
Cells were lysed with RIPA buffer (Beyotime, China) containing cocktail inhibitor (Roche, USA). The protein samples were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked and then incubated with primary antibodies overnight at 4°C. Specific antibodies used in present study are listed in [fig_ref] Table 1: Clinicopathological characteristics and expression of PGAM1 in ZZU NSCLC cohort [/fig_ref]. Subsequently, the membranes were incubated with corresponding secondary antibodies and visualized by ECL detection system as described in our previous study 34 .
## Luciferase activity assay and rna immunoprecipitation (rip)
The 3′-UTR fragment of PGAM1 containing the miR-3614-5p binding sequences were cloned into psiCHECK-2 vector (Promega, USA) with firefly luciferase reporter. Mutated plasmid was used as control. The cells were cotransfected with luciferase reporter construct, miR-3614-5p mimic or PGAM1 expression vector. Cells were collected after 24 h transfection and luciferase activity were measured using the Dual Luciferase Reporter Assay System (Promega, USA). The interaction between miR-3614-5p and PGAM1 was detected by performing RIP assay using anti-PGAM1 and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) according to its instructions as previous described [bib_ref] Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis..., Chen [/bib_ref].
## Immunohistochemical (ihc) staining
IHC was performed on 4 μm sections of paraffinembedded tissues to determine the expression level of PGAM1 protein as previous described [bib_ref] Cross talk between RNA N6-methyladenosine methyltransferaselike 3 and miR-186 regulates hepatoblastoma progression..., Cui [/bib_ref]. In brief, the TMA slides slides were incubated in PGAM1 antibody diluted 1:200 at 4°C overnight and carried out using the EnVision™ system (DAKO, Demark). The images of TMA slides were obtained using a the NanoZoomer system (Hamamatsu Photonics Inc., Germany).
Tumor formation assay in a nude mouse model A549 cells were subcutaneously injected into either side of the flank area of 6-week-old nude mice (n = 6 mice per group). Tumor volumes in mice were calculated (1/2 × length × width 2 ) on a weekly basis. After 5-week incubation, the mice were euthanized, and tumors were excised and subjected to IHC analysis. Tumor growth was monitored with tumor weight, and photographed by IVIS@ Lumina II system (Caliper Life Sciences, USA). Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Zhengzhou University.
RNA isolation and qRT-PCR analyses, knockdown and overexpression of PGAM1, oligonucleotides and transfection, cell proliferation assay, invasion and wound healing assay These assays were described in the Supplementary Materials and Methods section.
# Statistical analysis
Results are presented as means ± SD. Statistical analysis was performed in GraphPad Prism, version 5.0 (GraphPad Prism Software, CA), using the Mann-Whitney test for comparison of two groups. The analysis of correlation between factors was performed by Pearson's correlation coefficient rank test. Survival analyses were conducted using the Kaplan-Meier method, and the comparison was (see figure on previous page) MiR-3614-5p suppress the proliferation of NSCLC cells in vivo. A549 cells infected by lentiviral to achieve miR-3614-5p stable overexpression (Lenti-miR-3614-5p) or infected by negative control (Lenti-MOCK) were implanted into the nude mice and tumor growth was recorded. Tumor weight in nude mice was assessed at week 5 (a, b) and tumor volume (c) was determined based on tumor size measured every week in nude mice from Lenti-miR-3614-5p or Lenti-MOCK group. Representative bioluminescent images and quantification of bioluminescent imaging signal intensities in nude mice from Lenti-miR-3614-5p or Lenti-MOCK group (d). Representative images of HE staining, Ki-67 and PGAM1 IHC staining of tumor tissues obtained from nude mice from Lenti-miR-3614-5p or Lenti-MOCK group. Scale bar = 100 μm (e). Expression levels of TGF-β, BMP4, ICAM1 and VCAM1 in A549 cells transfected with Lenti-miR-3614-5p or Lenti-MOCK were analyzed by IHC staining (f). A proposed regulated axis between miR-3614-5p/PGAM1 axis driving TGF-β-induced EMT in NSCLC progression (g). The results are presented as the mean ± SD for each group (n = 6). *p < 0.05, **p < 0.01 by Mann-Whitney U-test.
performed using the log-rank test. Cox proportional hazards regression models were adopted for the univariate and multivariate analyses. p < 0.05 was considered to indicate a significant difference.
[fig] Figure 1: PGAM1 mRNA is upregulated in NSCLC based on TCGA analysis. PGAM1 mRNA expression levels in NSCLC tissues or non-tumor control tissues were analyzed by QT-PCR in 30 paired NSCLC specimens (a). PGAM1 mRNA expression levels were analyzed in LUAD and LUSC tissues from TCGA databases (b, c), and three independent GEO datasets (d). Expression of PGAM1 in different TNM stage (e). Overall survival (OS) and disease-free survival (DFS) rates analysis of the NSCLC patients in GSE42127 (f), GSE31210 (g), GSE37745 (h) and TCGA cohort (i, j). [/fig]
[fig] Figure 2: PGAM1 is upregulated in NSCLC tissues and associated with poor prognosis in TMA cohort. a Protein levels of PGAM1 in 8 paired NSCLC and matched adjacent non-tumor tissues were determined by Western blot assay. (N, matched adjacent non-tumor tissues; T, tumor tissues). [/fig]
[fig] Figure 5: PGAM1 promotes NSCLC progression through activating TGF-β signaling pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (a) and Gene set variation analysis (GSVA) (b) were conducted in TCGA dataset to explore PGAM1-related downstream pathways. c-e GSEA analysis the enrichment of the TGF-β signaling pathway related genesets in NSCLC tissues with different PGAM1 expression level. Expression levels of TGF-β, BMP4, ICAM1 and VCAM1 in A549 cells transfected with shCtrl or shPGAM1 were analyzed by real-time PCR (f), western blot (g) in vitro. h The xenografts were embedded in paraffin blocks and paraffin sections were examined by IHC staining. Scale bars, 100 μm. *p < 0.05, **p < 0.01. [/fig]
[table] Table 1: Clinicopathological characteristics and expression of PGAM1 in ZZU NSCLC cohort. LUAD lung adenocarcinoma, LUSC Lung squamous cell carcinoma. Bold values indicate statistical significance, p < 0.05. [/table]
[table] Table 2: Correlation of clinic-pathological features with PGAM1 expression in ZZU NSCLC cohort.Univariate and multivariate analysis of overall survival in ZZU LUSC cohort (n = 85)Bold values indicate statistical significance, p < 0.05. [/table]
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De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways
# Introduction
Chronic myeloid leukemia (CML) is a myeloproliferative disorder with an incidence of 1-2 cases per 100,000/year. It is characterized by the presence of the BCR-ABL1 fusion gene, the product of the reciprocal translocation between chromosomes 9 and 22. [bib_ref] Structural organization of the bcr gene and its role in the Ph'..., Heisterkamp [/bib_ref] After the translocation, the coding regions of BCR and ABL1 genes are juxtaposed, leading to an enhanced ABL1 tyrosine kinase activity. [bib_ref] Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of..., Daley [/bib_ref] CML is a multi-step disease, evolving from a mild form that is easy to control, called chronic phase (CP), to a very aggressive and incurable acute phase called blast crisis (BC). The majority of CML-CP patients are successfully treated with drugs able to impair BCR-ABL1 kinase activity (tyrosine kinase inhibitors, TKI), thus confirming the central role of the oncogenic fusion protein in CML pathogene-sis. [bib_ref] Multicenter independent assessment of outcomes in chronic myeloid leukemia patients treated with..., Gambacorti-Passerini [/bib_ref] [bib_ref] Long-Term Outcomes of Imatinib Treatment for Chronic Myeloid Leukemia, Hochhaus [/bib_ref] However, a fraction of these patients fail to respond to the treatment (primary resistance) or become resistant after an initial response. [bib_ref] Long-Term Outcomes of Imatinib Treatment for Chronic Myeloid Leukemia, Hochhaus [/bib_ref] [bib_ref] Chronic myeloid leukemia: mechanisms of blastic transformation, Perrotti [/bib_ref] [bib_ref] Molecular mechanisms of resistance to imatinib in Philadelphia-chromosome-positive leukaemias, Gambacorti-Passerini [/bib_ref] The persistence of BCR-ABL1 activity typically drives the progression to the advanced phase of the disease within 3-5 years. One of the open issues in CML concerns the dissection of the molecular mechanisms driving the transformation to BC, commonly considered as a heterogeneous disease at the molecular level. [bib_ref] A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML)..., Grossmann [/bib_ref] [bib_ref] Highdensity single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in..., Boultwood [/bib_ref] [bib_ref] Cytogenetic and molecular genetic evolution of chronic myeloid leukemia, Johansson [/bib_ref] BC is mainly characterized by the rapid expansion of the differentiation-arrested BCR-ABL1-positive blast cells, 10 therefore resembling an acute leukemia. In most cases of BC cases (approx. 70%), blasts maintain myeloid features, while in 20-30% blast lineage is lymphoid. BCR-ABL1 expression, which increases during CML progression in conjunction with BCR transcription, seems to have a prominent role in this process, hyperactivating proliferative and anti-apoptotic signals and inducing genetic instability. [bib_ref] Chronic myeloid leukemia: mechanisms of blastic transformation, Perrotti [/bib_ref] [bib_ref] BCR and BCR-ABL regulation during myeloid differentiation in healthy donors and in..., Marega [/bib_ref] Previous reports showed the existence of a heterogeneous molecular signature among distinct BC patients. [bib_ref] Chronic myeloid leukemia: mechanisms of blastic transformation, Perrotti [/bib_ref] [bib_ref] A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML)..., Grossmann [/bib_ref] [bib_ref] Highdensity single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in..., Boultwood [/bib_ref] However, these data were limited by the scarcity of matched CP/BC samples, due to the infrequent progression to BC after the advent of TKI. Here we analyzed ten paired CP/BC samples through a wholeexome sequencing (WES) approach, identifying somatic variants specific for BC progression since these were not present in the autologous CP controls. Along with several mutations previously identified as BC driver events, [bib_ref] Chronic myeloid leukemia: mechanisms of blastic transformation, Perrotti [/bib_ref] [bib_ref] A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML)..., Grossmann [/bib_ref] [bib_ref] BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros, Mullighan [/bib_ref] we detected, for the first time, recurrent BC-specific mutations occurring on the UBE2A gene. These data suggest that the appearance of UBE2A variants in CML cells could contribute to BC progression through the impairment of myeloid differentiation.
# Methods
## Cell lines
The BA/F3-BCR-ABL1 and 32Dcl3-BCR/ABL1 cell lines were generated and maintained as described by Puttini et al. [bib_ref] In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,..., Puttini [/bib_ref] K562 and 293FT were purchased from DSMZ (Braunschweig, Germany) and Thermo-Fisher-Scientific (Waltham, MA, USA) respectively, and were maintained according to the manufacturers' instructions.
## Patients
Diagnosis and staging were performed according to the World Health Organization WHO-2008 classification. [bib_ref] The 2008 revision of the World Health Organization (WHO) classification of myeloid..., Vardiman [/bib_ref] Peripheral blood (PB) or bone marrow (BM) of ten matched CML chronic phase/blast crisis samples, 31 CP-CML, 14 AP/BC-CML, 38 atypical-CML (aCML), and 40 AML were collected at diagnosis and after obtaining written informed consent approved by the institutional ethics committee. The study was conducted in accordance with the Declaration of Helsinki. Samples were prepared as described by Whole exome sequencing Genomic DNA (gDNA) was extracted from purified cells with PureLink Genomic DNA kit (Thermo-Fisher-Scientific). 1 μg of gDNA from each sample was fragmented (500bp) with a Diagenode-Bioruptor sonicator system (Diagenode, Belgium) and processed according to the standard Illumina protocol. The Illumina TruSeq Exome Enrichment kit (Illumina Inc., San Diego, CA, USA) was used to enrich the genomic libraries for the exonic regions and samples were sequenced as described in the Online Supplementary Appendix.
## Plasmids, transfections and lentiviral infections
BA/F3_BCR-ABL1-positive cells were transfected with pMIGR1_UBE2A vectors (Online Supplementary Appendix) as by Puttini et al. [bib_ref] In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,..., Puttini [/bib_ref] and were analyzed for GFP positivity with a FACSAria flow cytometer (BD Bioscience, San Jose, USA) and FACS-sorted when transfection efficiency was lower than 85%.
32Dcl3-BCR/ABL1 cells were electroporated using a Gene Pulser® II Electroporation System (BIORAD) with pMIGR1_UBE2A WT and I33M vectors as described by Puttini et al. [bib_ref] In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,..., Puttini [/bib_ref] To obtain stable UBE2A WT or I33M cell lines, GFP positive population was FACS-sorted with a MoFlo Astrios cell sorter equipped with Summit 6.3 software (both from Beckman Coulter, Miami, FL, USA).
For UBE2A silencing, K562 cells were infected with lentivirus obtained from MISSION-shRNA pLKO.1-based vectors (TRCN0000320625) (Sigma-Aldrich, Missouri, USA) and packaged using 293FT cell line. As a control, a pLKO.1MISSION nontarget control vector (SHC002) (Sigma-Aldrich) was used. After infection K562 cells were maintained in 2 µg/mL puromycin for selection of silenced (K562_shUBE2A) and control cells (K562_shNC).
## Quantitative real-time polymerase chain reaction
Total RNA was extracted using Trizol (Thermo-Fisher-Scientific) following the manufacturer's instructions. 1 μg of total RNA was used to synthesize cDNA using reverse transcription reagents (Thermo-Fisher-Scientific) after pre-treatment with DNAseI (Thermo-Fisher-Scientific) to avoid contamination from genomic DNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using TaqMan® Brilliant II QPCR Master Mix (Agilent Technologies, CA, USA) on a Stratagene-MX3005P (Agilent-Technologies) under standard conditions. The housekeeping gene glucoronidase β gene (GUSB) was used as an internal reference. [bib_ref] BCR and BCR-ABL regulation during myeloid differentiation in healthy donors and in..., Marega [/bib_ref] TaqMan® Gene Expression Assays (Thermo-Fisher-Scientific) were used (Online .
## In vitro translation and ubiquitination assay
In vitro translation of UBE2A proteins was performed with 1-
Step Human Coupled IVT Kit-DNA (Thermo-Fischer-Scientific) following the manufacturer's instructions. For ubiquitination assay we incubated 15 μg of UBE2A proteins with 1 μg of GST-Ubiquitin (Enzo-Life-Sciences, NY, USA), 0.2 ng of ubiquitin activating enzyme (E1) (Enzo-Life-Sciences), 2 mM ATP, energy regeneration solution (BostonBiochem, MA, USA), 2 mM MgCl2, 2 mM KCl, 16 µg of BA/F3_BCR-ABL1 whole cell lysate in 50 mM TrisHCl (ph7.5). The reactions were incubated for 20 minutes at 37°C. The products were analyzed by western blotting.
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machinery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and 50 µM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
## Neutrophilic differentiation
For induction of neutrophilic differentiation, 32Dcl3-BCR/ABL1 UBE2A WT and I33M cells were treated as previously described. [bib_ref] The effects of Bcr-Abl on C/EBP transcriptionfactor regulation and neutrophilic differentiation are..., Schuster [/bib_ref] 32Dcl3-BCR/ABL1 cells expressing UBE2A WT or I33M were seeded at a density of 2x10 5 cells per milliliter and cultured in the presence of imatinib mesylate (1 µM final concentration) in combination with human recombinant GCSF (10 ng/mL) or IL3 (0.5 ng/mL).
At days 3 and 6, cells were analyzed using FACS for CD11b surface expression and imaged with confocal microscopy (Online Supplementary Methods).
# Results
## Single nucleotide variants acquired during chronic myeloid leukemia progression
Genomic DNA (gDNA) from matched CP/BC samples was obtained for each patient at diagnosis (CP) and after [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref]. In one patient (patient #7) no acquired exonic SNV could be detected during CML progression. Analysis of SNV data showed the presence of two recurrently mutated genes in this cohort: ABL1, with mutations F486S, E255V and T315I occurring on the BCR-ABL1 fusion gene and leading to TKI resistance (30%, 95%CI: 0.574, 0.026), and UBE2A (Xq24), an E2-ubiquitin conju-gating enzyme required for post-replicative DNA damage repair 15 (20%, 95%CI: 0.447, 0.000), which has never been previously reported as mutated in CML patients. UBE2A mutations occurred on two non-contiguous residues: D114V and I33M [fig_ref] Table 2: UBE2A single nucleotide variants and indels identified in blast crisis samples and... [/fig_ref]. Patient #3 (male) showed an UBE2A variant frequency of 93%, as expected given that the gene is localized on the X chromosome. Patient #8 (female) carried a heterozygous UBE2A mutation (mutation ratio: 39%). The high mutation ratio observed in both patients suggests that UBE2A is present in the dominant BC clone .
## Ube2a mutations are recurrent and acquired in late chronic myeloid leukemia
The evidence of recurrent, somatic UBE2A mutations has never been reported in BC cases; however, they had been previously found in other clonal disorders both of solid and hematopoietic origin, confirming their potential role in tumor progression. [bib_ref] Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived..., De Miranda [/bib_ref] To further characterize the pattern and the frequency of UBE2A mutations in a larger cohort of patients, we sequenced 31 additional CML CP haematologica | 2019; 104(9)
## Ube2a mutations affect protein activity
Polyphen-2 (http://genetics.bwh.harvard.edu/pph/), 20 DANN 11 and FATHMM-MKL 21 analyses revealed that all the UBE2A variants identified were potentially damaging, as also suggested by the presence of a N-terminal frameshift variant (M34fs) in one of the patients [fig_ref] Table 2: UBE2A single nucleotide variants and indels identified in blast crisis samples and... [/fig_ref]. To gain insight into the functional role of UBE2A mutations, we stably transfected the BA/F3_BCR-ABL1 cell line [bib_ref] In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,..., Puttini [/bib_ref] with the wild-type (WT) and the mutated UBE2A variants, I33M and D114V. The level of UBE2A expression in stable transfectants was verified both at protein [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref] and mRNA [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref] levels.
The analysis of the levels of ubiquitin-conjugated H2A, a known UBE2A substrate, [bib_ref] The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain..., Sung [/bib_ref] in total cell lysate revealed a decreased H2A ubiquitination for both UBE2A variants compared to WT [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref] , with the effect of I33M being more prominent. In line with these findings, suggesting a decreased UBE2A activity for both variants, ubiquitination assay performed with in vitro translated WT and mutated UBE2A proteins confirmed a decrease in ubiquitin-conjugating activity for mutants compared to the WT form [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref]. To further support this indication, we developed a new in vitro assay based on the measurement of the AMP concentration as a proxy to assess the overall level of ubiquitination. This test was performed in the presence of GST-ubiquitin and of the E1 ubiquitin activating enzyme UBA1 together with WT or mutated UBE2A; this revealed a significant decrease in
## Transcriptome analysis of ube2a cellular models shows significant perturbation of downstream pathways related to myeloid development
To identify the gene networks perturbed by the UBE2A knock-out, stable lentiviral UBE2A silencing models were generated [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref] in the human myeloid K562 cell line (K562-shUBE2A and K562-shNC cells for UBE2A silencing and scrambled control, respectively) (Online Supplementary [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref]. Whole-transcriptome analysis (RNA-Seq) highlighted the presence of 168 differentially expressed genes, with 117 of them being down-regulated and 51 up-regulated [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref]. Gene set enrichment analysis (GSEA) showed significant enrichment for ontologies related to myeloid differentiation [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref] and neural development (Online Supplementary [fig_ref] Figure 3: Induction of erythroid differentiation in UBE2A-silenced K562 cell line [/fig_ref]. RT-qPCR on K562shNC/shUBE2A cell lines on a set of five differentially expressed genes (ITGB4, RDH10, CLEC11A, CSF3R, RAP1GAP) confirmed RNA-Seq data [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref]. Interestingly, the colony stimulating factor 3 receptor (CSF3R) was potently down-regulated in shUBE2A both at mRNA (12.5-fold downregulation; [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref] and protein [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref] levels, hence suggesting that its downmodulation may play a role in the differentiation block that is ultimately responsible for the onset of V. Magistroni et al. haematologica | 2019; 104(9)
## A b c d
the BC. Immunoblot analysis on CP/BC mononuclear cells from patient #3, which acquired the D114V-UBE2A mutation in BC phase, confirmed CSF3R downmodulation [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref]. To confirm the expression signature identified in the UBE2A silencing models, we stably overexpressed UBE2A WT and I33M in the 32Dcl3-BCR/ABL1 murine myeloid cell line (Online Supplementary [fig_ref] Figure 4: Induction of neutrophilic differentiation in UBE2A wild-type [/fig_ref]. In line with the expression profile shown in K562 UBE2A silenced cells, also in these cell lines we observed a comparable modulation in the previous analyzed set of five differentially expressed genes (ITGB4 [fig_ref] Figure 2: UBE2A silencing in K562 cells [/fig_ref] ; data are reported as fold-change in UBE2A I33M compared to UBE2A WT), therefore supporting the hypotheses that: 1) UBE2A mutations modulate the activity of the target protein in a loss of function manner; and 2) UBE2A mutations probably act as dominant negative variants. Comparison of our signature with known BC data (GEO _GSE47927 -HSC data were used for BC vs. CP calculation) indicated the presence of a moderate positive linear correlation (R2 = 0.234) (Online Supplementary . Notably, CSF3R expression level was seen to be markedly decreased also in the reference BC database, with a Log2 fold-change of -2.19. Globally, these data indicate that UBE2A mutations are directly responsible for the modulation of CSF3R, ITGB4, RDH10, CLEC11A and RAP1GAP expression. This hypothesis is also corroborated by the 32Dcl3 cell models.
## Ube2a activity is involved in myeloid differentiation
Erythrocytes and megakaryocyte differentiation can be induced in K562 cells by treating with hydroxyurea or phorbol 12-myristate 13-acetate (PMA), respectively. [bib_ref] Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factorkappa B pathway mediates phorbol 12-myristate..., Kim [/bib_ref] [bib_ref] Involvement of p38 kinase in hydroxyureainduced differentiation of K562 cells, Park [/bib_ref] Treatment of UBE2A-silenced K562 cells with hydroxyurea showed a significant delay in the ability to differentiate into erythrocytes, as assessed by glycophorin A (GYPA-CD235a) expression levels when compared with the scrambled control [fig_ref] Figure 3: Induction of erythroid differentiation in UBE2A-silenced K562 cell line [/fig_ref] (relative CD235a expression compared to shNC fold-change at day 0: 0.54±0.13, data are reported as fold-change in UBE2A I33M compared to UBE2A WT<0.001; day 1: 0.70 ± 0.12, data are reported as fold-change in UBE2A I33M compared to UBE2A WT <0.05; day 3: 0.61 ± 0.15, data are reported as fold-change in UBE2A I33M compared to UBE2A WT <0.05). Fluorescence-activated cell sorting analysis (FACS) showed a 45% decrease in GYPA surface expression in silenced cells compared to controls after 24 hours (h) of treatment [fig_ref] Figure 3: Induction of erythroid differentiation in UBE2A-silenced K562 cell line [/fig_ref]. In line with these findings, induction of hemoglobin-subunit-β (HBB) production was almost completely suppressed in shUBE2A cells [6.6-fold relative decrease of HBB mRNA level at 24 (h) of treatment: data are reported as fold-change in UBE2A I33M compared to UBE2A WT<0.001] further confirming the negative effect of UBE2A silencing on erythroid differentiation [fig_ref] Figure 3: Induction of erythroid differentiation in UBE2A-silenced K562 cell line [/fig_ref]. Similarly, treatment of K562 cells with the megakaryocytic-inducing agent PMA showed significant impairment of megakaryocyte differentiation in shUBE2A cells, as assessed by the expression levels of CD41 (33% downreg- . Neutrophilic differentiation was similarly tested in the 32Dcl3 BCR/ABL1 cell lines over-expressing UBE2A WT or I33M. Treatment of the UBE2A I33M cell line with GCSF + IL-3 showed a delay in neutrophilic differentiation, as assessed by CD11b expression levels when compared with both UBE2A WT or control [fig_ref] Figure 4: Induction of neutrophilic differentiation in UBE2A wild-type [/fig_ref]. Cells treated with IL-3 alone were used as an internal control. FACS showed no difference in CD11b surface expression in UBE2A I33M cell line compared to controls after three days of treatment but showed a 37% decrease at day 6 which was also confirmed by confocal microscopy analysis [fig_ref] Figure 4: Induction of neutrophilic differentiation in UBE2A wild-type [/fig_ref].
# Discussion
In line with previous results, 5,7,9 our analysis performed on matched CP/BC CML samples showed considerable somatic heterogeneity in BC phase. In all the samples, we detected a very low number of acquired SNV, corresponding to an average of 4.1 non-synonymous mutations per patient, a frequency far below the average reported for other hematopoietic neoplasms, such as acute myeloid leukemia (AML: 7.8) and chronic lymphocytic leukemia (CLL: 11.9). [bib_ref] Cancer genome landscapes, Vogelstein [/bib_ref] This can in part be explained by the characteristics of our analysis, where somatic variants occurring in BC were filtered against those in CP, therefore filteringout all the driver and passenger variants pre-existing the evolution to BC. All BC samples showed the prevalence of transition events and, in particular, of C:G>T:A substitutions, accounting for 66.7% of all the SNV (Online Supplementary [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref]. Approximately 85% of the C:G>T:A transitions were part of a CpG dinucleotide. Cytosines in CpG sites are known to be affected by a high mutation rate, caused by a spontaneous deamination of methylated cytosines. [bib_ref] Mutagenic deamination of cytosine residues in DNA, Duncan [/bib_ref] This mutation pattern is also in accordance with a BCR-ABL1 dependent mutation signature, characterized by inhibition of the mismatch repair system (MMR) and by accumulation of reactive oxygen species (ROS), as previously reported. [bib_ref] BCR/ABL inhibits mismatch repair to protect from apoptosis and induce point mutations, Stoklosa [/bib_ref] Mutations in RUNX1 and IKZF1, both involved in hematopoietic differentiation, have already been detected in the advanced stages of CML7 and are confirmed here as specific markers for BC progression. Along with this, the XPO1 gene (exportin-1) mutated here in a single patient with the E571K substitution, is also frequently mutated in clonal hematologic disorders, with the E571K mutation widely represented in chronic lymphocytic leukemia. [bib_ref] Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia, Puente [/bib_ref] SNV analysis showed the presence of a recurrent mutation affecting the UBE2A gene (Xq24) (pt#3 and pt#8). UBE2A is an E2-ubiquitin conjugating enzyme that has never been found mutated in CML. Interestingly, the two patients harboring UBE2A mutations lacked any recognizable copy number alteration [fig_ref] Table 2: UBE2A single nucleotide variants and indels identified in blast crisis samples and... [/fig_ref]. WES and targeted resequencing of a broader cohort showed that somatic UBE2A mutations are found in a significant fraction (16.7%) of advanced CML phases, thus confirming the initial exome analysis and suggesting a driver role for UBE2A loss of function during disease progression.
The Saccaromyces Cerevisiae UBE2A homolog Rad6 participates in DNA repair, sporulation and cell cycle regula-tion; [bib_ref] Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation, Shekhar [/bib_ref] in mammals a role for UBE2A in the regulation of transcription and chromatin reorganization through posttranslational histone modifications has recently been hypothesized. [bib_ref] The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for..., Roest [/bib_ref] Germline mutations of the UBE2A gene in humans have been associated with the X-linked Nascimento-type intellectual disability syndrome. [bib_ref] Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair..., Haddad [/bib_ref] [bib_ref] Novel missense mutations in the ubiquitination-related gene UBE2A cause a recognizable X-linked..., Budny [/bib_ref] [bib_ref] UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked..., Nascimento [/bib_ref] In order to understand the effect of UBE2A mutations in a BCR-ABL1-positive model, we tested the activity of exogenous UBE2A both in the WT or mutated forms (D114V and I33M) in BA/F3 BCR-ABL1-positive cell lines. We observed a reduced amount of mono-ubiquitinated histone H2A, a known UBE2A substrate, after overexpression of mutated UBE2A compared to the WT [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref] , which indicates that the UBE2A mutations analyzed in this study decrease the activity of the enzyme. This result has been further confirmed by in vitro assays for ubiquitination and enzymatic activity on total cell lysates [fig_ref] Figure 1: Activity of UBE2A mutants [/fig_ref] and E), thus providing evidence of a damaging effect of the two mutations on UBE2A function. Accordingly, one of the four variants identified in our cohort is a N-terminal frameshift mutation, thus supporting this hypothesis. This evidence is further strengthened by the distribution of UBE2A mutations throughout the entire protein, a pattern that is more common for genes undergoing inactivation. Mutations in the UBE2A paralog UBE2B were not detected in this study, which suggests a specific role for UBE2A in chronic myeloid leukemia. Stable silencing of UBE2A in the BCR-ABLpositive K562 cell line or overexpression of the I33M mutated form in a BCR-ABL1-positive 32Dcl3cl3 myeloid cell line showed profound downmodulation of CSF3R, a critical regulator of myeloid lineage differentiation and development. [bib_ref] Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis..., Yang [/bib_ref] [bib_ref] The hematopoietin receptor superfamily, Cosman [/bib_ref] CSF3R, also known as granulocyte colony-stimulating factor receptor (GCSFR), is a member of the hematopoietin receptor superfamily [bib_ref] The hematopoietin receptor superfamily, Cosman [/bib_ref] and plays a key role in promoting neutrophilic differentiation but may also support the development of different types of hematopoietic progenitors. [bib_ref] Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis..., Yang [/bib_ref] This suggests a potential role for CSF3R modulation in the suppression of myeloid differentiation in BC. Although the precise mechanism by which UBE2A controls CSF3R expression is still unknown, our data suggest that UBE2A-mediated CSF3R regulation occurs at transcriptional level. Alteration of CSF3R transcription could occur either by a direct activity of UBE2A on CSF3R promoter through epigenetic mechanisms 36 or indirectly by UBE2A-mediated ubiquitination of specific transcription factors. Further studies will be needed to clarify this process and to establish the relevance of CSF3R deregulation in the impairment of CML cell differentiation. In line with these findings, we showed that impairment of UBE2A function induces a delay in the differentiation of K562 and 32Dcl3-BCR/ABL1 cells after PMA, hydroxyurea or GCSF treatment, suggesting an important role for UBE2A as a modulator of myeloid differentiation.
In conclusion, in this work we identified recurrent, somatic UBE2A mutations occurring in a significant proportion of advanced CML cases. We propose that the acquisition of somatic UBE2A mutations affects myeloid developmental pathways, promoting a differentiation blockade. Further studies will be required to thoroughly dissect the molecular mechanisms responsible for these effects and to define possible therapeutic strategies for UBE2A-mutated BC-CML cases.
[fig] Figure 1: Activity of UBE2A mutants. (A) Western blot analysis of total cell lysates from BA/F3_BCR-ABL cell lines stably transfected with pMIGR-UBE2A vectors encoding for wild-type (WT) or mutated (D114V or I33M) UBE2A. Empty vector has been used as negative control. (B) Real-time quantitative polymerase chain reaction (RT-qPCR) of total RNA extracted from BA/F3_BCR-ABL_pMIGR/UBE2A cell lines. The values are normalized on the EMPTY cells (***P<0.001). (C) Western blot of total cell lysates from BA/F3_BCR-ABL_pMIGR/UBE2A cell lines. The signal at 14KDa corresponds to histone H2A. The signal at ~23KDa corresponds to monoubiquitinated histone H2A (mUbH2A) according to Wu et al. 37 (D) Western blot analysis of the in vitro ubiquitination reaction performed with in vitro translated UBE2A (WT and mutated forms) and GST-ubiquitin on total BA/F3_BCR-ABL lysate. (Right) The densitometric analysis of GST-ub signal (>170KDa) from three independent experiments obtained with ImageJ software. 38 The fold change is obtained normalizing the signal on the WT sample (WT vs. D114V *P=0.022; WT vs. I33M **P=0.0069). (E) Histogram showing the enzymatic activity of in vitro expressed UBE2A using the AMP Glow assay (WT vs. D114V *P=0.0056; WT vs. I33M **P=0.0036). [/fig]
[fig] Figure 2: UBE2A silencing in K562 cells. (A) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of total RNA extracted from K562 cell lines infected with a lentiviral based system for UBE2A silencing (shNC: scrambled negative control; shUBE2A: UBE2A silenced cells). Values are normalized on shNC cells (***P<0.0001). (B) Western blot analysis of total cell lysates from K562_shNC and K562_shUBE2A cells. (C) Heat map of RNA-sequencing data showing colorcoded expression levels of differentially expressed genes in three distinct populations of K562-shUBE2A compared to control (shNC). (D) RT-qPCR analysis in K562 cell lines of a subset of differentially expressed genes identified by RNA-sequencing. (E) Gene set enrichment analysis of the shUBE2A transcriptome. (F) RT-qPCR analysis in the 32Dcl3 cell line of a subset of differentially expressed genes identified by RNA-sequencing. (G and H) CSF3R protein levels in total cell lysate of K562 cells (G) and of BC/CP samples from patient #3, carrying UBE2A mutation in the BC phase (H).ATP consumption, and therefore in ubiquitin-conjugating activity, for UBE2A mutants compared to the WT form [seeFigure 1E: enzyme specific activity assay, 1.55-fold (P<0.01) and 1.53-fold (P<0.01) decrease in UBE2A D114V and I33M AMP concentration compared to UBE2A WT]. [/fig]
[fig] Figure 3: Induction of erythroid differentiation in UBE2A-silenced K562 cell line. K562 cells were treated with 400 μM hydroxyurea. (A) CD235a immunofluorescence staining for UBE2A-silenced K562 (shUBE2A) and control (shNC) cells after hydroxyurea or mock (-) treatments for the indicated times. (scale bar: 25 μm). (B) Average intensity of CD235a signal obtained acquiring ten fields from two independent experiments for each sample (approx. 80 cells each). (C) Fluorescenceactivated cell sorting analysis (FACS) analysis of CD235a levels in K562 cells in presence (red line) or absence (black line) of hydroxyurea. (D) Quantification of CD235a and hemoglobin mRNA relative levels (HBB: Hemoglobin-subunit-β) through real-time quantitative polymerase chain reaction (RT-qPCR) after hydroxyurea treatment. [/fig]
[fig] Figure 4: Induction of neutrophilic differentiation in UBE2A wild-type (WT) or I33M 32Dcl3 cell line. Cells were treated with IL-3 or granulocyte-colony stimulating factor (GCSF). (A) Fluorescence-activated cell sorting analysis (FACS) analysis of CD11b staining after induction of differentiation at days 3 and 6. (B) CD11b immunofluorescence staining for 32Dcl3 control (CTRL), UBE2A WT and I33M at day 6 showing a clear reduction in UBE2A I33M CD11b staining (scale bar: 20 μm). [/fig]
[table] Table 2: UBE2A single nucleotide variants and indels identified in blast crisis samples and absent in the paired chronic phase control. [/table]
[bib_ref] Cytogenetic and molecular genetic evolution of chronic myeloid leukemia, Johansson [/bib_ref] |
Micronutrients and Anaemia
Micronutrient deficiencies and anaemia remain as major health concerns for children in Bangladesh. Among the micronutrient interventions, supplementation with vitamin A to children aged less than five years has been the most successful, especially after distribution of vitamin A was combined with National Immunization Days. Although salt sold in Bangladesh is intended to contain iodine, much of the salt does not contain iodine, and iodine deficiency continues to be common. Anaemia similarly is common among all population groups and has shown no sign of improvement even when iron-supplementation programmes have been attempted. It appears that many other causes contribute to anaemia in addition to iron deficiency. Zinc deficiency is a key micronutrient deficiency and is covered in a separate paper because of its importance among new child-health interventions.
# Introduction
Almost a third of the world's population suffer from micronutrient deficiency, also known as 'hidden hunger', which mostly affects those living in developing countries. The public-health importance of vitamin and mineral deficiencies has been underscored through significant investment by national governments and donors in combating micronutrient deficiency to achieve "Millennium Development Goals" relating to mother and child health. In the following section, the deficiency of vitamin A has been reviewed in the context of Bangladesh. Beginning from historical perspectives of the control programmes, the most recent and ongoing activities of the Government have been highlighted with emphasis on successful interventions that need to be continued for sustained outcome. Attempts have been made to identify weaknesses of the programmes with recommendations on how to overcome them with reference to other successful programmes in similar settings.
## Vitamin a deficiency situation in bangladesh
Vitamin A deficiency, the leading cause of preventable blindness in children, increases the risk of disease and death from severe infections. Vitamin A deficiency has long been identified as a serious public-health problem in Bangladesh, especially since the first national nutrition survey conducted in the then East Pakistan in . In this survey, 0.2% of the total population was found to have Bitot's spots, and 4.2% had conjunctival xerosis. A historical perspective of vitamin A deficiency assessed by the nationwide nutrition surveys are summarized in [fig_ref] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 [/fig_ref].
With the decline in the prevalence of severe clinical manifestations of vitamin A deficiency, nightblindness was found to be the most useful indicator for the assessment of vitamin A deficiency. In addition,
- The vitamin A programmes of the past have been extremely effective and need to be sustained.
- Despite the legal requirement of iodized salt, a large proportion of the salt does not contain sufficient iodine, and a large proportion of the population is iodine-deficient.
- Anaemia rates remain high. Iron supplements and/or fortificants may help, but much of the anaemia is not due to iron deficiency. [fig_ref] Table 3: Prevalence of vitamin A deficiency among preschool children of Bangladesh [/fig_ref] provides data on the prevalence of vitamin A deficiency among preschool children based on clinical signs and symptoms. provides data on retinol levels in a similar age-group. A summary of data on serum retinol levels in populations of different demographic groups in Bangladesh is given in [fig_ref] Table 5: Serum retinol levels in population of different demographic groups in Bangladesh [/fig_ref].
## Vitamin a capsule programmes
The Government of Bangladesh started the supplementation of vitamin A capsules in 1973 under the National Blindness Prevention Programme, and it remains one of the most successful programmes [bib_ref] Vitamin A deficiency in Bangladesh: a review and recommendations for improvement, Ahmed [/bib_ref]. A detailed account of the coverage of the programme is enumerated in [fig_ref] Table 6: Supplementation of vitamin A capsule to preschool children in Bangladesh during [/fig_ref] and 7.
In the mid-1990s, supplementation of vitamin A to children aged less than 12 months was integrated into the Expanded Programme on Immunization (EPI), resulting in an increased coverage for this group. In 1995, vitamin A administration among 1-6 year(s) old children was integrated with the National Immunization Day (NID) which also succeeded in increasing the coverage for preschool children. Subsequently, the Government initiated the week-long nationwide mobilization campaign-'National Vitamin A Week'for distribution of vitamin A to children aged 12-71 months. To include mothers, postpartum vitamin A supplementation was then started by the Government with the support of United Nations Children's Fund. Later, the Bangladesh Integrated Nutrition Project (BINP) started distribution of vitamin A capsules among mothers within 14 days
## Dietary intake of vitamin a
All the three successive national nutrition surveys [bib_ref] Production of fruits and vegetables at the homestead is an important source..., Bloem [/bib_ref]. More recent programmes by HKI included animal food production, e.g. poultry, in addition to home-gardening. A programme evaluation using direct indicators, such as measurement of serum retinol or assessment of vitamin A pool size when possible, will be needed to determine the
## Increasing vitamin a in diet
- Improve food diversification, including greens and fruits.
- Introduce new foods, such as orange sweet potatoes.
- Introduce fortified foods where possible, e.g. cooking oil or atta.
## Recommendations for vitamin a programmes in bangladesh
- Continue distribution of vitamin A capsules to preschool children.
- Although dependent on external donor support, the vitamin A capsule programme is highly effective and needs continuation by the Government of Bangladesh.
- Improve coverage of postpartum vitamin A capsule distribution-it is lagging.
- Include supplementation of vitamin A capsules to pregnant women in the National Nutrition Programme. A weekly supplement of up to 25,000 IU (8,500 µg) is an alternative to daily supplementation.
## Recommendations for improving vitamin a status
Nutrition education should be provided through government outreach centres to increase the intake of vitamin A-rich foods by increasing those from animal origin together with adequate intakes of fruits and vegetables. Cooking techniques need to preserve the bioavailability of vitamin A in foods [bib_ref] Daily consumption of Indian spinach (Basella alba) or sweet potatoes has a..., Haskell [/bib_ref]. Studies conducted in Africa have shown that consumption of orange-fleshed sweet potato (OFSP), which is rich in beta-carotene, can effectively reduce vitamin A deficiency in children [bib_ref] Beta-carotene-rich orange-fleshed sweet potato improves the vitamin A status of primary school..., Van Jaarsveld [/bib_ref]. The same variety of OFSP is being grown in Bangladesh at research centres of Bangladesh Agricultural Research Institute. The low cost of production and high growth potential of OFSP make it an ideal food, meeting the requirements of vitamin A and total energy. Continued research is needed to determine if this can be successfully introduced into diets in Bangladesh and to determine the extent to which requirements of vitamin A can be met with a staple food, like this one which is rich in vitamin A.
Fortification of food is yet another effective means to increase the intake of vitamin A by the entire population. At present, only one private company in Bangladesh produces edible oil fortified with vitamin A. Government efforts must be made to promote food fortification to combat vitamin A deficiency in Bangladesh. Alternatively, micronutrient 'sprinkles' can be added to foods in the home, but additional operations research is needed to determine if this newer technology will be acceptable in Bangladesh [bib_ref] Controlling iron deficiency anemia through the use of home-fortified complementary foods, Zlotkin [/bib_ref].
## Iodine deficiency disorders in bangladesh introduction
Iodine deficiency is considered to be the most com-mon preventable cause of mental disorders in the world today, having manifestations at different stages of human life. A large proportion of people with severe iodine deficiency are women of reproductive age, who are at a higher risk of pregnancyrelated problems, including abortion, stillbirth, low-birthweight infants, brain damage or cretinism in infants even before birth, and lower chance of survival. Iodine deficiency can cause goitre and brain damage in neonates whereas manifestations in children include goitre, loss of energy, impaired school performance, and retarded physical development. In adults, iodine deficiency can lead to goitre and related complications, loss of energy, and impaired mental function. According to a 1990 WHO report, some 26 million people suffer from brain damage associated with iodine deficiency disorder, which includes six million cretins. All these have resulted in a growing awareness of the problem all over the world.
## Iodine deficiency and goitre in bangladesh
Results of surveys conducted since the 1960s have shown that high levels of iodine deficiency are prevalent in Bangladesh. The Nutrition Survey of East Pakistan 1962-1964 reported a goitre rate of 28.9% in former East Pakistan, now Bangladesh (1). The 1981-1982 National Goitre Prevalence Survey reported levels of iodine deficiency disorder nationwide, with the goitre rate at 10.5%. This result was, however, criticized because the health officers and workers who were assigned to identify goitre cases were not adequately trained. The National Iodine Deficiency Disorder Survey 1993 revealed a goitre rate of 47.1%. Another survey, using the 'EPI-30 cluster'-sampling methodology, found a prevalence of cretinism of 0.5%; 69% of subjects had low urinary concentrations of iodine (urinary iodine excretion [UIE] <10 mg/dL). Women and children were more affected than men, in terms of prevalence of both goitre and UIE. The presence of widespread severe iodine deficiency in all ecological zones indicates that the country as a whole is an iodine-deficient region [bib_ref] Iodine deficiency disorders in Bangladesh, Yusuf [/bib_ref].
## Salt iodization in bangladesh
To combat iodine deficiency disorder, the Government of Bangladesh, in 1989, passed the Iodine Deficiency Disease Prevention Act. The Act proclaimed universal iodization of edible salt for human and animal consumption and included prevention, enforcement, and education efforts. Under this act, the Bangladesh Council of Scientific and Industrial Research (BCSIR) and other institutions would be responsible for monitoring the quality of iodized salt manufactured and sold from that time onwards. Most salt-crushing units (which produce pure white sea salt from impure coloured products of salt croppers) have been provided with iodization equipment, and UNICEF supplies the iodizing agent-potassium iodate-free of charge. Despite this law and this assistance, much of the salt used by the people is not iodized.
A survey in 1995 showed that only 30% of iodized salt manufactured in Bangladesh contained an acceptable level of iodine [bib_ref] Salt iodization in Bangladesh-problems and a suggestion, Khorasani [/bib_ref]. Surprisingly, 10% of commercial brands contained no iodine at all. Only 30% of producers were using the recommended level of iodine, 10% were using mixtures of fortifying agents, and 10% were not using any iodine at all. Another survey conducted with UNICEF support in 1997 showed that the situation had not improved [bib_ref] Iodine deficiency disorders in Bangladesh, Yusuf [/bib_ref]. This survey found that only 57% of salt factories with iodization facilities were in regular production, 7% produced iodized salts only irregularly, and 36% were closed. Of 379 samples collected from 138 factories, only 5% contained adequate amounts of iodine, 46% contained too little, 1% contained no iodine at all, and 49% contained too much; some contained significantly more than it should, i.e. up to 20 times the recommended amount of iodine. Of 1,104 samples collected from retail outlets, 7% contained no iodine, and only one contained the recommended amount whereas 44% contained too little, and 56% contained a very large excess. Another cross-sectional study conducted in a coastal area in southern Bangladesh, during 1997-1998, comprising 21,190 households revealed that only 1.9% of the households used iodized salt in daily cooking [bib_ref] Policy of universal salt iodization in Bangladesh: do coastal people benefit?, Rasheed [/bib_ref].
In the Baseline Survey of the National Nutrition Programme in 2004, 39.5% of households were consuming table salt containing an inadequate concentration of iodine (<15 ppm).
The barriers limiting the use of iodized salt include the wide availability of coarse salt, lack of knowledge about the link between iodized salt and iodine deficiency disorders, and the high cost of iodized salt. These data show that the salt-iodization programme in Bangladesh is not making headway. The reasons may include: lack of quality-control measures in production units, lack of skills among production personnel, and failure on the part of the government regulatory agencies.
## Anaemia: a public-health problem in bangladesh introduction
Anaemia, a major public-health problem, was iden-tified about four decades ago in Bangladesh. In the following section, the trend in the prevalence of anaemia among different age-and sex-groups is presented. Information was collected from national nutrition surveys and from intervention and observational studies. Besides, the prevalence of iron deficiency was also documented from available sources. This section reviews the consequences of anaemia on health outcomes in children and women. The section also highlights on the aetiological factors causing anaemia and reviews the strategies that can be adopted to prevent the magnitude and extent of the condition.
## Prevalence of anaemia in bangladesh
Anaemia is defined as a reduction in the oxygencarrying capacity of blood. It is observed by reduced levels of haemoglobin concentration and red cell mass (haematocrit). At an individual level, however, anaemia is said to exist when haemoglobin concentration falls below a threshold: standard deviation of ±2 below the median for a healthy population of the same age, sex, and stage of pregnancy. The criteria for assessing the magnitude of the anaemia problem in relation to public-health significanceand the recommended cut-off points of haemoglobin levels for defining the presence of nutritional anaemia (41) are shown in .
Although anaemia has been recognized as a public-health problem for many years, there has been little progress towards improvement and the global prevalence of anaemia remains unacceptably high. It has been estimated that around two billion people in the world are anaemic, mostly in the lowerincome countries of Africa and Asia. In Bangladesh, anaemia is common among all age-groups, and both sexes are affected, especially children and women-both pregnant and non-pregnant.
Although there are many causes of anaemia, three main aetiologic categories are of concern in developing countries. These are: (a) nutritional deficiencies, (b) chronic infection, and (c) haemoglobinopathies.
Over the last four decades, data on the prevalence of anaemia have been gathered from national nutrition surveys conducted during this period and from a number of studies, which have been carried out to investigate the prevalence of anaemia, or from baseline information of a number of intervention or observational studies. Many of these studies have been done with small numbers and/or samples that were not representative of the populations of the country. However, all information is important in indicating the magnitude and trend of this public-health problem in Bangladesh.
The anaemia-prevalence data in preschool children are summarized in [fig_ref] Table 9: Summary of prevalence data on anaemia among preschool children in Bangladesh [/fig_ref]. All the surveys indicated a high prevalence of anaemia, without any trend for improvement. [fig_ref] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 [/fig_ref] shows similar information for older children. Overall, it would seem that rates of anaemia in this age-group may be decreasing, although the rates are still very high at 30-40% for school-age children. The rates for girls are slightly higher than for boys. For pregnant women, the available prevalence data are summarized in [fig_ref] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 [/fig_ref]. The general findings of these surveys suggest that nearly half of the pregnant women have anaemia. Somewhat surprisingly, anaemia is also highly prevalent in non-pregnant women and in adult males in Bangladesh. The prevalence data on anaemia for adult males and non-pregnant women . Recommended cut-off points of haemoglobin levels to define anaemia in population groupsand criteria for assessing the magnitude of the anaemia problem in relation to publichealth significance (40) Group Cut-off points of haemoglobin level (g/L)
Public-health significance [fig_ref] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 [/fig_ref]. Most of these surveys showed rates exceeding 50% even in men.
## Prevalence of iron deficiency in bangladesh
Many equate anaemia with iron deficiency. While iron is certainly crucial for normal levels of haemoglobin, iron deficiency is not the only cause of anaemia. Several studies have been carried out to determine the trends in iron deficiency. Of five studies that estimated serum transferrin receptor and/or serum ferritin levels, two studies were conducted in school settings, one in a garment factory, one in antenatal care centres in rural area, and one in a rural community setting [fig_ref] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 [/fig_ref]. [Iron deficiency was defined by serum ferritin (sFt) levels of <12 µg/L or <20 µg/L, and serum transferrin receptor (sTfR) levels of >8.5 mg/L or >5.0 mg/L.] Hyder et al. found an iron-deficiency prevalence of 42% (sFt <12 µg/L) among pregnant woman in rural areas [bib_ref] Anaemia and iron deficiency during pregnancy in rural Bangladesh, Hyder [/bib_ref]. The prevalence of iron deficiency was 55% and 29% respectively for women who were anaemic (Hb <110 g/L) and who were non-anaemic. When sTfR was considered, 30% of the anaemic and 21% of the non-anaemic pregnant women had tissue iron deficiency with an overall prevalence of 25%. In one study, a prevalence of deficient iron store of 30% has been observed among school children aged 6-12 years [bib_ref] Relationships between vitamin A, iron status and helminthiasis in Bangladeshi school children, Persson [/bib_ref]. The high prevalence of deficient iron stores of 81% (sFt <12 µg/L) has been observed in anaemic (Hb 80-120 g/L) adolescents working in the garment factory [bib_ref] Concomitant supplemental vitamin A enhances the response to weekly supplemental iron and..., Ahmed [/bib_ref]. A recent study in a school setting has reported the prevalence of iron-deficiency status of 29.8% in 14-18 years agegroup [bib_ref] Efficacy of twice-weekly multiple micronutrient supplementation for improving the hemoglobin and micronutrient..., Ahmed [/bib_ref]. A study conducted in a rural community found a prevalence of low iron status at 21.5% using a cut-off level for sFt <20 µg/L among schoolage children of 6-15 years [bib_ref] Randomized double-blind controlled trial of wheat flour (chapatti) fortified with vitamin A..., Rahman [/bib_ref]. However, when sTfR >5 mg/L was used for indicating tissue iron deficiency, the prevalence was 6.9%.
Based on the findings of all these studies, it can be concluded that, although iron deficiency is common in Bangladesh, it certainly does not totally explain the burden of anaemia. Further studies are needed to better understand the causes of anaemia. Iron fortification may help, but it will not solve the problem.
## Health consequences of anaemia
## Child health outcomes
Hospital-based data principally from malaria-en-demic regions have revealed that severe anaemia (Hb <50 g/L) in children is associated with an increased risk of death, but evidence for an increased mortality risk from moderate anaemia are inconclusive [bib_ref] An analysis of anemia and child mortality, Brabin [/bib_ref]. Iron deficiency and anaemia are associated with poor cognition and motor development, and anaemic infants may continue to have poorer school achievement and behavioural problems in later childhood [bib_ref] A review of studies on the effect of iron deficiency on cognitive..., Grantham-Mcgregor [/bib_ref].
## Maternal health and pregnancy outcomes
There is evidence of a relationship between severe anaemia (Hb <47 g/L) and increased maternal mortality [bib_ref] An analysis of anemia and pregnancy-related maternal mortality, Brabin [/bib_ref]. However, the distinction between anaemia as a primary or a contributory factor in death is related to its acute or chronic pattern. Acute onset of severe anaemia (Hg <80 g/L) during pregnancy can lead to rapid cardiac decompensation and can be a primary cause of death relating to acute haemolysis due to an underlying disease, e.g. sicklecell disease. Chronic anaemia, on the other hand, is considered to be a frequent contributory factor in death as a consequence of haemorrhage and infection. Iron-deficiency anaemia may contribute to increased morbidity and mortality by increasing maternal susceptibility to infection. However, evidence is scanty and inconsistent for the implication of moderate anaemia in excess maternal mortality or morbidity [bib_ref] An analysis of anemia and pregnancy-related maternal mortality, Brabin [/bib_ref] [bib_ref] Nutrition and maternal mortality in the developing world, Rush [/bib_ref]. There is some evidence of an association between maternal anaemia and low birthweight and preterm birth [bib_ref] Is there a causal relationship between iron deficiency or iron-deficiency anemia and..., Rasmussen [/bib_ref]. However, Steer et al. reported a minimal association between low birthweight and anaemia with maternal haemoglobin values of 96-105 g/L [bib_ref] Relation between maternal haemoglobin concentration and birth weight in different ethnic groups, Steer [/bib_ref]. Other studies have reported other haemoglobin values as the minimal rate for low birthweight: 104-132 g/L in one study [bib_ref] Relation of haemoglobin levels in first and second trimesters to outcome of..., Murphy [/bib_ref] and 105-125 g/L in Caucasian women in another study [bib_ref] Hematological status and pregnancy outcomes, Garn [/bib_ref]. A review of these studies pointed out that, during pregnancy, a haemoglobin value of <100 g/L is likely to reflect inadequate maternal nutritional status with respect to iron and other nutrients [bib_ref] Is there a causal relationship between iron deficiency or iron-deficiency anemia and..., Rasmussen [/bib_ref].
In a rural community in Bangladesh, an observational study has indicated a significant association of maternal haemoglobin levels of <80 g/L at first trimester and low birthweight [bib_ref] Factors associated with low birthweight in rural Bangladesh, Hosain [/bib_ref]. It may be predicted that 47.7% of pregnant women (Hg <80 g/L) in this study had inadequate nutritional status, including micronutrients, before pregnancy. Despite iron supplementation during pregnancy (73% good compliance), the prevalence of anaemia remained high. This study also suggested concomitant presence of other nutrient deficiencies, other than iron, and health risk factors.
## Haemoglobinopathies
Genetically-inherited disorders of haemoglobin or haemoglobinopathies may be causal factors for anaemia; however, their prevalence is among the least explored. Abnormal haemoglobin may be produced as a result of an alteration in the amino acid structure of the polypeptide chains of the globin fraction of haemoglobin, such as sickle-cell disease. In another form, the amino acid sequence is normal but the polypeptide chain production is impaired or absent for various reasons; these are thalassaemias. In Bangladesh, no screening programmes for thalassaemias are available; thus, there are no prevalence data regarding carrier status; however, a WHO report estimates that about 3% and 4% of populations are carriers of beta-thalassaemia and Hb-E-associated disease respectively in Bangladesh.
## Strategies to prevent anaemia
Three major interventions that may prevent anaemia include dietary diversification, food fortification, and supplementation. Dietary diversification involves promotion of a diet that contains a wider variety of naturally iron-containing foods with high bioavailability. Foods, such as meat, poultry, fish, and diary products, contain haem iron, which is more bioavailable than non-haem iron present in cereals, vegetables, and fruits. The principal diet of the people in poor countries consists mainly of cereals that are high in phytate, which is a known inhibitor of iron absorption. In many developing countries, the principal reason for iron-deficiency anaemia is poor dietary quality, and the intake of bioavailable iron is low. The diet of rural women of Bangladesh is principally based on cereal staples and has low iron and high phytate content [bib_ref] Dietary intakes and socioeconomic factors are associated with the hemoglobin concentration of..., Bhargava [/bib_ref]. Efforts to improve dietary quality through home-gardening and animal husbandry will presumably increase the intake of many essential micronutrients; such programmes can also help combat poverty by generating income.
Fortification of a staple food item with iron is one of the effective strategies to prevent iron-deficiency anaemia in a population who regularly eat the staple food and, thus, increase their iron intake. Efficacy studies with iron salts (added to wheat flour) fortified with encapsulated ferrous sulphate and ferric pyrophosphate respectively demonstrated a reduction in the prevalence of iron-deficiency anaemia from 35% to 8% and 30% to 5% among Moroccan school children [bib_ref] Dual fortification of salt with iodine and microencapsulated iron: a randomized, double-blind,..., Zimmermann [/bib_ref]. Micronutrient Iron-supplementation programmes aim to prevent anaemia or improve haemoglobin status in target population groups. The objective of such programmes in pregnant mothers is to improve anaemia status and its health consequences during pregnancy and the post-delivery period in mothers and their babies. Clinical trials have indicated the efficacy of iron supplementation in raising iron stores or haemoglobin levels [bib_ref] Efficacy and trial effectiveness of weekly and daily iron supplementation among pregnant..., Ekström [/bib_ref]. However, evidence is lacking that population-based iron-supplementation programmes have a notable impact on haemoglobin levels, iron status, or any other indices of maternal or perinatal health [bib_ref] Efficacy of weekly compared with daily iron supplementation, Cook [/bib_ref]. The increased requirement of iron during pregnancy justifies supplementation for pregnant women where poor dietary intake cannot supply adequate iron during that period. However, in poor settings, the levels of micronutrients other than iron, including vitamin A, zinc, calcium, riboflavin, and vitamin B12, are also low in poor diets, and some of these micronutrients also contribute to anaemia. Thus, addressing only iron and/or folic acid may not be effective in correcting nutritional anaemia and may address only part of the problem concerning nutritional deficiencies. Therefore, where deficiencies of multiple micronutrients are common, a more appropriate formulation of multiple micronutrients may be considered [bib_ref] Significance of an abnormally low or high hemoglobin concentration during pregnancy: special..., Yip [/bib_ref]. However, before such a programme is planned, aetiologies of anaemia, especially of nutritional origin in a particular area, need to be identified.
# Discussion
Anaemia is still a severe public-health problem in Bangladesh. Available data indicate that, over the last four decades, the situation has not improved. The cause of anaemia among young children is multi-factorial, including the low intake of bioavailable iron and high rates of infection. The intake of iron from complimentary foods is critical for the infant from six months as breastmilk alone cannot provide for the infant's increased need for iron for accelerated growth during that period. A WHO/UNICEF review of complimentary foods in developing countries concluded that requirements of iron might be difficult to meet from non-fortified complimentary foods, especially if animal products are not widely consumed. Home fortification of complimentary foods with sprinkles that contain iron plus other micronutrients could be an effective strategy to improve iron-deficiency anaemia in infants and young children. Community trials conducted in different countries, including Bangladesh, with sprinkles have demonstrated an impact in improving haemoglobin status in infants and young children.
Although iron deficiency is common, not all anaemias can be explained by iron deficiency alone. Iron-supplementation trials in controlled settings have shown to be efficacious in raising haemoglobin levels in pregnant women in Bangladesh and elsewhere. However, evidence is lacking that population-based iron-supplementation programmes have a notable impact on haemoglobin level and iron status or any other indices of maternal or perinatal health. In a rural community in Bangladesh, an observational study has indicated that, despite supplementation of iron during pregnancy to these women (73% good compliance), the prevalence of anaemia is still high. A significant association of maternal haemoglobin level of <80 g/L at first trimester and low birthweight was found in this study.
Fortification of a staple food item with iron could be an effective strategy to prevent iron-deficiency anaemia in this population who regularly eat the staple food and, thus, increase their intake of iron. Clearly, the choice of iron salt in the fortificant is critical when using this approach.
For anaemia, the lack of progress over 40 years makes one cautious about being optimistic for the success of the programme unless the aetiology of the problem is better understood. The perception about iron deficiency is common that iron fortification and iron supplements are a worthwhile recommendation wherever feasible. However, objective improvements may not be obvious. Thus, if anaemia is to be addressed as a serious issue, there needs to be much more basic research to understand the causes and physiology of anaemia in Bangladesh. Expecting iron supplements or fortificants to solve the problem is unlikely to be successful by itself.
Two evidence-based micronutrient interventions are now being proposed for widespread scaling up in Bangladesh: treatment of diarrhoea with zinc and the home-fortification of sprinkles micronutrient sachet. This will lead to questions from health planners and leading health professionals about their relative impact and the potential for programmatic interaction. Since these are leading nutritional programmes, these two interventions will need to be studied together and in combination.
[table] Table 1: Prevalence of vitamin A deficiency among the general population during 1962-1982 (based on clinical signs) Vitamin A status has been assessed by clinical examination and identification of signs of deficiency in the national nutrition surveys. However, studies conducted by other investigators have used biochemical indicators of vitamin A deficiency among various population groups. [/table]
[table] Table 3: Prevalence of vitamin A deficiency among preschool children of Bangladesh (based on clinical signs and symptoms) [/table]
[table] Table 5: Serum retinol levels in population of different demographic groups in Bangladesh [/table]
[table] Table 6: Supplementation of vitamin A capsule to preschool children in Bangladesh during [/table]
[table] Table 9: Summary of prevalence data on anaemia among preschool children in Bangladesh [/table]
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New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness
Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2 + breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin) and bevacizumab (Avastin), respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient's own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.
Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2 + breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin) and bevacizumab (Avastin), respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient's own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.
Keywords: cancer, antibody combinations, oncology, therapeutic antibodies, immunotherapy iNTRODUCTiON Nowadays, the therapeutic activity of antibodies in oncology has been widely demonstrated [bib_ref] State of the art in anti-cancer mAbs, Chiavenna [/bib_ref] [bib_ref] Cancer immunotherapy: the beginning of the end of cancer, Farkona [/bib_ref] [bib_ref] Therapeutic antibodies: past, present and future, Leavy [/bib_ref] [bib_ref] Antibody therapy of cancer, Scott [/bib_ref] [bib_ref] Monoclonal antibodies: versatile platforms for cancer immunotherapy, Weiner [/bib_ref] , being these proteins, after chemotherapy, radiotherapy, and small molecule inhibitors, one of the most used drugs for oncological treatments [bib_ref] The expanding role of immunotherapy, Martin-Liberal [/bib_ref]. Most of the antibodies used on antitumor immunotherapies had positive health effects as long as the antibody is present in the patient's blood. The clinical use of antibodies directed against antigens not present on the tumor cells, but on cells of the immune system (i.e., anti-immune checkpoint antibodies), evidenced the beneficial effects of the treatment, which persisted even after it was finished [bib_ref] At the bedside: CTLA-4-and PD-1-blocking antibodies in cancer immunotherapy, Callahan [/bib_ref]. These findings allowed to demonstrate that the anti-checkpoint antibodies were able to reprogram the organism's response, re-directing the antitumor immune response, and skewing the balance on the tumor microenvironment toward immune destruction of the tumor. Thus, allowing to envisage a cure for cancer.
The aim of this review is to discuss the information on the possible anti-cancer treatments using monoclonal antibodies (mAbs; in clinical trials or already in the market) in combinations, either with other antibodies or with other biological agents [bib_ref] Editorial overview: special section: new concepts in antibody therapeutics: what's in store..., Schuurman [/bib_ref] [bib_ref] Evolving synergistic combinations of targeted immunotherapies to combat cancer, Melero [/bib_ref] [bib_ref] The use of combinations of monoclonal antibodies in clinical oncology, Henricks [/bib_ref] [bib_ref] Combinatorial approach to cancer immunotherapy: strength in numbers, Vilgelm [/bib_ref] [bib_ref] Immunotherapy and novel combinations in oncology: current landscape, challenges, and opportunities, Morrissey [/bib_ref] [bib_ref] Cancer immunotherapy: strategies for personalization and combinatorial approaches, Sathyanarayanan [/bib_ref] [bib_ref] The future of cancer treatment: immunomodulation, CARs and combination immunotherapy, Khalil [/bib_ref]. The clinical trials are mentioned throughout this review only as examples of the different types of combinations being currently analyzed for cancer treatment. Thus, for most of the studies details and results will be shown on Supplementary Tables S1 and S2. The use of radiation therapy, chemotherapy, small-molecule compounds, or stem cell autotransplants will be mentioned only, if the information is strictly necessary in our attempt to dissect the reasons behind the use of these combinations. Similarly, there is no specific section describing combinations including checkpoint inhibitory mAbs, since this subject has been recently reviewed [bib_ref] New checkpoints in cancer immunotherapy, Ni [/bib_ref] [bib_ref] New frontiers in oncology: immune checkpoint inhibitors in combination therapy, Romano [/bib_ref] [bib_ref] Immune checkpoint blockade for hematologic malignancies: a review, Pianko [/bib_ref] [bib_ref] PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas, Goodman [/bib_ref] [bib_ref] PD-1 and PD-L1 antibodies in cancer: current status and future directions, Balar [/bib_ref] , including the reviews by Xu-Monette and Young and Aris et al., in this issue [bib_ref] Immunomodulatory monoclonal antibodies in combined immunotherapy trials for cutaneous melanoma, Aris [/bib_ref]. Similarly, combinations directed against cancer stem cells will not be discussed since they have been treated elsewhere in this research topic.
Antibodies are generated by the immune system's adaptive arm to defend the organism from pathogens and malignant cells. The antibodies are basically secreted molecules involved in mediating interactions on the extracellular compartment; they are made by a variable part that gives its binding specificity, and a constant region that is able to interact with other molecules or cells of the innate and adaptive immune system, to give them molecular information regarding their interaction with antigen. Thus, it should come to no surprise that antibodies' functions promote health and that treatments based on antibodies might, therefore, be curative [bib_ref] Harnessing the immune system to improve cancer therapy, Papaioannou [/bib_ref] [bib_ref] Active and passive immunotherapy for lymphoma: proving principles and improving results, Brody [/bib_ref]. Very few therapeutic antibodies are able to directly kill the tumor cells, either by interacting with a signal pathway (i.e., as a receptor antagonist or sequestering the ligand), or by direct triggering of apoptosis. Most of them kill the tumor cells through the interaction with other molecules or cells of the immune system, acting as molecules mediating interactions on the extracellular compartment. Although they originated as receptors on the surface of cells from the acquired immune system, they became secreted on mature B cells, and through either engagement with Fc-receptor-bearing cells or by interaction with the complement system, they can exert a broad spectrum of effector functions, coordinating the immune response (see .
A current goal of antitumor immune therapies is to trigger, from the beginning, all the possible host body defense mechanisms. Aiming to destroy, as early as possible, the highest number of tumor cells, decreasing the possibilities of the tumor developing escape mechanisms, to obtain a more effective therapy. The defense mechanisms include (i) to directly kill the tumor cells; (ii) to switch the immune system from an antitumor immunosuppressed status to another that allows to attack the tumor, i.e., through stimulating the secretion of cytokines or modulating cell to cell interactions; (iii) to attract the immune system cells to the tumor; (iv) to decrease the tumor-directed neo-vascularization; and (v) to inhibit migration, metalloprotease secretion, and tumor cell invasion, among others.
The current trends for the use of antibodies in oncology as therapeutic agents are to employ them either alone or, more often, as combinations with (i) cytotoxic agents; (ii) radiotherapy; (iii) molecularly targeted drugs interfering with tumor cell survival or proliferation; (iv) other antibodies against the same target; (v) other antibodies against molecules implicated in the same signaling pathway; (vi) other antibodies, each one of them specific for unrelated targets (including targets in immune system cells and neo-vascularization); (vii) vaccines or oncolytic virus; (viii) cells that would either act as immunogens or as effector cells; or (ix) adjuvants, liposomes, nanoparticles, etc.
For the treatment of cancer, the FDA and the EMA (United States and European Union Drug Administrations), have approved (or are reviewing) a total of 32 therapeutic antibodies or their derivatives. Interestingly, the number has doubled between 2012 and 2017 , concomitant with a 100% increase in phase III clinical trials using mAb on a similar time-period [bib_ref] Antibodies to watch in 2017, Reichert [/bib_ref]. Twenty of these antibodies are indicated for treatment of patients with solid tumors [fig_ref] TAble 1 |: Antibodies approved [/fig_ref] , identifying 13 different targets; whereas 12 are indicated for neoplasias of hematological origin [fig_ref] TAble 2 |: Antibodies approved [/fig_ref] , identifying eight different targets. The targets identified by these antibodies are described in [fig_ref] TAble 3 |: Characteristics of the main target molecules identified by therapeutic antibodies used in... [/fig_ref]. Other therapeutic antibodies, not yet approved, but mentioned on this review are summarized on [fig_ref] TAble 4 |: Summary of the therapeutic antibodies not yet approved for clinical treatments [/fig_ref]. From the antibodies approved (or under review) for the treatment of solid tumors, seven of them recognize tumor cell surface tyrosine kinase receptors involved in proliferation and survival pathways. These receptors (see [fig_ref] TAble 3 |: Characteristics of the main target molecules identified by therapeutic antibodies used in... [/fig_ref] are PDGFRα (targeted by the antibody olaratumab), HER2 (pertuzumab, trastuzumab and emtansine, ado-trastuzumab) and epidermal growth factor receptor (EGFR; necitumumab, panitumumab, and cetuximab). Two antibodies inhibit tumor angiogenesis by binding to the soluble ligand VEGF (bevacizumab) or to the endothelial cell receptor VEGFR2 (ramucirumab). Six of the mAb disrupt inhibitory immune checkpoint signals by binding to the programmed cell death protein 1 (PD-1) receptor on the T cells (nivolumab and pembrolizumab), to PD-L1 on the tumor cells (atezolizumab, durvalumab, and avelumab) or to CTLA-4 on T cells (ipilimumab). Two of these mAb block the binding of cytokines that are involved in the growth of some tumors, including antibodies against RANK-L (denosumab) and IL-1α (MABp1). The last three mAb recognize antigens overexpressed on the surface of tumor cells. They identify GD2 (dinutuximab) and EpCAM (edrecolomab and catumaxomab) [fig_ref] TAble 1 |: Antibodies approved [/fig_ref].
From the antibodies approved (or under review) for the treatment of hematopoietic neoplasias, eight antibodies recognize B cell antigens. Among those, one recognizes CD19 (blinatumomab), five mAb recognize CD20 (obinutuzumab, ofatumumab, rituximab, ibritumomab tiuxetan, and 131I tositumomab), one mAb binds to CD22 (inotuzumab ozogamicin), and the last one recognizes CD52 (alemtuzumab). Other two antibodies identify antigens expressed by B cells and by other cells of the immune system, including an anti-CD30 mAb (shared between B and T cells, brentuximab vedotin) and an anti-SLAMF7 (present on activated B cells and natural killer (NK) cells among others, elotuzumab). In addition, there are two antibodies against other immune cells, an anti-CD33 (myeloid lineage, gemtuzumab ozogamicin); and the non-lineage-restricted CD38 (daratumumab) [fig_ref] TAble 2 |: Antibodies approved [/fig_ref].
We will describe in the following paragraphs a set of clinical trials using antibodies in combination for oncological treatments, giving a systematic description of the antibody combinations with biological agents and their rationale. Describing the current aims of antibody-mediated cancer therapy and to envisage where its future lies. In addition, there will be a section where the therapeutic effects and toxicities for selected clinical trials will be discussed, which will help us to envisage the future of therapeutic antibodies for cancer treatments. Before starting with this systematic analysis, we will describe, with one example, in this case for the treatment of GD2 + -neuroblastomas, the complexity of the clinical trials being carried out.
## Evolution of treatment complexity with antibody in combinations
In this section, we will discuss, as an example, the use of anti GD2 antibodies for the treatment of GD2-positive solid tumors, including neuroblastoma [bib_ref] Dinutuximab for the treatment of pediatric patients with high-risk neuroblastoma, Mora [/bib_ref] [bib_ref] GD2-targeted immunotherapy and radioimmunotherapy, Dobrenkov [/bib_ref] [bib_ref] Disialoganglioside GD2 as a therapeutic target for human diseases, Suzuki [/bib_ref] [bib_ref] Anti-GD2 mAbs and next-generation mAb-based agents for cancer therapy, Perez Horta [/bib_ref]. Near 50 clinical assays have been started using two different mouse antibodies and their corresponding chimeras or humanized antibodies. The clinical use of dinutuximab (ch14.18) was approved in 2015, whereas the therapeutic efficacy of the other antibody, 3F8 has been demonstrated with many patients [bib_ref] Targeted immunotherapy for highrisk neuroblastoma -the role of monoclonal antibodies, Parsons [/bib_ref] [bib_ref] Murine anti-GD2 monoclonal antibody 3F8 combined with granulocyte-macrophage colony-stimulating factor and 13-cis-retinoic..., Cheung [/bib_ref] [bib_ref] Dinutuximab: first global approval, Dhillon [/bib_ref]. These antibodies, recognizing the neuroblastoma tumor-associated antigen GD2, are being used to (i) kill the tumor with either the naked antibody alone, apparently through Fc-mediated effector actions [antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP)] or apoptosis (NCT00002458, NCT00072358, NCT01418495, NCT01419834, NCT01704872, NCT02258815, NCT02743429); (ii) kill the tumor by the naked antibody in combination with chemotherapeutic agents (busulfan, carboplatin, cisplatin, cyclophosphamide, doxorubicin, etoposide, lomustine, melphalan, or vincristine), small molecule drugs (crizotinib), external radiation, and/or conventional surgery (NCT03098030, NCT03126916); (iii) directly transport a radioelement toward the tumor, by conjugating the radioelement to the anti-GD2 mAb. This will induce radiolysis of the tumor cells, minimizing the effects on normal cells (NCT00058370, NCT00445965, NCT03126916); (iv) use them in combination with agents that modify cell expression patterns, inhibiting proliferation and inducing cell differentiation and apoptosis [i.e., isotretinoin (13-cis retinoic acid or RA)] (NCT00003022, NCT00030719, NCT01183416, NCT01183429, NCT01183884, NCT01526603, NCT01711554, NCT02100930, NCT03033303); or (v) use them in combination with agents able to burst the host immune response against the tumor. These include, granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) (NCT01704716, NCT01767194, NCT02484443, NCT02502786, NCT03189706); which increase the number of innate immune response cells by triggering the proliferation of granulocytes and macrophages; increasing both innate and adaptive responses by inducing the maturation/proliferation of NK cells and T lymphocyte proliferation with interleukin-2 (IL-2) alone or in combination with GM-CSF and/or RA (NCT00005576, NCT00026312, NCT01041638, NCT01592045, NCT01662804, NCT02169609, NCT02641782); regulating the threshold of the immune response with an adjuvant, changing the secreted cytokine expression pattern of cells bearing certain pattern recognition receptors (i.e., beta glucan that binds the C-type lectin receptor Dectin-1) (NCT00037011, NCT00492167, NCT00089258); increasing the pool of cytotoxic cells able to fight the tumor with allogeneic NK cells (NCT00877110, NCT01857934, NCT02573896, NCT02650648); using in vitro activated T cells coated with bispecific OKT3-hu3F8 mAb, together with IL-2 and GM-CSF to redirect T lymphocyte cell lysis (NCT02173093); and combining the anti-GD2 antibody with nivolumab, an anti-immune checkpoint (PD-1) mAb able to block the immunosuppressor activity induced by the tumor (NCT02914405). From these "basic" aims further combinations arose, for example one where the aim is to induce radiolysis of the tumor cells with 131 I-3F8, simultaneously bursting the innate immune response with filgastrim (G-CSF), inhibiting neo-vascularization with bevacizumab (anti-VEGF), together with autologous stem cell rescue of irradiated patients (NCT00450827). We believe that this example gave a rough idea of the complexity that clinical trials for one antibody (two in this case) can reach. The chimeric, human-murine, anti-GD2 mAb dinutuximab has been approved in combination with GM-CSF, IL-2, and retinoic acid for the treatment of pediatric patients with high-risk neuroblastoma [bib_ref] Dinutuximab: an anti-GD2 monoclonal antibody for high-risk neuroblastoma, Ploessl [/bib_ref]. Interestingly, the overall survival and event-free survival of patients treated with dinutuximab increased 2 years when compared to standard treatment during phase III clinical trials [bib_ref] Dinutuximab: an anti-GD2 monoclonal antibody for high-risk neuroblastoma, Ploessl [/bib_ref].
## Combination of antibodies with non-biological agents
Chemotherapeutic drugs are cytotoxic agents affecting unspecifically cell proliferation and survival, which inhibit topoisomerases I or II (doxorubicin, etoposide, irinotecan, topotecan, etc.), produce DNA breaks interfering with DNA replication, RNA transcription and cell division through changes in DNA alkylation, DNA methylation, and DNA cross-linking or intercalating between base pairs in the DNA helix (busulfan, melphalan, cyclophosphamide, carboplatin, cisplatin, lomustine, thiotepa, etc.). These chemotherapeutic drugs are being used in combination with mAbs for many cancer treatments [bib_ref] Review of cisplatin and oxaliplatin in current immunogenic and monoclonal antibody treatments, Mehmood [/bib_ref].
In addition to surgery, treatment with antibodies and external irradiation has also been used. Localized external irradiation allows, by destroying tumor cells, better exposure of the tumor antigens to the immune system cells, this combination is also working well and is being used in numerous clinical trials [bib_ref] Immune effects of targeted radiation therapy for cancer, Sridharan [/bib_ref] [bib_ref] Combinations of immunotherapy and radiation in cancer therapy, Vatner [/bib_ref] [bib_ref] The abscopal effect of radiation therapy: what is it and how can..., Hu [/bib_ref] [bib_ref] Radiation-induced effects and the immune system in cancer, Kaur [/bib_ref] [bib_ref] Systemic effects of local radiotherapy, Formenti [/bib_ref].
Small molecule drugs that inhibit molecular interactions or enzymatic activity of proteins involved in cell signaling, or inhibitors of protein kinases overexpressed in tumor cells (including erlotinib, ibrutinib, imatinib, lapatinib, olaparib, regorafenib, ruxolitinib, sorafenib, sunitinib, etc.), are also being used in combination with antibodies [bib_ref] Targeted inhibition of kinases in cancer therapy, Baker [/bib_ref] [bib_ref] Targeted therapies combined with immune checkpoint therapy, Prieto [/bib_ref]. There are numerous examples of treatments with this type of combinations that, by simultaneously inhibiting ligand-receptor interactions and kinases belonging to the same signaling pathway, have led to very positive therapeutic results [bib_ref] Targeting cancer with kinase inhibitors, Gross [/bib_ref] [bib_ref] Antibody therapy alone and in combination with targeted drugs in chronic lymphocytic..., Robak [/bib_ref] [bib_ref] Evolution of anti-HER2 therapies for cancer treatment, Parakh [/bib_ref] [bib_ref] Emerging therapies for acute myeloid leukemia, Saygin [/bib_ref] [bib_ref] Emerging strategies for the dual inhibition of HER2-positive breast cancer, Konecny [/bib_ref].
## Combination of antibodies with biological agents
These are therapies that use a combination of antibodies or antibody-based molecules with other biological substances, for example, recombinant proteins, genetic material, virus, bacteria, and cells [bib_ref] Immunotherapy and novel combinations in oncology: current landscape, challenges, and opportunities, Morrissey [/bib_ref]. Most of these strategies are designed to stimulate the host immune system to act against the cancer cells.
In the following paragraphs, we describe antibodies in combinations, where (i) one of the antibodies identifies a tumorassociated antigen (an antigen overexpressed in tumor cells), used either naked, as an antibody-drug conjugate (ADC) or as an immunotoxin; (ii) antibodies against the tumor cell are used in combination with cytokines or immunocytokines to burst the immune response against the tumor, or conversely use anti-cytokine antibodies when the expressed cytokines can be harmful for the antitumor response, aiming to disrupt their balance; (iii) the antibodies directly target the angiogenesis process, aiming to inhibit new vascularization required for tumor growth; (iv) the mAb can also be combined with effector cells to increase the immune response against the tumor; or (v) combined with antibodies against immunomodulatory or immunostimulatory proteins to disrupt the inhibitory signals sent by the tumor to the host immune system to inhibit the antitumor response. Although several of the examples we will describe could be included more than one subheading, each one of them is described only in one of them.
## Antibodies against tumor-associated antigens
The rationale of using antibodies as therapeutic agents was to kill the tumor cells either directly or through activating the patient's immune system effector functions (ADCC, CDC, or phagocytosis) with antibodies specific for tumor-associated antigens. Mucin 1 (MUC-1), an antigen present on the surface of many adenocarcinomas, which is recognized by mAb m170 [bib_ref] High-dose radioimmunotherapy combined with fixed, low-dose paclitaxel in metastatic prostate and breast..., Richman [/bib_ref]. This mAb has been used radiolabeled as [bib_ref] Analytical characterization of ch14.18: a mouse-human chimeric disialoganglioside-specific therapeutic antibody, Soman [/bib_ref] In-m170 or 90 Y-m170, in combination with chemotherapy and the immunosuppressor cyclosporine to treat patients with metastatic prostate cancer that did not respond to hormone therapy. This treatment was followed by peripheral stem cell transplantation (NCT00009750). The rationale is to kill the tumor with the combination of chemotherapy and the mAb coupled to the radioisotope in the presence of cyclosporine. Afterward peripheral stem cell transplantation will allow to refurbish the hematopoietic compartment.
Other approaches have been used on hematological neoplasias, one of them, a combination of two anti-transferrin receptor (TfR) antibodies A27.15 and E2.3 [bib_ref] Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in..., White [/bib_ref] [bib_ref] Effects of transferrin receptor blockade on cancer cell proliferation and hypoxia-inducible factor..., Jones [/bib_ref] was used for the treatment of chronic myeloproliferative disorders (NCT00003082). The anti-TfR mAb block the binding of (Fe 3+ )2transferrin to TfR, resulting in decreased tumor cell growth. Other targets used in hematopoietic malignancies are CD20 and CD74. In this case, a combination of the anti-CD20 mAb veltuzumab (IMMU-106) (288) and milatuzumab (anti-CD74) (239) was used to treat relapsed or refractory B cell non-Hodgkin lymphoma (NCT00989586). CD74, a surface receptor of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) [bib_ref] Macrophage migration inhibitory factor: a key cytokine and therapeutic target in colon..., Gordon-Weeks [/bib_ref] [bib_ref] Tumor growth-promoting properties of macrophage migration inhibitory factor, Bifulco [/bib_ref] [bib_ref] Macrophage migration inhibitory factor (MIF): its potential role in tumor growth and..., Nishihira [/bib_ref] , is an MHC class II chaperone and an accessorysignaling molecule [bib_ref] CD74: a new candidate target for the immunotherapy of B-cell neoplasms, Stein [/bib_ref]. Milatuzumab induces apoptosis, related to inhibition of CD74 activation by MIF, ADCC, or CDC (293), while veltuzumab triggers CDC and ADCC in cells that overexpress CD20 [bib_ref] Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and Its use..., Stein [/bib_ref]. Other combinations include rituximab (anti-CD20) in combination with CC-90002 (anti-CD47) [bib_ref] Cancer immunotherapy targeting the CD47/SIRPalpha axis, Weiskopf [/bib_ref] [bib_ref] Targeting CD47: the achievements and concerns of current studies on cancer immunotherapy, Huang [/bib_ref] for the treatment of advanced solid and hematologic cancers (NCT02367196). CC-90002 selectively binds to CD47 expressed on tumor cells, blocks CD47 interaction with signal regulatory protein alpha (SIRPa), a protein expressed on phagocytic cells, which prevents CD47/SIRPa-mediated signaling and abrogates the CD47/SIRPa-mediated inhibition of phagocytosis. The result is an induction of pro-phagocytic signaling, resulting in macrophage activation and the specific phagocytosis of tumor cells. In addition, CD47 signaling blockade activates both, an antitumor T lymphocyte immune response and T cell-mediated killing of CD47-expressing tumor cells. CD47, also called integrin-associated protein (IAP), is a tumor-associated antigen (TAA) expressed on normal, healthy hematopoietic stem cells (HSC) and overexpressed on the surface of a variety of cancer cells. Expression of CD47 and its interaction with SIRPa leads to the inhibition of macrophage activation and protects cancer cells from phagocytosis, resulting in cancer cell proliferation [bib_ref] Cancer immunotherapy targeting the CD47/SIRPalpha axis, Weiskopf [/bib_ref] [bib_ref] The interaction between signal regulatory protein alpha (SIRPalpha) and CD47: structure, function,..., Barclay [/bib_ref] [bib_ref] The CD47-SIRPalpha signaling axis as an innate immune checkpoint in cancer, Matlung [/bib_ref] [bib_ref] Macrophages are critical effectors of antibody therapies for cancer, Weiskopf [/bib_ref].
## Adc and immunotoxins
Some antitumor treatments use, rather than naked antibodies, antibody-toxin fusion proteins (immunotoxins) or antibodies linked to drugs [bib_ref] Challenges and advances in the assessment of the disposition of antibody-drug conjugates, Kamath [/bib_ref] [bib_ref] Preclinical pharmacokinetic considerations for the development of antibody drug conjugates, Kamath [/bib_ref] [bib_ref] Antibody-drug conjugates-a new wave of cancer drugs, Bouchard [/bib_ref] [bib_ref] New developments for antibody-drug conjugate-based therapeutic approaches, De Goeij [/bib_ref]. In some cases, to increase the cellkilling potential of antibodies, they can be covalently linked to potent cytotoxic or cytostatic agents, including small molecule drugs or inactive forms of a biological toxin. The antibody directs the toxin toward the tumor cell. When the cell endocytoses the ADC, it undergoes enzymatic cleavage and the drug is released, gets activated, and exerts its cytotoxic action, killing the tumor cell. The endocytic process works for antigens that can be internalized. Most of the current antibodies that are being used as ADC identify cell surface receptors that are efficiently endocytosed. However, many cell surface proteins are not internalized and a large amount of work is being carried out to develop alternatives, such as making an ADC where the antibody is coupled to the drug through a linker that can be cleaved by tumor cell-surface proteases, when in close contact with the tumor cell; conversely the antibody may carry a tumor receptor antagonist to direct it toward the tumor cell surface [bib_ref] Antibody drug conjugates for cancer therapy, Polakis [/bib_ref] [bib_ref] Triggered drug release from an antibody-drug conjugate using fast "click-to-release" chemistry in..., Rossin [/bib_ref].
One example includes a combination of an antitumor-associated antigen mAb and an ADC. Trastuzumab in combination with an antibody against the zinc transporter LIV-1 (SLC39A6) conjugated to the cytotoxic agent monomethyl auristatin E (MMAE) [bib_ref] SGN-LIV1A: a novel antibody-drug conjugate targeting LIV-1 for the treatment of metastatic..., Sussman [/bib_ref] for the treatment of patients with metastatic breast cancer (NCT01969643). In this particular combination, trastuzumab inhibits the tyr-kinase receptor HER2, while through the potent microtubule disrupting agent MMAE, which is coupled to the anti-LIV-1 antibody, induces cell cycle arrest in the G2/M phase and apoptosis of LIV-1 + cells [bib_ref] SGN-LIV1A: a novel antibody-drug conjugate targeting LIV-1 for the treatment of metastatic..., Sussman [/bib_ref]. This type of combination can be made more potent by adding to the equation, antibodies against immune-checkpoints to burst antitumor immune responses. For example, the combination of nivolumab, ipilimumab, and rovalpituzumab tesirine has been used in extensive-stage small cell lung cancer (SCLC) (NCT03026166). Rovalpituzumab tesirine is an anti-delta-like protein 3 (DLL3) antibody conjugated to the cytotoxic pyrrolobenzodiazepine dimer D6.5 [bib_ref] Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a..., Rudin [/bib_ref] [bib_ref] Rovalpituzumab tesirine and DLL3: a new challenge for small-cell lung cancer, Rossi [/bib_ref]. This antibody recognizes the membrane protein DLL3, which is overexpressed in certain tumors, binds to Notch receptors, and regulates Notch-mediated signaling [bib_ref] Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a..., Rudin [/bib_ref]. Thus, this combination should kill the cells overexpressing DLL3, while the anti-checkpoint antibodies nivolumab and ipilimumab redirect the host immune response to attack the tumor.
Another example shows the combination of mAb CR011, against the transmembrane protein GPNMB (glycoprotein nonmetastatic B) coupled to MMAE [bib_ref] Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate..., Pollack [/bib_ref]. This ADC was used in combination with anti-PD-1 mAb nivolumab or pembrolizumab and varlilumab [bib_ref] Characterization of the human T cell response to in vitro CD27 costimulation..., Ramakrishna [/bib_ref] [bib_ref] Safety and activity of varlilumab, a novel and first-in-class agonist anti-CD27 antibody,..., Burris [/bib_ref] , an agonistic anti-CD27 mAb, for the treatment of advanced melanoma (NCT02302339). The rationale is, in addition to targeting the GPNMB + cells with the antibodycoupled to the toxin, the anti-PD-1 antibodies suppress the tumor-promoted inhibition of the antitumor immune response, while the anti-CD27 triggers an activation of the cytotoxic T lymphocytes (CTL).
The following example combines the ADC mirvetuximab soravtansine with either bevacizumab (VEGF) or pembrolizumab (PD-1) in primary peritoneal, fallopian tube, or endometrial cancer (NCT02606305). Mirvetuximab soravtansine is an immunoconjugate consisting of a folate receptor 1 (FOLR1) mAb (M9346A) conjugated to the cytotoxic maytansinoid DM4 [bib_ref] Mirvetuximab soravtansine (IMGN853), a folate receptor alpha-targeting antibody-drug conjugate, potentiates the activity..., Ponte [/bib_ref]. DM4 is released after internalization, binds to tubulin, and disrupts microtubule dynamics. FOLR1 is a member of the folate receptor family, overexpressed on a variety of epithelial-derived cancer cells [bib_ref] Mirvetuximab soravtansine (IMGN853), a folate receptor alpha-targeting antibody-drug conjugate, potentiates the activity..., Ponte [/bib_ref] [bib_ref] IMGN853, a folate receptor-alpha (FRalpha)-targeting antibody-drug conjugate, exhibits potent targeted antitumor activity..., Ab [/bib_ref]. Other studies combine nivolumab (anti-PD-1) with the ADC BMS-986148 [bib_ref] Novel antibody therapeutics targeting mesothelin in solid tumors, Zhao [/bib_ref] [bib_ref] Mesothelin immunotherapy for cancer: ready for prime time?, Hassan [/bib_ref] , composed of a mAb against the cell surface glycoprotein mesothelin (MSLN), conjugated to an as of yet undisclosed cytotoxic drug, for the treatment of mesothelioma, NSCLC, ovarian cancer, pancreatic cancer, and gastric cancer (NCT02341625). The rationale here is to block with the anti-PD-1 mAb the binding of PD-L1 (present on the tumor cells) to its receptor PD-1 (present on T cells), avoiding the suppression of antitumor responses triggered by the PD-L1/PD-1 interaction, while targeting mesothelin + cells with the BMS-986148 mAb. Since the mAb is an ADC, upon internalization the cytotoxic agent kills the tumor cells. The mAb also activates ADCC. Another combination, for the treatment of MSLN-expressing NSCLC uses a combination of the anti-VEGF antibody bevacizumab with the single-chain anti-MSLN mAb SS1 (dsFv) linked to the exotoxin PE-38 from Pseudomonas (258) (NCT01051934). Since MSLN is not shed in significant amounts into the bloodstream, the dsFv-toxin can be concentrated onto the tumor cell surface. Once the dsFv toxin is internalized, the toxin is released and inactivates eukaryotic translation elongation factor 2, disrupting tumor cell protein synthesis. Concomitantly, the anti-VEGF antibody inhibits angiogenesis [bib_ref] Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic..., Hassan [/bib_ref].
## Antibodies combined with cytokines and immunocytokines
Another way to burst the host immune response against the tumor involves the use of either exogenous cytokines or fusion proteins that include a cytokine, administered either systemically or directly in the tumor. In some cases, due to cytokine toxicity, it could be envisaged to directly couple the cytokine to a mAb specific for a tumor-associated antigen, as a recombinant fusion protein [bib_ref] Immunocytokines: a review of molecules in clinical development for cancer therapy, List [/bib_ref] [bib_ref] Immunocytokines for cancer treatment: past, present and future, Neri [/bib_ref]. These combinations allow, decreasing the dose, to reach higher local concentrations at the tumor site and to exert its therapeutic function avoiding systemic toxicity, while increasing the cytokine's half-life, since it is coupled to the antibody, which prevents renal clearance [bib_ref] Immunocytokines: a novel class of potent armed antibodies, Pasche [/bib_ref].
In another example for the treatment of advanced or metastatic solid tumors, a combination of nivolumab and the Aldesleukin Prodrug NKTR-214 was used (NCT02983045). NKTR-214 is a recombinant human IL-2 conjugated to six releasable polyethylene glycol chains (PEG) [bib_ref] NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure,..., Charych [/bib_ref]. When the cytokine is released, binds to CD122 (IL-2 receptor beta subunit) and the mAb may act synergistically with NKTR-214 by blocking PD-1 activation through the mAb and simultaneously stimulating growth and cytotoxic activity against the tumor of the patient's T and NK cells by the exogenous IL-2. The advantages of using this conjugated form of the IL-2 are that, on the one side, is released in a controlled way in the tumor's proximity, avoiding systemic toxic effects; and on the other side, PEG conjugation prevents IL-2 binding to the IL2Ralpha subunit (and the subsequent activation of CD4-positive regulatory immunosuppressive T cells), while IL2Rbeta activation plays a key role on the proliferation and activation of effector T cells [bib_ref] NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure,..., Charych [/bib_ref]. In another clinical trial, the NKTR-214 immunocytokine was also administered, using a similar therapeutic strategy for the treatment of patients with metastatic urothelial bladder cancer or metastatic NSCLC, in combination with atezolizumab (74-76) (NCT03138889). Another trial for advanced or metastatic solid tumors expressing the carcinoembryonic antigen (CEA), combined atezolizumab and cergutuzumab amunaleukin [CEA-IL-2 variant (IL2v)] (218), alone or together with a pretreatment with the anti-CD20 mAb obinutuzumab (NCT02350673). The immunocytokine CEA-IL2v is a fusion protein between a recombinant IL2v, unable to bind CD25, fused to the C-terminus of a high affinity, bivalent CEAspecific antibody [bib_ref] Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy:..., Klein [/bib_ref]. The strategy is to inhibit the PD-1/PD-1L checkpoint, increasing locally IL-2 activity on the tumor cells, allowing its binding to CD122. A similar strategy uses nivolumab in combination with the superagonist ALT-803 for the treatment of advanced and unresectable NSCLC (NCT02523469). ALT-803 is a fusion protein containing a mutated IL-15 (IL-15N72D) cytokine and a soluble, dimeric IL-15 receptor alpha Fc fusion protein (IL-15Ra-Fc) [bib_ref] The IL-15-based superagonist ALT-803 promotes the antigen-independent conversion of memory CD8+ T..., Wong [/bib_ref]. The rationale of this study is to suppress the signaling through negative checkpoints, while activating and increasing NK levels and memory CD8 + T cells, through the binding of ALT-803 to the IL-2/IL-15 receptor beta gamma chain, strengthening the patient's immune response.
Another example is the anti-HER2 mAb trastuzumab, which has been used in combination with IL-12 in treating patients with recurrent solid tumors [breast cancer, endometrial carcinoma, gastric cancer, non-small cell lung cancer (NSCLC), SCLC, and ovarian epithelial cancer] (NCT00028535). IL-12 stimulates IFNgamma production and enhances T-and NK-cell proliferation, differentiation, and activation [bib_ref] Interleukin 12: still a promising candidate for tumor immunotherapy?, Lasek [/bib_ref]. A more complex example combines two mAb-cytokine fusion proteins, consisting of L19, a human single-chain variable fragment directed against the extra-domain B (ED-B) of fibronectin, linked to either the human pro-inflammatory cytokine tumor necrosis factor alpha (TNFa, L19-TNF) or to human IL-2 (L19-IL-2). These combinations have been used on patients with malignant melanoma (317-320) (NCT02076633). The rationale of the trial is that the L19 moiety binds to the ED-B domain of a fibronectin isoform selectively expressed in the tumor neovasculature during neo-angiogenesis. TNFa may locally induce an immune response against ED-B + tumor cells, while the IL-2 moiety may locally activate CTL, NK cells, and macrophages.
In another clinical trial, atezolizumab was combined with (i) ipilimumab, (ii) interferon alfa-2b, (iii) PEG-interferon Alfa-2a, (iv) bevacizumab and PEG-interferon alfa-2a, and (v) obinutuzumab, for the treatment of locally advanced or metastatic solid tumors (NCT02174172). The aim is to compare on the same trial the results from these different strategies. The rationale is to restore the antitumor immune response by blocking immune checkpoints, while inducing cell cycle arrest, apoptosis or differentiation, which will led to tumor growth inhibition, concomitant with T cell and NK cell activation, inhibition of angiogenesis and induction of cytokine expression, through the administration of interferon alpha (321).
## Anti-cytokine antibodies
On the previous section, the aim was to provide exogenous cytokines to burst the antitumor immune response. Here, the aim is to disrupt the balance of other cytokines such as IL-17 or IL-1, that may hinder the antitumor immune response.
An example, for the treatment of patients with multiple myeloma, uses the anti PD-1 mAb PDR001 in combination with the mAb CJM112 (targeting IL-17) or with the Smac Mimetic LCL161 drug (an IAP inhibitor) (NCT03111992). This strategy aims to restore the cellular immune response inhibiting checkpoint signaling, changing the cytokine balance by decreasing the available IL-17 and favoring tumor cell apoptosis. A more complex clinical trial was used for the treatment of colorectal cancer, triple-negative breast cancer, NSCLC and adenocarcinoma, where the anti-PD-1 mAb PDR001 (13) was used in combination with either (i) the anti-interleukin-1 beta (IL-1b) mAb canakinumab (214), (ii) the anti-IL-17 mAb CJM112 (219), (iii) the small molecule inhibitor trametinib (MAPKK1 and MAPKK2 inhibitor) or (iv) the EGFR antagonist nazartinib (NCT02900664). The aim is to inhibit the immune checkpoint, while either simultaneously suppressing the inflammatory responses (blocking IL-1b or IL-17), or inhibiting tumor cell proliferation with mitogen-activated protein kinase and EGFR inhibitors.
## Antibodies targeting angiogenesis
Unlike to what happens with hematologic tumors, the growth of a solid tumor is concomitant with a local increase in nutrient and oxygen consumption and secretion of metabolites, requiring neovascularization for its growth. Therefore, some of the antitumor therapies aim to interfere with the neo-vascularization process, either by including antibodies against the soluble ligands, or against their receptors present in the cell surface of endothelial cells [bib_ref] Targeting angiogenesis in cancer therapy: moving beyond vascular endothelial growth factor, Zhao [/bib_ref].
The anti-VEGF-A mAb bevacizumab, able to inhibit angiogenesis is currently being tested in combination with other therapeutic agents to determine its usefulness for cancer treatment. These combinations include: cetuximab, in advanced lung cancer (NCT00368992); MEDI3617 (anti-Ang-2) (238), for advanced solid malignancies (NCT01248949); the mAb drozitumab (PRO95780) (223) against death receptor 5 (DR5/ TRAIL-R2), in metastatic colorectal cancer (NCT00851136); NK immunotherapy, in recurrent solid tumors (NCT02857920); and atezolizumab (NCT03038100, NCT02659384), nivolumab (NCT02873962), or pembrolizumab (NCT02853318) for the treatment of ovarian, fallopian tube, or primary peritoneal cancer. Bevacizumab has also been combined with MNRP1685A (242, 323), a mAb against membrane-bound endothelial cell coreceptor neuropilin-1 (NRP1), overexpressed in certain tumor cells, for advanced or metastatic solid tumors (NCT00954642). MNRP1685A prevents angiogenesis by blocking binding of VEGF, VEGF-B, and placental growth factor 2 to neuropilin-1, resulting in vessel immaturity. Other combinations of bevacizumab include parsatuzumab (247), a mAb against the vascularrestricted extracellular matrix protein epidermal growth factor-like domain multiple 7 (EGFL7), upregulated during angiogenesis and overexpressed on the cell surface of different solid tumors, for the treatment of metastatic colorectal cancer (NCT01399684). Parsatuzumab inhibits vascular development regulated by EGFL7, affecting to the survival and migration of endothelial cells during angiogenesis. An additional combination used bevacizumab and anti-VEGFC/Flt4 (VGX-100) (265) for metastatic solid tumors (NCT01514123). The rationale is to simultaneously inhibit vascular and lymphatic endothelial cell proliferation and angiogenesis.
A different approach is to target angiogenesis with the Anti-VEGFR-2 mAb ramucirumab, in combination with the anti-c-MET [hepatocyte growth factor receptor (HGFR)] mAb emibetuzumab (227), on advanced refractary solid tumors (NCT02082210). The rationale of the trial is to inhibit angiogenesis and MET signaling on the tumor cells [bib_ref] A first-in-human phase I study of a bivalent MET antibody, emibetuzumab (LY2875358),..., Rosen [/bib_ref]. A similar strategy has been used where instead of using an anti-HGFR mAb, uses ficlatuzumab (229, 324), a mAb against the c-MET ligand (HGF), in combination with cetuximab (NCT02277197).
A similar strategy targets molecules with an expression highly restricted to the vascular endothelium. An example is the combination of pembrolizumab with demcizumab (222), a mAb that blocks the interaction of anti-delta-like ligand 4 (DLL4) with Notch-1 and Notch-4, inhibiting Notch-mediated signaling and gene transcription, impairing the productive growth of new blood vessels (325) (NCT02722954). Pembrolizumab avoids the immunosuppression by immune checkpoint signaling while demicizumab prevents angiogenesis.
In K-ras wild-type metastatic colorectal cancers, cetuximab mAb was used in combination with the alphaVbeta3 (vitronectin receptor) integrin inhibitor EMD 525797, an anti-alphaV integrin subunit mAb (226) (NCT01008475). AlphaVbeta3 integrin is a cell adhesion and signaling receptor expressed on the surface of tumor endothelial cells, with a crucial role in their adhesion and migration. The aim of the trial is to inhibit angiogenesis and endothelial cell interaction(s) with other cells or with the extracellular matrix, required for tumor angiogenesis and metastasis. A similar study in solid tumors combines bevacizumab with intetumumab (235, 326), a pan alpha-v human mAb that blocks both alpha-v beta-3 and alpha-v beta-5 integrins, resulting in inhibition of integrin-mediated tumor angiogenesis and tumor growth (NCT00888043).
The remaining clinical trials on this section, all of them combine an anti-checkpoint antibody (either anti-CTLA-4, PD-1 or PD-L1) with anti-angiogenic antibodies such as the anti-Angiopoietin 2 (Ang-2) mAb (MEDI3617) in metastatic melanoma (NCT02141542); with the antibody carotuximab (TRC105) (215) that recognizes the endothelial cell surface protein endoglin, essential for angiogenesis, in metastatic NSCLC (NCT03181308); with antibodies specific for Ang-1 and Ang-2, which prevent their interaction with their target tie2 receptors (NCT00861419); with vanucizumab (bispecific anti-VEGF/ Combinations Using Therapeutic Antibodies for Cancer Frontiers in Immunology | www.frontiersin.org
December 2017 | Volume 8 | Article 1804
Ang-2 antibody) (263), in advanced or metastatic solid tumors (NCT01688206). The bispecific mAb targets both VEGF-A and Ang-2, which are upregulated in a variety of tumor cell types, play key roles in tumor cell proliferation, angiogenesis and metastasis. The anti-VEGF-A arm is based on bevacizumab and the anti-Ang-2 arm is based on the anti-Ang-2 antibody LC06 [bib_ref] Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2..., Kienast [/bib_ref]. It simultaneously binds and neutralizes both VEGF-A and Ang-2. This prevents the activation of both VEGF-A/VEGFR-and Ang-2/Tie2-mediated signaling pathways, resulting in the inhibition of proliferation of VEGF-A-and/or Ang-2-overexpressing tumor cells [bib_ref] Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2..., Kienast [/bib_ref] [bib_ref] Ang-2/ VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype..., Kloepper [/bib_ref]. Another strategy is to combine the anti-EGFR mAb panitumumab with an anti-hepatocyte growth factor mAb rilotumumab [bib_ref] a mAb against human hepatocyte growth factor for the treatment of cancer, Rilotumumab [/bib_ref] or ganitumab [bib_ref] Efficacy of ganitumab (AMG 479), alone and in combination with rapamycin, in..., Beltran [/bib_ref] , an anti-insulin-like growth factor 1 receptor (IGF-1R) mAb in metastatic colorectal cancer with wild-type KRAS (NCT00788957) [bib_ref] Randomized phase Ib/II trial of rilotumumab or ganitumab with panitumumab versus panitumumab..., Van Cutsem [/bib_ref]. The rationale here is to simultaneously inhibit strong proliferative signals triggered by EGFR and c-MET.
## Antibodies combined with effector cells
In some patients, the number of cells from the innate or adaptive immune system could be decreased by the effects of previous treatments. In these cases, treatments with antibodies, whose mechanisms of action depend on immune system cell effector functions (i.e., ADCC, ADCP, etc.), could be compromised. In these cases, either autologous (harvested prior to the treatment) or allogeneic cells (NK cells, T cells, CTL cells, dendritic cells (DC), etc.) can be administered concomitantly with the therapeutic antibodies.
A combination of mAb and cells for the treatment of hematological malignancies combines the anti-CD22 mAb epratuzumab [bib_ref] Preclinical and clinical evaluation of epratuzumab (anti-CD22 IgG) in B-cell malignancies, Leonard [/bib_ref] with haploidentical NK cells and low-dose exogenous IL-2, for the treatment of relapsed acute lymphoblastic leukemia (NCT00941928). CD22 is a cell surface glycoprotein present on mature B cells and on many B cell malignancies. In this example, since epratuzumab action involves ADDC, the exogenous administered haploidentical NK cells strengthens its effects. The exogenous IL-2 induces NK cell proliferation, activates cytotoxic immune responses against the tumor and induces expression of certain cytotoxic cytokines, such as interferon-gamma (IFNgamma) and transforming growth factor-beta. In another example, NK cells were used in combination with nivolumab for the treatment of recurrent solid tumors (NCT02843204), strengthening the endogenous immune response with the anti-checkpoint antibodies and increasing the NK cell load. Another combination used for the treatment of recurrent solid tumors uses NK cells in combination with bevacizumab (NCT02857920), increasing the NK cell load and simultaneously targeting tumor neo-vascularization. The last combinations to be mentioned with effector cells use pembrolizumab, administrated with autologous dendritic cells-cytokine induced killer cell (DC-CIK), for advanced solid tumors (NCT03190811), or the anti-PD-1 mAb, that was used in vitro to activate and expand DC-CIK from the patient's peripheral blood, before infusion (NCT02886897). These clinical trials aim to target the immune checkpoint and increasing the load of cytolytic cells with the DC-CIK.
## Bispecific antibodies
Nowadays the FDA and EMA allow clinical trials where the therapeutic agent is a combination of two antibodies. Bispecific antibodies may be considered as a particular combination where both antibodies are in a single molecule. This type of antibodies allows to put in close proximity the tumor cell with an effector cell, a cytokine, etc., or to re-direct the immune response of cytotoxic T cells bypassing antigen recognition through the TCR [bib_ref] Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection, Zhukovsky [/bib_ref].
On all the trials on hematological tumors using bispecific mAb reported here, the same bispecific mAb blinatumomab (biespecific CD19-CD3) was used in different combinations. An example combines blinatumomab with the anti-CD20 mAb rituximab, for non-receptor tyrosine kinase (ABL)-negative B lineage acute lymphoblastic leukemia (NCT02003222). The rationale is to target the CD20 + B cells, while putting in close contact T cells (CD3 + ) with CD19 + B cells, to mount a strong cytotoxic T cell response. The rest of the trials from this group, all of them combine mAb targeting immune-checkpoints (CTLA-4, PD-1, or PD-L1) with the bispecific antibodies. These include blinatumomab in relapsed or refractory precursor B-lymphoblastic leukemia (NCT02879695) or for relapsed or refractory B cell acute lymphoblastic leukemia (NCT03160079). The rationale for these trials is to avoid the suppression of antitumor responses, while putting in close contact T cells (CD3 + ) with CD19 + B cells.
Bispecific antibodies in combination have also been used for the treatment of solid tumors. For most of the examples, one of the arms of the bispecific mAb identifies a tumor-associated antigen, such as the bispecific antibody RO6958688 (bispecific CD3-CEA) combined with atezolizumab, for the treatment of advanced and metastatic solid tumors (NCT02650713). The bispecific antibody RO6958688 [bib_ref] CEA TCB: a novel head-to-tail 2:1 T cell bispecific antibody for treatment..., Bacac [/bib_ref] [bib_ref] A novel carcinoembryonic antigen T-cell bispecific antibody (CEA TCB) for the treatment..., Bacac [/bib_ref] recognizes, on the one side, the CD3 molecule of the TCR and, on the other side, the CEA, an antigen overexpressed in several tumors. The rationale of the trial is to block the binding of PD-L1 to its receptor avoiding the suppression of antitumor responses, while putting in close contact T cells (CD3 + ) with the CEA + tumor cells, inducing a strong T cell activation which may result in a potent antitumor CTL response. Similarly, solid tumors were treated with atezolizumab in combination with bevacizumab or with vanucizumab, a bispecific mAb that simultaneously targets VEGF and Ang-2 (NCT02715531). This treatment aims to block the immune checkpoint suppression while inhibiting angiogenesis.
Another example of targeting tumor-associated antigens is the use of in vitro-activated T cells armed with GD2Bi-aATC, a bispecific antibody that recognizes CD3 and GD2 [bib_ref] Clinical pharmacology and translational aspects of bispecific antibodies, Trivedi [/bib_ref] [bib_ref] Bispecific antibodies and their applications, Fan [/bib_ref] , in combination with IL-2 and GM-CSF in patients with neuroblastoma or osteosarcoma (NCT02173093). The rationale of this study is to generate in vitro activated T cells that are infused in the patient after binding to the bispecific mAb, which will direct them to the tumor, generating a potent CTL response to kill tumor cells. Exogenous IL-2 and GM-CSF are added to maintain these cells and generate an inflammatory environment surrounding the tumor. In addition, another approach that has been used is to combine the anti-HER2 antibody trastuzumab with a bispecific MM-11 mAb anti-ErbB2/anti-ErbB3 mAb [bib_ref] Antitumor activity of a novel bispecific antibody that targets the ErbB2/ ErbB3..., Mcdonagh [/bib_ref] for the treatment of HER2 + breast cancer (NCT01097460). The aim is to simultaneously inhibit signaling through this family of tyrosine kinase receptors. Another approach is to use bispecific mAb to inhibit angiogenesis in solid tumors. The two examples we describe use the bispecific mAb vanucizumab (VEGF-A/Ang-2). In one of them, it is used in combination with RO7009789 (255), an antibody with immunostimulatory effects that recognizes CD40, a member of the TNF receptor superfamily, for the treatment of metastatic solid tumors (NCT02665416). In the other example, it is used either alone or in combination with atezolizumab (PD-L1) in advanced or metastatic solid tumors (NCT01688206). The rationale for both cases is to either use an anti-CD40 agonist or an anti-checkpoint antibody to burst the antitumor immune response, while inhibiting simultaneously angiogenesis by blocking the VEGF-A/VEGFR-and Ang-2/ Tie2-signaling pathways.
## Antibodies with immunomodulatory effects
In a clinical trial for metastatic colorectal cancer, imalumab (BAX69) (233), a mAb that identifies MIF is used in combination with panitumumab (NCT02448810). BAX69 abrogates MIF signaling and MIF-mediated secretion of cytokines (IL-1β, TNFα, etc.) and inhibits proliferation of MIF overexpressing tumor cells, together with the antiproliferative effects of panitumumab (anti-EGFR mAb).
Other examples, used for solid tumors, combine nivolumab with cabiralizumab, an anti-colony-stimulating factor 1 receptor (CSF1R) mAb, which inhibits binding of its ligands (CSF-1 and IL-34), blocking the production of inflammatory mediators by macrophages and monocytes and preventing osteoclast activation (NCT02526017; NCT03158272). The aim is to inhibit the tumorinduced immune suppression with nivolumab, and to block with cabiralizumab the recruitment of CSF1R-dependent tumorassociated macrophages (TAMs). Cabiralizumab also enhances T cell infiltration and antitumor T cell immune responses.
Another immunomodulatory antibody, anti-CD73 (BMS-986179) [bib_ref] Anti-CD73 in cancer immunotherapy: awakening new opportunities, Antonioli [/bib_ref] [bib_ref] Anti-CD73 immunotherapy: a viable way to reprogram the tumor microenvironment, Antonioli [/bib_ref] [bib_ref] CD73: a potent suppressor of antitumor immune responses, Beavis [/bib_ref] , has been used in combination with nivolumab on advanced or spread solid cancers (NCT02754141). In this case, the use of an antibody against the cell surface enzyme CD73 turns out to be very interesting. CD73 is overexpressed in many tumors and catalyzes the conversion of extracellular nucleotides into nucleosides, generating adenosine [bib_ref] CD73: a potent suppressor of antitumor immune responses, Beavis [/bib_ref]. The anti-CD73 antibody prevents the conversion of AMP to adenosine, which releases the inhibition of T cell, DC, and NK activities, induces the activation of macrophages, and reduces the activity of both myeloid-derived suppressor cells and regulatory T cells [bib_ref] Anti-CD73 in cancer immunotherapy: awakening new opportunities, Antonioli [/bib_ref] [bib_ref] Anti-CD73 immunotherapy: a viable way to reprogram the tumor microenvironment, Antonioli [/bib_ref] [bib_ref] CD73: a potent suppressor of antitumor immune responses, Beavis [/bib_ref]. This treatment was designed to abrogate the immunosuppressor effects of both, the immune checkpoint with nivolumab and the metabolic checkpoint with BMS-986179.
In addition to antibodies that release the inhibitory effects of immune-checkpoints, there are other antibodies that are able to directly activate the immune response. The following examples represent clinical trials where these immunostimulatory antibodies are used. One of them combines avelumab, which suppresses the signaling through negative immune checkpoints, with either the anti-cytokine antibody PD-0360324 (anti-CSF-1 mAb) [bib_ref] Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage..., Masek-Hammerman [/bib_ref] ; or with the immunostimulatory antibodies PF-04518600 (anti-OX40 mAb), or utomilumab (an anti-CD137 mAb) (262) (NCT02554812); whereas another combines the anti-OX40 mAb (MOXR0916) with atezolizumab in locally advanced or metastatic solid tumors. (NCT02410512). The aim is to inhibit the PD-1/ PD-L1 axis (avelumab or atezolizumab), simultaneously bursting the immune response through OX-40, CD137, or blocking TAMs generation with the anti-CFS-1 . A similar strategy uses atezolizumab in combination with varlilumab (an agonistic anti-CD27 mAb), which results in an increase of the CTL response against CD27 ligands expressed on tumor cells (NCT02543645).
The anti-CD40 mAb RO7009789 activates and triggers proliferation of antigen-presenting cells (APC) and activates B and T cells, resulting in an enhanced immune response. When CD40 is expressed in solid tumor cells, RO7009789 leads to apoptosis and decreased tumor growth. This antibody in combination with the CSF1R inhibitory antibody emactuzumab has been used for the treatment of advanced solid tumors (225) (NCT02760797). Related examples combine anti-CD40 mAb either with nivolumab for the treatment of metastatic pancreatic adenocarcinoma (NCT03214250) or with the bispecific antibody vanucizumab (anti-VEGF-A and anti-Ang-2) (NCT02665416). The rationale is to activate the immune response through CD40 while inhibiting the angiogenesis blocking the binding of VEGF-A and Ang-2 to their receptors.
## Adjuvants and other immunostimulatory agents
Another possible strategy is to combine therapeutic antibodies with molecules carrying repeated structural motifs that cannot be synthesized by vertebrates, and bind to pattern recognition receptors present in cells from the innate arm of the immune system (i.e., beta glucan that binds the C-type lectin receptor Dectin-1). These molecules are able to regulate the threshold of the immune response as an adjuvant, changing the secreted cytokine expression pattern. A particular example of this type of agents is the use of an attenuated preparation of the BCG (Bacille Calmette-Guerin) strain of Mycobacterium bovis, with potential immunostimulatory activity for the treatment of patients with bladder cancer [bib_ref] BCG immunotherapy for bladder cancer -the effects of substrain differences, Gan [/bib_ref].
An example, combining mAb and adjuvants is the use of BTH1704 [bib_ref] Novel approaches in the management of pancreatic ductal adenocarcinoma: potential promises for..., Goel [/bib_ref] , an mAb against MUC1, an aberrantly glycosylated antigen overexpressed on the surface of a variety of cancer cells, in combination with a polysaccharide beta 1,3/1,6 glucan derived from the cell wall of Saccharomyces cerevisiae (PGG Beta-Glucan), for the treatment of patients with advanced pancreatic cancer (NCT02132403). The rationale of this trial is to directly target the tumor with the anti-MUC1 mAb, while unspecifically stimulate the immune response with beta glucan by binding to an alternate site on the neutrophil complement receptor 3 (CR3), priming the neutrophil to become cytotoxic after binding to complement on tumor cells via CR3. In addition, this agent may induce hematopoietic progenitor cell mobilization.
## Antibodies in combination with vaccines
Tumor cells carry antigens which can be recognized as non-self by the immune system. In some cases, however, the microenvironment in which these tumor antigens are presented do not allow to evoke an immune response. There is a plethora of possibilities to burst the antitumor immune response, one of them is to use tumor antigens as a vaccine. In the context of antitumor therapies, anti-idiotipic antibodies represent a particular type of vaccines.
Since the interaction of antibodies with other molecules is based on structural complementarity, antibodies that recognize the region of an antibody that interacts with its antigen (antiidiotipic antibodies) might mimic the structure of this antigen (i.e., a tumor marker). Thus, the anti-idiotipic antibodies might act as an antitumor vaccine able to trigger a host immune response to kill tumor cells. Combining chemotherapy and radiation therapy with vaccine therapy may help to kill tumor cells more effectively.
An example of anti-idiotipic antibodies used as vaccines in antitumor therapy is abagovomab, an IgG1 anti-idiotype mAb, that functionally mimics the 3D structure of a specific epitope on the ovarian cancer tumor-associated antigen CA-125 [bib_ref] Phase I study of abagovomab in patients with epithelial ovarian, fallopian tube,..., Sabbatini [/bib_ref]. Its variable region acts as a surrogate antigen for CA-125, bestowing potential antineoplastic activity; it has been used in ovarian epithelial, fallopian tube, or peritoneal cancer (NCT00058435). Another example of vaccine therapy combines two anti-idiotipic antibodies, 11D10 (201) an mAb that mimics an epitope of the high molecular weight human milk fat globule glycoprotein, expressed at high levels by human breast and other tumor cells and 3H1 an mAb that mimics an epitope of the tumor-associated protein CEA [bib_ref] Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen, Pervin [/bib_ref]. This combination has been used for the treatment of colorectal cancer metastatic to the liver (NCT00033748). The 11D10 mAb has also been used in other combinations, for example, with a GD2 anti-idiotype mAb vaccine, together with chemotherapy and radiotherapy, for the treatment of limitedstage SCLC (NCT00045617).
A study combining the anti-idiotipic mAb abagovomab, which mimics a specific epitope on CA-125 with stereotactic body radiation therapy (SBRT), chemotherapy, and the synthetic antiviral agent nelfinavir mesylate, which selectively binds to and inhibits human immunodeficiency virus protease, has been used for the treatment of locally advanced pancreatic cancer (NCT01959672).
Currently, other types of vaccines are being used with mAb in combination with agents that allow to evoke an immune response against tumor antigens. The mAb in these combinations may strengthen the evoked immune response. For the examples described below, the combination contains antibodies that either disrupt the PD-1/PDL-1 axis or that block the binding of B7-1 and B7-2 to CTLA-4 allowing T cell co-stimulatory signals and activation, unless otherwise specified. One example is the treatment with a peptide from Wilms tumor 1 antigen on recurrent ovarian cancer (NCT02737787). Another, more complex example is to administrate the peptide vaccine PVX-410 (derived from X-box-binding protein 1-unspliced XBP1-US, XBP1-spliced syndecan-1, and CS1), to treat triplenegative breast cancer tumors (NCT02826434). A third example uses a HER2 intracellular domain peptide in combination with the polysaccharide-K as adjuvant, in HER2 + recurrent breast cancer patients, which are receiving pertuzumab or trastuzumab (NCT01922921). The rationale for this trial is to combine the effects of the anti-HER2 mAb with using a HER2 peptide to switch the B and T cell responses through APC activation. Otherwise, peptides could be presented as a fusion protein, such as in CIMAvax vaccine (EGF-rP64K/Montanide ISA 51), which triggers a strong humoral immune response against EGF and has been used in NSCLC (NCT02955290). There are also personalized neoantigen cancer vaccines, such as NeoVax for the treatment of high-risk renal cell carcinoma (NCT02950766).
Another approach to generate vaccines is to use modified virus such as Ad-CEA vaccine, an oncolytic adenovirus encoding an epitope of human CEA (331), used for the treatment of patients with previously untreated metastatic colorectal cancer (NCT03050814). This vaccine may induce both humoral and cellular immune responses against CEA + tumor cells.
A different example uses the CV301 (CEA-MUC-1-TRICOM Vaccine) viral vaccine, which contains a version of the recombinant vaccinia viral vector and a recombinant fowlpox viral vector encoding both CEA and MUC-1, in combination with TRICOM [co-stimulatory molecules, B7-1, intracellular adhesion molecule 1 (ICAM-1), and LFA-3] (332). It may enhance presentation of CEA and MUC-1 to APC and subsequently a CTL response against the tumor cells, it has been used in previously treated NSCLC (NCT02840994). A similar approach has been evaluated for the treatment of prostate cancer using a recombinant vaccinia virus encoding a modified peptide of the prostate-specific antigen and TRICOM (NCT00113984). This viral vaccine may enhance antigen presentation and may activate a CTL response.
Attenuated bacteria have also been used as carriers for antitumor vaccines. For example, the attenuated Listeria ADXS11-001 encoding a papillomavirus type 16 E7 fused to a non-hemolytic listeriolysin O protein, it has been used for the treatment of cervical and Head and Neck Cancer (HNSCC) (NCT02291055). The rationale is to mount a CTL response against cancer cells overexpressing the cell surface glycoprotein HPV 16 E7, overexpressed in the majority of cervical cancer cells.
More sophisticated approaches use tumor vaccines, such as GM.CD40L, which is a cell-based vaccine composed of irradiated tumor cells transduced with GM-CSF and CD40-ligand (CD40L) genes [bib_ref] A phase-I trial using a universal GM-CSF-producing and CD40L-expressing bystander cell line..., Dessureault [/bib_ref]. Upon administration, this vaccine may stimulate an antitumoral DC-mediated immune response, it has been used in lung adenocarcinomas (NCT02466568). Another example that does not use anti-checkpoint antibodies but combines the anti-HER2 mAb trastuzumab with a cell-based vaccine, consisting of two irradiated allogeneic mammary carcinoma cell lines genetically modified to secrete human GM-CSF, has been used for the treatment of HER2 + metastatic breast tumors (NCT00399529). An additional example of cell-based vaccines uses GVAX (334), a pancreatic cancer vaccine and IMC-CS4, a macrophage targeting mAb (CSF1R inhibitor) for the treatment of pancreatic adenocarcinomas [bib_ref] Antibodies directed against receptor tyrosine kinases: current and future strategies to fight..., Fauvel [/bib_ref]. GVAX is composed of irradiated, whole tumor cells (autologous or allogeneic), genetically modified to secrete GM-CSF (NCT03153410).
The immune response against the tumor can be busted using also DNA, RNA, or liposome-based vaccines. As an example, triple-negative breast cancers have been treated with neoantigen DNA vaccine combined with anti-immune-checkpoint antibodies (NCT03199040). Other vaccines use autologous DC loaded in vitro with Cytomegalovirus pp65-lysosomal-associated membrane protein mRNA as a vaccine, in combination with the anti-IL2R alpha mAb basiliximab, in glioblastoma multiform (NCT00626483). The rationale is to restore the number of immunosuppressive T regulatory cells during recovery from therapeutic temozolomide-induced lymphopenia, together with a synergistic enhancement of vaccine-driven CTL responses. Another study describes the use of the immuno-modulating mAb varlilumab (anti-CD27, TNFR family) in combination with a liposome-based vaccine consisting of two peptides from MUC1 and the toll-like receptor 4 encapsulated in liposomes (NCT02270372). This immunization stimulates both cellular and humoral responses.
Another clinical trial uses trastuzumab and an allogeneic GM-CSF-secreting whole cell breast cancer vaccine for HER-2 + breast tumors. This study will also test whether cyclophosphamide can eliminate the suppressive influence of regulatory T cells. The vaccine consists of two irradiated allogeneic mammary carcinoma cell lines genetically modified to secrete human GM-CSF (NCT00399529).
Pidilizumab (anti-PD-1) (250) in combination with a DC fusion vaccine, following autologous stem cell transplantation has been used on multiple myeloma (NCT01067287). The rationale is to disrupt the PD-1/PD-L1 axis, while vaccinating the patients with de DC fusion vaccine, which consists of DC fused to the patient's myeloma, where the myeloma antigens will be presented by HLA class I to CD8 + T cells, allowing their activation and mounting a CTL response. This is done on patients after an autologous transplantation with HSCs. A similar approach uses nivolumab vaccine with autologous DCs pulsed with tumor lysate antigen, in patients with recurrent glioblastoma (NCT03014804).
Another study combines lymphodepletion with anti-CD45 mAb with a vaccine generated from autologous DC and Epstein Barr virus (EBV)-infected lymphoblastoid cell lines transduced with an LMP1/LMP2-expressing adenoviral vector, which are irradiated, and then used to stimulate and expand autologous CTL to produce LMP1-/LMP2-specific CTL ex vivo, for the treatment of EBV + -nasopharyngeal carcinoma (NCT00515957). The rationale of this trial is to deplete autologous CD45 + cells, then generate a cell-based vaccine to activate ex vivo specific CTL that are then infused into the patient.
## Other strategies
In some approaches, a fusion protein between the antibody and a tumor antigen is used in combination with other therapies. For example, on the treatment of NY-ESO 1 + NSCLC, where atezolizumab was combined with both the adjuvant poly-ICLC (a synthetic complex of carboxymethylcellulose, polyinosinicpolycytidylic acid and poly-L-lysine double-stranded RNA) and DEC-205/NY-ESO-1 [CDX-1401, a fusion protein between a mAb directed against the endocytic DC receptor DEC-205, linked to the tumor-associated antigen (NY-ESO-1)] (NCT02495636) [bib_ref] Induction of antigen-specific immunity with a vaccine targeting NY-ESO-1 to the dendritic..., Dhodapkar [/bib_ref]. Atezolizumab will inhibit immune checkpoints negative signals, while the internalization by DC of the mAb-antigen fusion protein may specifically deliver the NY-ESO-1 molecule and trigger a CTL response against cancer cells expressing this antigen. Simultaneously, the adjuvant may stimulate the release of cytotoxic cytokines by inducing IFNgamma production. A similar approach, for the treatment of melanoma patients, using CDX-1401 combined with a neoantigen-based melanoma-poly-ICLC vaccine and a recombinant Flt3 Ligand (CDX-301) (NCT02129075). This treatment should boost the immune system to mount a CTL response against cancer cells expressing NY-ESO-1. In addition, the adjuvant may induce IFNgamma production and the recombinant Flt3 ligand may stimulate the proliferation and mobilization of bone marrow precursor cells, including CD34 + cells, and DCs.
An additional strategy would be to use scavengers of the ligand with low immunogenicity. For example, sEphB4-HAS, a human serum albumin (HAS) fused with the extracellular domain of tyrosine kinase ephrin type-B receptor 4 (sEphB4) is combined with pembrolizumab, for the treatment of NSCLC or HNSCC (NCT03049618). Pembrolizumab inhibits negative immune checkpoint signals, whereas EphB4-HSA is expected to decrease angiogenesis and cell growth of Efnb2 and/or EphB4 overexpressing tumor cells, while the albumin moiety will avoid renal clearance of the fusion protein, increasing its half-life without affecting immunogenicity.
Another strategy would be to target matrix enzymes required for tumor invasiveness. An example is the use of nivolumab combined with andecaliximab (GS-5745) [bib_ref] Selective allosteric inhibition of MMP9 is efficacious in preclinical models of ulcerative..., Marshall [/bib_ref] [bib_ref] Biochemical characterization and structure determination of a potent, selective antibody inhibitor of..., Appleby [/bib_ref] , an inhibitory mAb of matrix metalloproteinase 9 (MMP-9) in recurrent gastric or gastroesophageal junction adenocarcinomas (NCT02864381). Since MMP-9 activity is associated with tumor invasion and metastasis [bib_ref] Tumor cell-produced matrix metalloproteinase 9 (MMP-9) drives malignant progression and metastasis of..., Mehner [/bib_ref] , the rationale is that andecaliximab will inhibit extracellular matrix protein degradation and angiogenesis, while nivolumab will interfere with the PD-1/PD-L1 axis.
Pembrolizumab (PD-1) was used in combination with CVA21 (CAVATAK™), coxsackievirus A21, a naturally occurring enterovirus with potential antitumor activity. This combination was used for advanced NSCLC (NCT02824965). CVA21, intratumor administered, targets and binds the ICAM-1 and decay acceleration factor, cell surface molecules, both overexpressed on certain malignant cells [bib_ref] Errington-Mais F. Applications of coxsackievirus A21 in oncology, Bradley [/bib_ref] [bib_ref] Systemic targeting of metastatic human breast tumor xenografts by coxsackievirus A21, Skelding [/bib_ref]. After entering the cells, the virus replicates causing cell lysis. This, together with the inhibition of the immune checkpoint, results in a reduction of tumor cell growth.
Another strategy combines conatumumab [bib_ref] Conatumumab, a fully human mAb against death receptor 5 for the treatment..., Rosevear [/bib_ref] , an agonist mAb directed against the extracellular domain of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), also known as DR5, with the anti-IGF-1R mAb ganitumab, in patients with advanced solid tumors without disease progression whose previous studies were closed (NCT01327612). TRAIL-2 and IGF-1R are expressed by a variety of solid tumors and cancers of hematopoietic origin. Conatumumab mimics TRAIL activity, activating caspase cascades and inducing tumor cell apoptosis, while ganitumab inhibits IGF-1 binding and, therefore, the PI3K/Akt pathway. This treatment may result in the inhibition of tumor cell proliferation and the induction of tumor cell apoptosis. Another clinical trial combining tigatuzumab, a mAb targeting the death receptor TRAIL-R2 with abraxane, an albumin-stabilized nanoparticle containing paclitaxel, noncovalently coated with the anti-CD20 mAb rituximab, in patients with metastatic, triple-negative breast cancer (NCT01307891). The relevance of the trial is that combines an anti-TRAIL-R2 mAb that induces death, while albumin stabilizes the complex, whereas rituximab allows to target paclitaxel to CD20 + cells, minimizing toxicity on normal cells. A strategy being used on EGFR + tumors is to combine an anti-immune checkpoint antibody with the anti-EGFR mAb necitumumab (NCT02451930) or nimotuzumab (245) (NCT02947386). Or the same strategy, but using B-701, a neutralizing mAb directed against the fibroblast growth factor receptor type 3, in combination with atezolizumab in urothelial cell carcinoma (NCT03123055).
One of the most sophisticated clinical trials combines chemotherapy, bevacizumab, avelumab, ALT-803 (IL-15 super agonist), aNK (allogenic human NK-92 cell line, expressing CD16 and IL-2), and GI-4000 (a heat-killed recombinant Saccharomyces cerevisiae yeast transfected with mutated forms of Ras) with the NANT pancreatic cancer vaccine (ETBX-011) containing a replicationdefective adenoviral vector encoding a CEA epitope Ad5-CEA(6D), used for pancreatic cancer (NCT03136406). A clinical trial for colorectal cancer uses a similar combination of chemotherapy, nivolumab, avelumab bevacizumab, cetuximab, SBRT, haNK, ALT-803, and a cocktail of vaccines: ETBX-011, ETBX-021, ETBX-051, ETBX-061, GI-4000, GI-6207, and GI-6301 (NCT03169777). The rationale for these two clinical trials is to hit the tumor simultaneously with a wide spectrum of the available tools against the tumor, where the concentration of each one of the agents can be decreased to minimize the unwanted effects on normal cells.
## Questions and queries raised by the combinations
We hope that we have been able to depict up to here, the huge complexity inherent to the use of therapeutic antibodies in combination with other biological agents for the treatment of cancer. Since most of the clinical trials described in this review are relatively recent (started during the last 7 years), many of them lack results in public databases, including the clinical trials database from NCI, or as published scientific manuscripts. This burst of clinical trials using antibodies in combinations with other biologicals is based on the positive results found by some combinations (anti-HER2 or anti-GD2 mAb), although the complexity increase in these combinations also implies an increase in the possibility of adverse side effects/increased toxicity or lack of additive or synergistic effects of the therapeutic agents. Initially, the therapeutic doses used for combinations were taken from the monotherapeutic trials, although in many cases, the non-toxic concentrations used in monotherapy, turn to be toxic in combinations, generating new toxicity profiles [bib_ref] New drugs, new toxicities: severe side effects of modern targeted and immunotherapy..., Kroschinsky [/bib_ref]. This is of particular relevance when antibodies able to burst the antitumor immune response are used (either to inhibit the immune checkpoint proteins, to block inhibitory NK receptors or to trigger NK cells through activating receptors, etc.), which might lead to a dis-regulation of the immune response, an uncontrolled inflammatory response, and autoimmunity. This problem can also be related to an apparent lack of additive or synergistic effects of the therapeutic agents, where the potential clinical benefits of the combination could be overlooked by the initial toxicity of the mixture. Dose and schedule changes, however, can overcome the toxicity effects, allowing to demonstrate the enhanced clinical benefits of a particular combination [bib_ref] Combination immunotherapy: a road map, Ott [/bib_ref]. The use of CTLA-4 and PD-1 inhibitor antibodies in combination (nivolumab and ipilimumab), improved the treatment efficacy in advanced melanoma, as compared to monotherapies [bib_ref] Nivolumab plus ipilimumab in advanced melanoma, Wolchok [/bib_ref]. Indeed, this combination has been approved in 2016 by the US FDA for the treatment of metastatic melanoma (341), despite the higher frequency and severity of adverse reactions of the combination, as compared to the corresponding monotherapies [bib_ref] Nivolumab plus ipilimumab in advanced melanoma, Wolchok [/bib_ref] [bib_ref] The development of immunotherapy in older adults: new treatments, new toxicities?, Helissey [/bib_ref] [bib_ref] Combined immune checkpoint blockade (anti-PD-1/anti-CTLA-4): evaluation and management of adverse drug reactions, Hassel [/bib_ref] [bib_ref] Combination immune checkpoint blockade with ipilimumab and nivolumab in the management of..., Spain [/bib_ref].
It is interesting to note that a combination of TRC105 (carotuximab, anti-endoglin antibody) with bevacizumab was used on a clinical trial for the treatment of patients with advanced cancer, where the combination was well tolerated and clinical activity was observed in a VEGF inhibitor-refractory population (NCT01332721) (345), the same combination failed to improve progression-free survival on patients with refractory metastatic renal cell cancer (NCT01727089) [bib_ref] Bevacizumab alone or in combination with TRC105 for patients with refractory metastatic..., Dorff [/bib_ref]. These data clearly suggest that the problem does not strictly lie with the antibody combination, but rather it might be related to the tumor microenvironment, tumor type, the therapeutic approach used, or the clinical history of the patient.
Several examples of clinical trials where antibodies that have been used in combination with other biologicals, which were well tolerated and showed additive or synergistic therapeutic responses have been selected. These include a clinical trial for melanoma patients with low tumor infiltrating lymphocytes, an anti-PD-1 non-responsive phenotype. The combination of pembrolizumab with an intratumoral electroporation of a plasmid coding for interleukin 12 cDNA (pIL-12) showed a 40% clinical response with associated positive immune-based biomarker data and a safety profile (NCT02493361) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Althammer [/bib_ref] , where the combination of pembrolizumab with the plasmid pIL-12 renders half of the patients responsive to the anti-PD-1. Another example that combines mAb with cytokines is a study of immune activation and antitumor activity in renal cancer of PEGylated human IL-10 (AM0010) in combination with pembrolizumab or nivolumab, the combinations were well tolerated, and CD8 + T cell activation was detected (NCT02009449) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Ager [/bib_ref].
Other examples include a phase Ib study, otlertuzumab (TRU-016, an anti-CD37 mAb) in combination with rituximab and bendamustine, which was well tolerated and induced therapeutic responses in the majority of patients with relapsed indolent B-non-Hodgkin lymphoma (NCT01317901) [bib_ref] Phase 1b study of otlertuzumab (TRU-016), an anti-CD37 monospecific ADAPTIR therapeutic protein,..., Gopal [/bib_ref]. Similarly, the combination of pidilizumab plus rituximab was well tolerated and therapeutically active in patients with relapsed follicular lymphoma [bib_ref] Safety and activity of PD1 blockade by pidilizumab in combination with rituximab..., Westin [/bib_ref]. On a phase Ib study of utomilumab (PF-05082566, a 4-1BB/ CD137 agonist), in combination with pembrolizumab (MK-3475) in patients with advanced solid tumors had a confirmed complete or partial response in 26.1% of them. Pharmacokinetics and immunogenicity of both mAb were similar when administered alone or in combination. A trend toward higher levels of activated memory/effector peripheral blood CD8 + T cells was observed in responders versus non-responders, supporting further investigation of this combination (NCT02179918) [bib_ref] Phase Ib study of utomilumab (PF-05082566), a 4-1BB/ CD137 agonist, in combination..., Tolcher [/bib_ref].
Other combinations include the anti-checkpoint antibody nivolumab in combination with an antibody that blocks the KIR inhibitory receptors in NK cells. On a phase I/II study of the NK-targeted antibody lirilumab (a fully human mAb that blocks inhibitory KIRs on NK cells) in combination with nivolumab in advanced HNSCC demonstrated, in preliminary results, clinical benefit, with deep and durable responses in some patients. This combination demonstrated a manageable safety profile similar to that observed with nivolumab monotherapy (NCT01714739) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Althammer [/bib_ref].
Another combination that might be interesting for the future of the field is the combination of oncolytic virus with anti-checkpoint antibodies. Indeed, preliminary results from several clinical trials using the oncolytic virus coxsackievirus A21 (CVA21, CAVATAK) in combination with ipilimumab (NCT01636882) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Lundqvist [/bib_ref] or pembrolizumab (NCT02043665) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Ager [/bib_ref] , for the treatment of patients with advanced cancer, showed that these combinations were generally well tolerated and induced antitumor activity. A phase II trial using intratumoral injection of the HF10 oncolytic virus, an attenuated, replication-competent mutant strain of herpes simplex virus type 1, and ipilimumab in patients with unresectable or metastatic melanoma showed therapeutic activity and the treatment was well tolerated (NCT02272855) [bib_ref] Genomic signature of the natural oncolytic herpes simplex virus HF10 and its..., Eissa [/bib_ref].
For other clinical trials, on early phases, the combination is well tolerated, such as a dose escalation study of the OX40 agonist MOXR0916 and atezolizumab (anti-PD-L1 mAb) in patients with advanced solid tumors, using each agent at its recommended monotherapy dose, was well tolerated. (NCT02410512) [bib_ref] A phase lb dose escalation study of the OX40 agonist MOXR0916 and..., Infante [/bib_ref].
Other clinical trials, including anti-checkpoint antibodies detect clear tumor regression, although with a toxicity higher than reasonable. This is the case for a clinical trial were BMS-986016 (anti-LAG-3 mAb) in combination with nivolumab was administered to patients with hematologic and solid malignancies. Preliminary results demonstrated objective tumor regressions, concomitant with the toxicity characteristic of immune checkpoint blockers (NCT02061761, NCT01968109) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Ager [/bib_ref].
On another group of clinical trials, the main characteristic is that although they are well tolerated in general, they failed to provide significant additive/synergistic therapeutic effects. This is the case of a clinical trial where urelumab (a CD137 agonist), in combination with nivolumab was used for the treatment of hematologic and solid tumor malignancies. This combination did not provide significant additive/synergistic clinical benefits at the doses evaluated (NCT01471210, NCT02253992) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Lundqvist [/bib_ref]. In another, urelumab in combination with rituximab or cetuximab was used in patients with refractory lymphoma or selected advanced solid tumors. Although the combinations were safe and well tolerated, with minimal evidence of liver toxicity, they did not demonstrate substantial enhancement of clinical responses or lead to intratumoral immune modulation in these tumor settings (NCT01775631, NCT02110082) [bib_ref] 31st annual meeting and associated programs of the society for immunotherapy of..., Ager [/bib_ref].
Finally, there are a couple of selected clinical trials using combinations of antibodies and other biologicals that were toxic or had to be terminated on overall benefit-risk assessment. These include a clinical trial with patients with advanced solid tumors, which were treated with a combination of MDX-447 [a bispecific mAb directed to FcγRI (CD64) and EGFR] with G-CSF, although the bispecific mAb alone was well tolerated, the combination was not well tolerated and precluded meaningful dose escalation on a phase I clinical trial [bib_ref] A phase-I trial of the epidermal growth factor receptor directed bispecific antibody..., Fury [/bib_ref]. A second example is a phase II study of imalumab [BAX69, an anti-oxidized macrophage MIF (oxMIF)] and 5-FU/Leucovorin or Panitumumab (anti-EGRF mAb), versus the standard of care in metastatic colorectal cancer patients, which was terminated (February, 2017) based on overall benefit-risk assessment (NCT02448810), although it was initially reported that this combination was generally safe and well tolerated [bib_ref] Safety and efficacy analysis of imalumab, an anti-oxidized macrophage migration inhibitory factor..., Mahalingam [/bib_ref].
The problems that arose with using therapeutic antibodies in combinations has led the Society for Immunotherapy of Cancer to name a Combination Immunotherapy Task Force to identify and prioritize the most promising prospects for combinatorial approaches as well as to address the challenges associated with developing these strategies [bib_ref] Combination immunotherapy: a road map, Ott [/bib_ref]. Furthermore, it seems clear by now, that an improved understanding of pharmacodynamic effects of each agent within a combination will support the rational development of immune-based combinations for cancer treatment [bib_ref] Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinct immunologic changes in..., Das [/bib_ref].
# Conclusion
The broad variety of clinical trials summarized here presents the overwhelming complexity of the use of antibody in combinations for cancer treatments. The antibodies used either interfere with a ligand-receptor interaction, blocking a signaling pathway relevant for tumor growth, or identify tumor-associated antigens, where they somehow induce the death of the tumor cells by ADCC, CDC, and ADCP or directly inducing apoptosis. If the antibodies by themselves cannot kill the tumor cells, they can be conjugated to cytotoxic drugs to exert this function, or directly coupled to radio-labeled agents, where they trigger radiolysis. These antibodies can be combined with other antibodies that rather than directed against the tumor cells, identify targets on the tumor environment. These include antibodies that inhibit tumorinduced vascularization, or antibodies against cells or molecules involved in the immune response. A turning point was the use of mAb that block PD-L1/PD-1 interaction, or anti-CTLA-4 mAb; all of them disrupting the tumor-induced suppression of antitumor immune responses. In addition, mAb are used in combination with vaccines with the aim of evoking an antitumor response, either against a single tumor antigen or against a broad spectrum of antigens, for example when using irradiated tumor cells expressing pro-inflammatory cytokines.
It is obvious that there are many challenges to be solved regarding antibodies in combinations as antitumor therapeutic agents. One of the most relevant challenges is the possible increase of toxicity of the combinations, concomitant with an increased complexity of the trials. This has to be carefully evaluated for each combination to identify the conditions giving the highest efficacy/toxicity ratio. This is of particular interest when the aim is to burst the antitumor host immune response, where a small response could fail to kill the tumor, but an over-response could lead to unwanted inflammatory or autoimmune processes. Another relevant challenge to be solved is the identification of new/additional biomarkers that would allow a personalized follow-up of the patient's status, which would be required due to the tumor and the patient's genotypic and phenotypic differences.
The current clinical trials suggest that in the future anti-cancer therapies will combine antibodies that block signaling cascades, or identify tumor-associated antigens with others that disrupt the tumor-induced immuno-suppression, together with vaccines that evoke an antitumor immune response, activated effector cells or CAR T cells. These combinations could also contain anti-cytokine antibodies, or cytokines, to burst the immune response. The aim will be to directly target tumor antigens or signal pathways, and at the same time interfere with the "immune history" of the organism, to make the patient's own body aware of the tumor presence and simultaneously help it to make a strong antitumor response. Thus, it will be the patient's immune response against the tumor the ultimate mechanism responsible for the cure of cancer.
# Author contributions
All authors contributed to drafting, revising, and approving the final article.
# Acknowledgments
The authors thank all the members of their laboratories for critical reading of the manuscript and helpful suggestions.
# Funding
The work in the author's laboratories was partially supported by grants from the CSIC (PIE-201420E109, to LK), the PN2014-A from the ISCIII (PI14/00703, co-financed by FEDER funds from the EU, Operative program on Intelligent Growth 2014-2020, to LK), and from the Spanish Ministry of Economy, Industry and Competitiveness (RTC-2015-3786-1 to LK and JG-S, and RTC-2015-3846-1 to JG-S), both co-financed by FEDER funds from the EU.
# Supplementary material
The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2017.01804/ full#supplementary-material.
[table] TAble 1 |: Antibodies approved (or in review) by the FDA and/or EMA for the clinical treatment of solid tumors. a [/table]
[table] TAble 2 |: Antibodies approved (or in review) by the FDA and/or EMA for the clinical treatment of hematologic neoplasias. a [/table]
[table] TAble 3 |: Characteristics of the main target molecules identified by therapeutic antibodies used in oncology. [/table]
[table] TAble 4 |: Summary of the therapeutic antibodies not yet approved for clinical treatments. a [/table]
|
A novel de novo Myocilin variant in a patient with sporadic juvenile open angle glaucoma
Background: Glaucoma is a leading cause of irreversible blindness. Pathogenic variants in the Myocilin gene (MYOC) cause juvenile open angle glaucoma (JOAG) in 8-36 % of cases, and display an autosomal dominant inheritance with high penetrance. Molecular diagnosis is important for early identification as therapies are effective in minimizing vision loss and MYOC variants can be associated to severe glaucoma. MYOC variants are usually inherited, however a fifth of carriers do not report a family history. The occurrence of de novo MYOC variants is currently unknown. Case presentation: In this study we investigated a 14 year old male Caucasian patient diagnosed with JOAG, and no family history of glaucoma. A novel probably deleterious MYOC:p.(Pro254Leu) variant was identified in the index case. This variant was not present in the parents or the siblings. Conclusion: This is the second report of a de novo MYOC variant in a sporadic case of JOAG and it is currently unknown if this mechanism occurs more frequently. This finding emphasizes the importance of screening individuals with JOAG for MYOC mutations irrespective of a negative family history.
# Background
Glaucoma is one of the leading causes of irreversible blindness affecting over 60 million individuals worldwide [bib_ref] The number of people with glaucoma worldwide in 2010 and 2020, Quigley [/bib_ref]. Primary open angle glaucoma (POAG, MIM 137760) is the most common type and is characterized by changes in the optic nerve head with corresponding visual field loss in the presence of an open anterior chamber angle [bib_ref] Genetic risk of primary open-angle glaucoma. Population-based familial aggregation study, Wolfs [/bib_ref]. Juvenile open angle glaucoma (JOAG) refers to a younger age at diagnosis usually defined by an onset before 30-40 years old and associated with a more severe phenotype [bib_ref] The distinction between juvenile and adult-onset primary open-angle glaucoma, Wiggs [/bib_ref] [bib_ref] Clinical and genetic characteristics of primary juvenileonset open-angle glaucoma (JOAG), Turalba [/bib_ref]. Therapies for POAG aim at controlling intraocular pressure (IOP) and are usually effective in minimizing disease progression [bib_ref] Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest..., Heijl [/bib_ref]. However, the early stages are often asymptomatic and half of the cases remain undiagnosed, making it challenging to implement treatment before irreversible vision loss occurs.
Pathogenic sequence variants in the MYOC gene (MIM 601652) have been first described in association with JOAG in 1997 [bib_ref] Identification of a gene that causes primary open angle glaucoma, Stone [/bib_ref]. Since then, they have been consistently identified in 2-4 % of adult-onset POAG [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref] [bib_ref] Analysis of myocilin mutations in 1703 glaucoma patients from five different populations, Fingert [/bib_ref] and in 8-36 % of JOAG [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref] [bib_ref] Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma, Shimizu [/bib_ref] [bib_ref] Prevalence of mutations in TIGR/ Myocilin in patients with adult and juvenile..., Wiggs [/bib_ref] among different ethnicities. MYOC comprises three exons which encode a protein consisting of two major domains, an N-terminal myosin-like domain and a C-terminal olfactomedin-like domain [bib_ref] How significant is a family history of glaucoma? Experience from the Glaucoma..., Green [/bib_ref]. Most disease causing variants are clustered within exon 3 in the olfactomedin domain [bib_ref] Complex genetics of complex traits: the case of primary open-angle glaucoma, Hewitt [/bib_ref]. The pathophysiology is not fully understood but it has been postulated that the accumulation of misfolded proteins lead to endoplasmic reticulum stress, which compromises the trabecular meshwork cells regulating the IOP [bib_ref] A molecular mechanism for glaucoma: endoplasmic reticulum stress and the unfolded protein..., Anholt [/bib_ref]. MYOC pathogenic variants are inherited in an autosomal dominant fashion and are often associated with high IOP, younger age at diagnosis and strong family history and can result in severe glaucoma and blindness if left untreated [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref] [bib_ref] Analysis of myocilin mutations in 1703 glaucoma patients from five different populations, Fingert [/bib_ref] [bib_ref] Myocilin allele-specific glaucoma phenotype database, Hewitt [/bib_ref].
The majority of MYOC carriers report a family history of glaucoma, however sporadic cases still account for 20 % of mutation carriers [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref]. It is currently unknown whether sporadic cases could be explained by de novo variants. In this study, we report a JOAG sporadic case with a novel de novo MYOC variant, and discuss the occurrence of de novo variants in MYOC associated glaucoma and the implications for the patient and his family.
## Case presentation
## Clinical presentation
The pedigree of the family is shown in [fig_ref] Figure 1: See legend on next page [/fig_ref]. The index case and his family were referred to the Australian and New Zealand Registry of Advanced Glaucoma (ANZRAG) through his treating ophthalmologist [bib_ref] Australian and New Zealand Registry of Advanced Glaucoma: methodology and recruitment, Souzeau [/bib_ref]. The proband was a 14 year old Caucasian male patient (II-1). He was referred to an ophthalmologist following a routine optometrist review for his glasses prescription which revealed high IOP. Following examination, he was diagnosed with JOAG. His IOP at presentation were 31 mmHg in the right eye and 32 mmHg in the left. His vertical cup-todisc ratio was 0.85 right and 0.8 left, and he had central field loss involving fixation in the right eye (Humphrey Field Analyzer, Zeiss) [fig_ref] Figure 2: Clinical presentation of the index case [/fig_ref]. His visual acuity was 20/20 in both eyes. His IOP was initially under control with latanoprost and brimonidine/timolol. However he underwent bilateral trabeculectomies following his most recent IOP which were 40 mmHg. Optic nerve appearances and retinal nerve fiber layer loss (Spectralis®, Heidelberg Engineering) are depicted in [fig_ref] Figure 2: Clinical presentation of the index case [/fig_ref] and c. His parents and two siblings had normal eye examinations.
## Genetic testing
Genetic testing was performed through the National Association of Testing Authorities (NATA) accredited laboratories of SA Pathology at the Flinders Medical Centre in Adelaide, Australia. The proband was sequenced for the 3 coding exons of the MYOC gene as previously described [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref]. A heterozygous substitution of Thymine for Cytosine at nucleotide 761 of the MYOC exon 3 coding sequence was identified (MYOC:c.761C > T), encoding a missense substitution of Proline to Leucine at position 254 (p.(Pro254Leu)) [fig_ref] Figure 1: See legend on next page [/fig_ref]. No other variants were identified in the MYOC gene of the proband. JOAG can also be associated with CYP1B1 variants [bib_ref] Occurrence of CYP1B1 Mutations in Juvenile Open-Angle Glaucoma With Advanced Visual Field..., Souzeau [/bib_ref]. The coding region of the CYP1B1 gene was sequenced to exclude other causative genes. No disease-causing variants were identified in CYP1B1.
The p.(Pro254Leu) variant is novel since it was absent from the MYOC Database (www.myocilin.com), NCBI dbSNP (www.ncbi.nlm.nih.gov/SNP/), and the Exome Aggregation Consortium (http://exac.broadinstitute.org/) which comprises exome sequence data spanning 60 706 unrelated individuals. A search of the scientific literature also failed to identify any reference to this variant. However, a recent study reported a MYOC variant at the same residue p.(Pro254Arg) in a patient with JOAG and his affected mother [bib_ref] Identification of a novel MYOC mutation in a Chinese family with primary..., Yang [/bib_ref]. SIFT and Polyphen-2 both predicted this variant to be deleterious, with sequence alignment demonstrating this position to be highly conserved among vertebrates and other olfactomedin domain-containing proteins [fig_ref] Figure 1: See legend on next page [/fig_ref]. MYOC is a well characterized gene and codon position 254 resides in the core hydrophobic β-sheet belt of the olfactomedin domain, which is important in protein-protein interactions and is sensitive to aggregation in the presence of substitutions [bib_ref] Structural basis for misfolding in myocilin-associated glaucoma, Donegan [/bib_ref]. The p.(Pro254Leu) variant is likely pathogenic based on bioinformatics prediction, invariant conservation of this residue, and characterization of the protein structure. MYOC disease-causing variants can be associated with severe glaucoma and blindness [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref]. In the view of the genetic result and the patient's most recent IOP, bilateral trabeculectomies were performed to better control his IOP and minimize damage on his optic nerves.
This variant was not detected in either parent of the index case [fig_ref] Figure 1: See legend on next page [/fig_ref]. The marker profile comparison using the AmpFLSTR® Identifiler® PCR Amplification Kit confirmed a profile consistent with the proband being the biological child of the stated parents, indicating p.(Pro254Leu) has arisen de novo in the proband. A de novo MYOC pathogenic variant, p.(Val251Ala), has been previously reported once in a JOAG case [bib_ref] A de novo MYOC mutation detected in juvenile open angle glaucoma associated..., Kuchtey [/bib_ref]. Interestingly, this variant was located three amino acids from p.(Pro254Leu) which was identified in this study.
While the occurrence of de novo pathogenic variants in the genome vary considerably based on genomic location, they are estimated to be common and have been linked to many sporadic diseases [bib_ref] De novo mutations in human genetic disease, Veltman [/bib_ref]. Conditions with dominant inheritance and modest fitness effect are more commonly inherited than caused by de novo variants, and this is the situation for MYOC associated glaucoma which is usually inherited. For example, a founder effect with an origin prior to the European settlement of Australia has been suggested for the most common MYOC disease-causing variant, p.Gln368Ter, in some families [bib_ref] Analysis of 15 primary open-angle glaucoma families from Australia identifies a founder..., Baird [/bib_ref]. However, we previously reported that 20 % of MYOC carriers do not report a family history of the disease [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref]. Although this may be explained by individuals not being aware of a diagnosis in their families, or relatives being undiagnosed, it is possible that variants occur de novo in some families. MYOC variants are often identified in older individuals with parents usually unavailable for testing, making it difficult to evaluate whether variants are inherited or sporadic. This case is the second report of a de novo MYOC variant, emphasizing that a sporadic variant should be considered when evaluating the likelihood of MYOC variants in cases with no family history of JOAG or POAG.
De novo variants arise either in the germline or during embryogenesis. If present in the germline cells of one parent, they can represent a recurrence risk in siblings of the variant carrier. We have previously shown that MYOC genetic testing is important for early identification of atrisk individuals and appropriate interventions to minimize irreversible vision loss [bib_ref] Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less..., Souzeau [/bib_ref] [bib_ref] Predictive genetic testing experience for myocilin primary open-angle glaucoma using the Australian..., Souzeau [/bib_ref]. To exclude a recurrence risk resulting from germline mosaicism, both siblings of the proband were subsequently tested for the MYOC variant. Our testing revealed that neither sibling carried the MYOC p.(Pro254Leu) variant, eliminating an inherited risk of developing MYOC associated glaucoma.
# Conclusion
In conclusion, we report a novel de novo MYOC variant considered pathogenic in a patient with sporadic JOAG. This is the second report of a MYOC de novo variant, and it is currently unknown if this mechanism occurs more frequently. This case also highlights that MYOC testing should not be restricted to individuals with a positive family history of glaucoma.
## Consent
Ethics approval was obtained from the Southern Adelaide and Flinders University Clinical Research Ethics Committee. The study conformed to the tenets of the Declaration of Helsinski and follows the National Health and Medical Research Council statement of ethical conduct in research involving humans. Written informed consents were obtained from each participating family member. A copy of the written consent is available for review by the Series Editor of this journal.
[fig] Figure 1: See legend on next page.) [/fig]
[fig] Figure 2: Clinical presentation of the index case. Glaucomatous defects in index case. a Visual field pattern deviation showing a superior arcuate defect involving fixation in the right eye (Humphrey Field analyser, Zeiss). b Optic discs photos showing a right infratemporal notch and disc haemorrhage. c Optical coherence tomography showing inferior retinal nerve fibre layer loss more prominent in the right eye than the left as shown by the black arrow (Spectralis®, Heidelberg Engineering). RE: right eye, LE: left eye, TMP: temporal, SUP: superior, NAS: nasal, INF: inferior (See figure on previous page.) Fig. 1 Pedigree and genetic analysis. a Pedigree of the family. Round symbols indicate female; square symbols, male; fully filled symbols, open angle glaucoma; unfilled symbols, unaffected; arrow, proband; plus/minus, presence/absence of the MYOC:p.(Pro254Leu) variant. b Chromatogram showing the presence of MYOC:c.761C > T, p.(Pro254Leu) sequence variant in individual II-1 at the top (affected) and its absence in individual I-1 at the bottom (unaffected). The black arrow marks the heterozygous variant. c. Alignment of MYOC protein sequences corresponding to residues 248 through 262 (NP_000252.1), against different species, and of different human olfactomedin proteins. The residue of interest, p.(Pro254Leu), is highlighted in yellow. Reference sequences IDs of the genes/species aligned are shown in brackets [/fig]
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Mobile phone brief intervention applications for risky alcohol use among university students: a randomized controlled study
Background: Brief interventions via the internet have been shown to reduce university students' alcohol intake. This study tested two smartphone applications (apps) targeting drinking choices on party occasions, with the goal of reducing problematic alcohol intake among Swedish university students. Methods: Students were recruited via e-mails sent to student union members at two universities. Those who gave informed consent, had a smartphone, and showed risky alcohol consumption according to the Alcohol Use Disorders Identification Test (AUDIT) were randomized into three groups. Group 1 had access to the Swedish government alcohol monopoly's app, Promillekoll, offering real-time estimated blood alcohol concentration (eBAC) calculation; Group 2 had access to a web-based app, PartyPlanner, developed by the research group, offering real-time eBAC calculation with planning and follow-up functions; and Group 3 participants were controls. Follow-up was conducted at 7 weeks.Results: Among 28574 students offered participation, 4823 agreed to join; 415 were excluded due to incomplete data, and 1932 fulfilled eligibility criteria for randomization. Attrition was 22.7-39.3 percent, higher among heavier drinkers and highest in Group 2. Self-reported app use was higher in Group 1 (74%) compared to Group 2 (41%). Per-protocol analyses revealed only one significant time-by-group interaction, where Group 1 participants increased the frequency of their drinking occasions compared to controls (p = 0.001). Secondary analyses by gender showed a significant difference among men in Group 1 for frequency of drinking occasions per week (p = 0.001), but not among women. Among all participants, 29 percent showed high-risk drinking, over the recommended weekly drinking levels of 9 (women) and 14 (men) standard glasses. Conclusions: Smartphone apps can make brief interventions available to large numbers of university students. The apps studied using eBAC calculation did not, however, seem to affect alcohol consumption among university students and one app may have led to a negative effect among men. Future research should: 1) explore ways to increase user retention, 2) include apps facilitating technical manipulation for evaluation of added components, 3) explore the effects of adapting app content to possible gender differences, and 4) offer additional interventions to high-risk users.Trial registration: clinicaltrials.gov: NCT01958398.
# Introduction
Approximately 37-50 percent of college and university students consume alcohol at risky levels [bib_ref] Alcohol involvement in Swedish university freshmen related to gender, age, serious relationship..., Andersson [/bib_ref]. Several effective methods for reducing risky and hazardous drinking among students have been identified, using information and early intervention as well as screening and brief intervention (SBI) [bib_ref] Identification, prevention and treatment: a review of individual-focused strategies to reduce problematic..., Larimer [/bib_ref] [bib_ref] Identification, prevention, and treatment revisited: individual-focused college drinking prevention strategies 1999-2006, Larimer [/bib_ref] [bib_ref] Screening and brief interventions for alcohol use in college health centers: a..., Seigers [/bib_ref]. One central focus of these methods concerns an individual's intentions to drink and his or her behavioral control over alcohol consumption. The Theory of Planned Behavior (TPB) [bib_ref] The theory of planned behavior, Ajzen [/bib_ref] proposes that a person's intentions are the foremost determinants of whether the behavior is performed or not. Information about the behavior that is available at any given moment influences intentions as well as actions. A number of studies confirm that drinking intentions among college students are predictive of drinking behaviors, and that the intended degree of drinking influences the level of the actual drinking [bib_ref] Using the theory of planned behavior to explain the drinking motivations of..., Glassman [/bib_ref] [bib_ref] The theory of planned behavior as a model of heavy episodic drinking..., Collins [/bib_ref] [bib_ref] Alcohol consumption and the theory of planned behavior: an examination of the..., Conner [/bib_ref]. A second central focus of effective methods is the use of protective cognitive and behavioral strategies that aim to limit alcohol consumption and collateral consequences [bib_ref] Do protective behavioral strategies mediate the relationship between drinking motives and alcohol..., Martens [/bib_ref]. Interventions that include skill training for protective behavioral strategies have shown an association with less alcohol use and alcohol-related consequences [bib_ref] Social-cognitive correlates of protective drinking behaviors and alcohol-related consequences in college students, Ray [/bib_ref] [bib_ref] Changes in protective behavioral strategies and alcohol use among college students, Martens [/bib_ref] [bib_ref] Protective behavioral strategies and negative alcohol-related consequences in college students, Araas [/bib_ref]. Many alcohol-related prevention programs tailored to college students include a skills training component aimed at reducing intoxication when drinking [bib_ref] Identification, prevention, and treatment revisited: individual-focused college drinking prevention strategies 1999-2006, Larimer [/bib_ref] [bib_ref] Intervention for hazardous alcohol use and high level of stress in university..., Andersson [/bib_ref] [bib_ref] Efficacy of counselor vs. computer-delivered intervention with mandated college students, Barnett [/bib_ref].
## Technology and brief intervention
Students and young people often are reluctant to seek interventions for reducing their drinking behavior when such interventions are provided by health care professionals [bib_ref] Brief alcohol intervention-where to from here? Challenges remain for research and practice, Nilsen [/bib_ref]. Short, technology-based interventions for younger people might contribute to the availability and access to interventions that could increase healthoriented behavior change. Indeed, the beneficial effects of technology-based brief interventions for problematic alcohol use have been shown for student populations in several reviews and meta-analyses, where the interventions were delivered via computer-with and without internet access [bib_ref] Online alcohol interventions: a systematic review, White [/bib_ref] [bib_ref] Can stand-alone computer-based interventions reduce alcohol consumption? A systematic review, Khadjesari [/bib_ref] [bib_ref] Computer-based interventions for college drinking: a qualitative review, Elliott [/bib_ref] [bib_ref] Computer-delivered interventions to reduce college student drinking: a meta-analysis, Carey [/bib_ref].
Since the advent of the smartphone, i.e., mobile telephones providing advanced functionality in addition to that of regular telephony, more and more people have immediate access to fairly powerful computers close at hand. In 2013, 94 percent of individuals between 16 and 25 years of age had access to a smartphone and 88 percent of those 26-35 years old had such access. Smartphones make it possible to download and run software applications, commonly referred to as "apps," the use of which has exploded since 2008 when the two major venues for downloading apps opened-Apple's App store and Google's Play (formerly Android market).
One app sector targets health-related behaviors such as smoking and obesity, including problematic alcohol consumption. Although over 3000 apps focus on alcohol consumption, recent reviews indicate that many of the apps are intended to encourage drinking; and while apps offering support in reducing problematic alcohol use do exist, very little research evaluating their effects has been published [bib_ref] Let's get wasted! and other apps: characteristics, acceptability, and use of alcohol-related..., Weaver [/bib_ref] [bib_ref] Promoting behavior change from alcohol use through mobile technology: the future of..., Cohn [/bib_ref].
## Estimating blood alcohol content
One of the most prevalent components of smartphone apps related to alcohol consumption is the functionality of calculating and displaying an individual's estimated blood alcohol concentration (eBAC) [bib_ref] Let's get wasted! and other apps: characteristics, acceptability, and use of alcohol-related..., Weaver [/bib_ref] [bib_ref] Promoting behavior change from alcohol use through mobile technology: the future of..., Cohn [/bib_ref]. Learning to calculate the eBAC and relating it to its effects on the individual-both in terms of desirable and positive effects as well as harmful and negative effects-is an integral part of the Alcohol Skills Training Program (ASTP) [bib_ref] The alcohol skills training program -a group intervention for young-adult drinkers, Fromme [/bib_ref] and Brief Alcohol Screening and Intervention for College Students Program (BASICS), information and early intervention methods that have good documented support [bib_ref] Identification, prevention and treatment: a review of individual-focused strategies to reduce problematic..., Larimer [/bib_ref] [bib_ref] Identification, prevention, and treatment revisited: individual-focused college drinking prevention strategies 1999-2006, Larimer [/bib_ref] [bib_ref] An experimental test of three methods of alcohol risk reduction with young..., Baer [/bib_ref]. Calculating and displaying an individual's eBAC is a form of personalized feedback, and personalized feedback via mail or computer has been found to be an effective brief intervention [bib_ref] Curbing problem drinking with personalized-feedback interventions: a meta-analysis, Riper [/bib_ref].
The background motivations for conducting the present study were twofold. First, the Swedish government-owned alcohol monopoly (Systembolaget), which has an explicit mandate to limit health-related harm caused by alcohol, launched its own app, Promillekoll, in late 2012, offering users real-time feedback in the form of eBAC. The stated purpose of the app was to reduce risky and harmful alcohol drinking among university students, but its effects have hitherto not been studied scientifically. Second, the senior author of this article had conducted a study on an automated telephony and web-based intervention that offered university students eBAC calculation to reduce risky drinking [bib_ref] Comparison of automated technologies to deliver brief alcohol interventions to university students, Andersson [/bib_ref]. Our research group adapted this intervention into an app format that included an added planning and follow-up component under the name PartyPlanner.
## Aims
This study investigates the effects of two Swedish-language smartphone apps with real-time eBAC calculation and feedback among university students with established levels of risky drinking. Each app was compared to assessmentonly controls. We hypothesized that using each of these apps would lead to greater reductions in risky drinking than those seen in the assessment-only control group. Given the differing levels of alcohol consumption between men and women, we conducted a secondary analysis to explore whether there were any gender differences for these two apps in terms of alcohol outcomes. Earlier studies on gender effects for SBI outcomes are somewhat inconclusive, with some studies reporting gender differences [bib_ref] Meta-analysis of randomized control trials addressing brief interventions in heavy alcohol drinkers, Wilk [/bib_ref] [bib_ref] Brief interventions for alcohol problems -a review, Bien [/bib_ref] but later research showing no such differences [bib_ref] Brief interventions for hazardous drinkers delivered in primary care are equally effective..., Ballesteros [/bib_ref]. As far as we know, gender differences in the effects of smartphonedelivered SBIs have not been previously studied.
In summary, we report analyses of outcomes between groups with access to each app in comparison to a control group, additionally examining possible gender differences. We further discuss the implications of these results for university students as well as future research recommendations.
# Methods
## Participants
The student unions at Stockholm University and the Royal Institute of Technology in Stockholm, Sweden, provided our research group with e-mail addresses for their current members. We e-mailed study information and a web page link to all addresses on the lists provided. Potential participants were informed that completing baseline and follow-up questionnaires in the study would automatically include them in a lottery with three iPad devices as prizes. Those interested in participation clicked a link directing them to a web page where they received further information on the Swedish Personal Data Act, and where they could indicate their consent to participate in the study. Individuals giving informed consent participated in a data intake process requiring registration of their mobile phone number, gender, age, and weight. Participants were also asked whether they had access to a smartphone running either of the two operating systems, iOS or Android. Thereafter, participants filled out baseline questionnaires consisting of the Daily Drinking Questionnaire (DDQ) [bib_ref] Social determinants of alcohol consumption: the effects of social interaction and model..., Collins [/bib_ref] and the Alcohol Use Disorders Identification Test (AUDIT) [bib_ref] Development of the alcohol use disorders identification test (AUDIT): WHO collaborative project..., Saunders [/bib_ref]. Participants with an AUDIT score indicating at least hazardous consumption (≥6 for women and ≥ 8 for men) [bib_ref] Alcohol use among Swedes and a psychometric evaluation of the alcohol use..., Bergman [/bib_ref] and having a smartphone running either iOS or Android were randomized to one of the two application conditions or to an assessment-only control group. Those not fulfilling these criteria were excluded from randomization. One reminder e-mail was sent out 2 days after the first email to those who had not responded. Study registration was open for one week. Participants were informed that some students would be contacted with an e-mail containing a link to a smartphone app, and that all would be asked to fill in follow-up questionnaires 6 weeks after registration, a timeframe based on a study previously conducted by the research group and commonly used in student alcohol studies [bib_ref] Comparison of automated technologies to deliver brief alcohol interventions to university students, Andersson [/bib_ref]. No feedback on baseline consumption levels was given to any of the participants, regardless of group assignment.
## Randomization
All eligible participants were randomized to one of the two interventions or to a control group with the ratio (1:1:1), using the randomization function in the IBM SPSS Statistics for MacOS X, Version 19 (IBM Corp, Armonk, NY, USA). Participants randomized to an intervention were sent an e-mail 10 days after the first information e-mail was sent (3 days after study registration ended) with a link to access the app to which they had been randomized. Participants in the intervention groups were instructed to use the app during the following weeks, in association with events where alcohol would be consumed. There were no further prompts to use the application during the period leading up to the follow-up. Participants were not blind as to whether or not they were allocated to an intervention condition, but they were not informed that one of the interventions included a planning and follow-up component. The randomization process was fully automated.
Interventions 1: Promillekoll app (tr. "Check your BAC"): As noted above, this app was developed by the Swedish government's Systembolaget and is publicly downloadable for iPhone and Android smartphones. This app was released for public use on September 25, 2012. The user can register his/her alcohol consumption in real time, where the app displays the user's current eBAC. The Promillekoll app is theoretically based on the assumption that information about one's own real-time eBAC levels can contribute to one's protective cognitive and behavioral strategies. Promillekoll also offers a number of specific strategies to maintain alcohol consumption at a level that is not harmful-in this case, 0.06 percent BAC. A further mechanism, congruent with the Theory of Planned Behavior (TPB) [bib_ref] The theory of planned behavior, Ajzen [/bib_ref] , is that providing information and feedback on risky levels of eBAC modifies the intention to consume alcohol.
The application warns the user if the drink entered will result in an eBAC over 0.06 percent and only displays values up to 0.08 percent. It also provides information texts on alcohol and BAC. The study was conducted using a publicly available app, which is constructed as a standalone application that can be used offline. No user data are collected. The research group had no influence on the development or functionality of the app.
2: PartyPlanner app. In order to further develop and test the idea of modifying drinking intentions with an app, our research group developed a new app, "PartyPlanner," with the functionality of simulating or planning a drinking event beforehand and then comparing the simulation to the real-time event afterwards. Our hypothesis is that setting up a plan for personal eBAC levels before the drinking event might explicitly modify the user's drinking intentions by adapting user perceptions of risk to reality. The app user would then be able to pace his or her drinking based on a more realistic view of the amount of alcohol actually corresponding to a certain eBAC level. Comparing eBAC levels after the event could increase skillfulness in future protective behavioral strategies and increase control when drinking alcohol.
Thus, in addition to registering alcohol consumption with instant visual eBAC similar to Promillekoll, this app gives the user the opportunity to simulate an event where alcohol will be consumed ahead of time. The app displays the eBAC level at distinct time points throughout the drinking occasion, both for pre-party simulations and real-time registrations. Color codes indicate whether the eBAC is at a risky level. The real-time registration with feedback can be used as a standalone function; i.e., without having made a prior plan. However, if there is a plan, the user can visually compare the plan with the logged real-time event after the actual drinking occasion. In contrast to Promillekoll, the PartyPlanner app was launched as a so-called web app that requires internet connection using a web browser in order to facilitate additional development following this study and prior to possible future, wider, public accessibility. This app was developed by the authors in collaboration with Liquid Media AB.
## Control group
The third group was a control group that did not receive any intervention or feedback on risky drinking. Individuals allocated to this group did not receive any further information in the time between study registration and follow-up.
## Follow-up
Seven weeks after registration, all participants received an e-mail inviting them to answer the follow-up questionnaires. One reminder e-mail was sent 4 days after the first e-mail to initial nonresponders. The e-mails contained a link to an online questionnaire, where participants filled out the AUDIT, the DDQ, and answered questions on access to other interventions for reducing their alcohol use, such as speaking with someone else, using web-based services, or contacting professional treatment providers. Participants in the intervention groups were also asked whether they had actually used the app and how they liked it. The entire process was fully automated and no contact occurred with a human counterpart, but participants were provided with an e-mail address for technical support and questions regarding the study.
The follow-up e-mail was sent after 7 weeks rather than the originally planned 6 weeks due to unforeseen technical difficulties. The reason for the chosen timespan of 6 weeks was to facilitate comparison with an earlier study on digital interventions by the study group targeting the same population [bib_ref] Comparison of automated technologies to deliver brief alcohol interventions to university students, Andersson [/bib_ref].
## Seasonality
The study took place in March and April 2013. Swedish university programming is not based on the concepts of midterms or finals, so there were no uniform examination periods during this time. During the intervention period, two major public holidays occurred: Easter and Walpurgis Night (April 30). Walpurgis Night is traditionally connected to partying, with high alcohol consumption in Swedish culture.
## Ethics
The study was approved by the regional ethics vetting board in Stockholm (ref. nr. 2012/1126-31/1). Since Swedish universities are not permitted to organize lotteries, the iPad lottery was conducted by the charity organization, Save the Children. See [fig_ref] Figure 1: Consort diagram of the trial [/fig_ref] for a participant flowchart.
## Design
The study evaluated the effectiveness of access to one of two smartphone apps addressing risky alcohol use among university students in Stockholm, Sweden. A randomized, parallel, three-group, repeated-measures design was used in which alcohol-related outcomes of two smartphone intervention groups were separately compared to an assessmentonly control group. Participants were assessed at baseline before trial and at follow-up 7 weeks later. The trial was registered at clinicaltrials.gov (ref. nr. NCT01958398).
## Measures
Participants' alcohol consumption levels and BAC were investigated during the trial. To measure quantity and frequency of alcohol consumption, the DDQ [bib_ref] Social determinants of alcohol consumption: the effects of social interaction and model..., Collins [/bib_ref] was used. The instrument was translated into Swedish by Malmö University, in collaboration with the University of Washington. Participants were asked to consider a typical week during the past month and state how many standard glasses of alcohol they drank and over how many hours for each day of the current week. They were also asked to report their peak alcohol consumption event during the past month in terms of how many standard glasses they drank during a self-reported number of hours. This measure has demonstrated good testretest reliability in paper format [bib_ref] An experimental test of three methods of alcohol risk reduction with young..., Baer [/bib_ref].
The eBAC was calculated based on the values from the DDQ in conjunction with weight and gender for each individual. The formula used was the widely known Widmark formula, as modified and used by the United States National Highway Traffic Safety Administration: eBAC (in parts per mille, as is standard in Sweden) = ([number of standard glasses] × 12 grams)/([body weight in kg] × C) -[no. of hours] × 0.15), where C is a gender-specific constant of 0.68 for men and 0.55 for women. In order to convert the eBAC to percentage values for this article, the values were divided by 10. In the study, a peak eBAC value was calculated as the eBAC of the peak alcohol consumption event of the past month. The mean eBAC value was calculated as the mean of the eBAC values specified for each day during the typical week reported in the DDQ.
The AUDIT [bib_ref] Development of the alcohol use disorders identification test (AUDIT): WHO collaborative project..., Saunders [/bib_ref] consists of 10 questions measuring consumption and signs of harm and dependence in relation to alcohol. The instrument was used to assess whether problematic drinking was present and, if so, its severity. The Swedish version has shown good internal consistency. The paper version has yielded Cronbach's α values of 0.81-0.82 [bib_ref] Alcohol use among Swedes and a psychometric evaluation of the alcohol use..., Bergman [/bib_ref] [bib_ref] Undersökning av förändringar i svenskarnas alkoholvanor åren 1997-2001. [Swedish women show more..., Bergman [/bib_ref] , and the internet version has yielded Cronbach's α values of 0.80-0.93 [bib_ref] Population screening of risky alcohol and drug use via internet and interactive..., Sinadinovic [/bib_ref] [bib_ref] Internet-based assessment and self-monitoring of problematic alcohol and drug use, Sinadinovic [/bib_ref].
In order to assess the app usage, self-reported data were gathered at follow-up on whether the apps had been used and, if so, how many times. Questions on the users' perception of the apps were also asked at follow-up.
## Definitions
## Risky and problematic drinking
No level of alcohol drinking is known to be risk free, and there is no internationally agreed-upon amount defined as hazardous. Guidelines thus vary considerably, but risky use in terms of volume consumed is often defined either as a high weekly consumption or high consumption on one occasion (binge drinking). In Sweden, the Swedish National Institute of Public Health defines hazardous drinking for men as 14 or more standard glasses (in Sweden, 12 grams of pure alcohol) in a week, or five or more standard glasses per occasion. For women, the limits are nine or more standard glasses in a week or four or more standard glasses at any one occasion. While binge drinking can be defined as four or five drinks per occasion, it can also be defined using BAC, where the level of 0.08 percent is commonly used [bib_ref] Defining "binge" drinking as five drinks per occasion or drinking to a.08%..., Fillmore [/bib_ref]. In this study the slightly more conservative level of 0.06 percent was used, which is closer to the 0.055 percent BAC recommended in the BASICS program.
In this study, we defined problematic drinking based on an AUDIT score over the cutoff level for risky or hazardous drinking (≥6 for women and ≥ 8 for men), thus also including the categories harmful drinking (≥16 for men and women) and probable alcohol dependence (≥20 for men and women).
## Statistical analyses
Descriptive statistics were used to describe baseline characteristics. Analysis of variance (ANOVA) was used to identify any baseline differences in age, AUDIT, mean eBAC, peak eBAC, quantity, frequency, and number of binge drinking occasions between the groups. Pearson's chi-squared tests were used to determine differences between the groups in proportion of gender and the proportion of participants drinking more than the weekly recommendation. A linear mixed model analysis was used to identify changes over time in alcohol consumption outcomes: mean eBAC, peak eBAC, quantity, frequency, and number of binge drinking occasions. These analyses were conducted per protocol-that is, including only those participants who reported using the app they were assigned to-and controlling for three variables: the number of times the app was accessed, having spoken to someone about alcohol consumption during the past 12 months, and having accessed the publicly available Promillekoll app prior to the study. For comparison, intention to treat analyses were performed with all participants who were randomized to experimental groups and retaining baseline values for as many participants as possible. No data imputation procedures were applied.
Descriptive statistics, ANOVA, repeated-measures ANOVA, and Pearson's chi-squared analyses were performed using IBM SPSS Statistics for MacOS X, Version 22 (IBM Corp, Armonk, NY, USA). Linear mixed model analyses were performed using Stata 13 (StataCorp. 2013. College Station, TX: StataCorp LP.). Values for averages and standard deviations are presented to three decimal places in order to make differences visually discernible.
## Exclusions
Two participants were identified as extreme outliers at baseline, one who entered a value of 70 standard glasses for the peak consumption occasion and another who indicated an age of 90 years. A third participant had left incomplete data at baseline, and was included in followup due to a technical problem. These three participants were excluded from all analyses.
# Results
## Participant characteristics
Baseline participant characteristics did not differ overall among participants randomized to the three arms of the trial (see [fig_ref] Table 1: Baseline characteristics of students with risky alcohol use in a randomized brief... [/fig_ref]. In total, the sample consisted of approximately equal numbers of men and women (48.3% male, 51.7% female). The mean AUDIT score (10.7, SD = 3.9) indicated hazardous drinking levels, with almost onethird (29.7%) of the participants drinking more on a weekly basis than the recommended Swedish guidelines of less than nine drinks for women and less than 14 for men per week.
## Attrition
The overall attrition rate was 29.4 percent (n = 568); one participant who asked to terminate his study participation was included in the attrition group. An analysis comparing the attrition group with participants who completed the follow-up ("completers") showed no significant differences in gender or age. However, participants in the attrition group had significantly higher scores on all outcome variables related to alcohol consumption, except for peak eBAC per month (see .
Analyzed by group, attrition was not equal. The Party-Planner group had a higher attrition rate, at 39.
## App use
Over one-third of the participants in all three groups had tried Promillekoll before initiation of the study.
## Other interventions and prior experiences of promillekoll
Between 0.4 percent and 0.6 percent of all study participants reported having used pharmaceutical medications in order to reduce alcohol consumption during the 12 months preceding the follow-up, and between 0.8 percent and 2.6 percent reported having accessed help other than medications or speaking to someone about their use. Over 20 percent in each group reported at follow-up that they had spoken with someone about their alcohol consumption during the prior 12 months.
## Usability data
Users rated the two apps for ease of use, suitability, and likelihood of recommending it to a friend. The only significant difference between the two apps was that Promillekoll participants rated ease of use higher (4.0) than PartyPlanner participants (3.2) (see [fig_ref] Table 3: Self-reported data on prior use of Promillekoll, speaking to someone about use,... [/fig_ref].
## Outcome analyses
Time-by-group interactions were investigated using linear mixed models analyses, separately comparing each app group (per protocol) to the control group and controlling for the number of app-use occasions, earlier use of Promillekoll, and having spoken to someone about alcohol consumption over the past 12 months. A Bonferroni correction of p = 0.003 was applied throughout. For the Party-Planner group, no significant time-by-group interactions for any outcome measures occurred. For the Promillekoll group, app users showed a significant increase in drinking frequency compared to the control group [Z = 3.39, p = 0.001] (see [fig_ref] Table 4: Analyses comparing the two intervention groups to controls included only individuals who... [/fig_ref].
The three covariates were all significant: number of appuse occasions [Z = 8.24, p < 0.001]; having spoken to someone about alcohol consumption in the past 12 months [Z = 3.73, p < 0.001]; and having tried Promillekoll before the study [Z = 2.62, p = 0.001].
An intention-to-treat analysis including all users regardless of reported app use, and not controlling for any other factors, did not yield any significant differences between the groups.
At follow-up, the proportion of group members having a weekly consumption over the recommended level remained more than 25 percent (Control group: 26.4%, PartyPlanner: 26.7%, and Promillekoll: 28.8%).
## Secondary outcome analyses
Outcomes were also analyzed secondarily to identify any gender differences. Outcomes for male participants showed two significant time-by-group effects in the per-protocol analysis. Male participants in the Promillekoll group with reported app use increased their drinking frequency from baseline to follow-up, in comparison to control group participants (Z = 3.48, p = 0.001). One covariate was significant: number of app-use occasions [Z = 5.80, p < 0.001].
# Discussion
This study compared the effects of two smartphone apps for reducing overconsumption of alcohol at single-party occasions among Swedish university students to assessmentonly controls. Both apps relied on mathematical estimates of blood alcohol concentration. The apps differed in content. The Promillekoll app offered information texts on different eBAC levels as well as strategies for avoiding risky drinking. The PartyPlanner app did not offer any Baseline characteristics for participants completing follow-up ("completers") compared to participants who did not ("attrition group") explanatory texts but included a component allowing participants to plan their drinking in advance. Because of these differences in the apps, as well as difficulties in assessing which factors might be instrumental in any observed change in outcome, we chose to compare each app only to the control group and not to each other. Also, because the proportion of participants who actually used the app was significantly lower in the PartyPlanner group, we chose to focus on per-protocol analyses only, including participants who reported having used each app. We chose to control our analyses with three covariates. One concerned prior access to the Promillekoll app, which had been available for public access for over 5 months at the beginning of our study. Over one-third of app participants had used it before our study. The PartyPlanner was launched at the same time as this study and is not publicly available at this writing. The second covariate concerned access to other modes of help for alcohol consumption during the 12 months preceding the follow-up. We assessed three categories of help: medication, speaking to someone about alcohol consumption, and accessing other types of help such as internetbased interventions. About one in five participants reported having spoken to someone about their consumption in the previous 12 months. The third covariate concerned the number of times intervention group participants had used the app. The results showed only one time-bygroup interaction, where Promillekoll participants showed a significant increase in drinking occasion frequency in comparison to controls. The proportion of students drinking more than the weekly Swedish recommendation of nine drinks for women and 14 for men appeared to remain stable over time at approximately 25-30 percent of all study participants. It is not clear why Promillekoll users increased the frequency of their drinking at the same time that they did not, as a group, consume larger quantities of alcohol. We can only speculate that app users may have felt more confident that they could rely on the app to reduce negative effects of drinking and therefore felt able to drink more often.
The secondary gender-focused analyses suggest that male participants were the source of the increase in drinking frequency in the Promillekoll group. This finding suggests that it might be interesting to test gender-related hypotheses about the mechanisms steering the drinking behavior of male and female university students when using smartphone apps. One speculation is that real-time use of a smartphone app might trigger men to compete with their peers in a competitive "drinking game". However, Promillekoll does not display eBACs over 0.08 percent, thus effectively setting an upper limit to how far the "game" can go. Interestingly, we did not see the same phenomenon in the PartyPlanner group. One reason could well be that attrition was significantly higher in the Party-Planner group than in the other two groups, and where a higher proportion of men in the PartyPlanner group had dropped out. Moreover, these dropouts had higher levels of baseline alcohol consumption, a pattern not seen in the other two groups. It is also possible that individuals who might have been triggered to drink more frequently when having access to a smartphone app were present to a higher extent in the Promillekoll group.
Regarding the significance of covariates, the number of app-use occasions was associated with increases in outcomes in the analyses, possibly because those who drank more frequently had more opportunities to use the app than those who did not. This explanation could also apply to the other covariates, where an individual with higher levels of alcohol consumption might be more likely to try out apps for controlling it. They might also be more likely to speak to someone about their consumption.
The study took place over Easter and Walpurgis Night, both high-consumption holidays. These events may have led to seasonal spikes in consumption and may have affected the reported levels of consumption; however, this effect is most probably equal for all participants.
# Strengths and limitations
An important strength of this study is that it is, to our knowledge, one of the first effectiveness studies on apps for health-related behavior change for reducing risky alcohol consumption. A second strength is that this is the first randomized controlled study conducted with the Promillekoll app, which was released publicly by Systembolaget in the fall of 2012 following research-based development and a qualitative usability study. Thirdly, we studied university students, a highly important target group in that both risky alcohol drinking and smartphone app use are quite prevalent. It is possible to reach a large population of students easily and directly via email addresses, facilitating this study and future research with this group. The design also ensures a minimum of human interaction, a possible advantage given the stigma attached to overconsumption of alcohol [bib_ref] The stigma of alcohol dependence compared with other mental disorders: a review..., Schomerus [/bib_ref] , and which potential participants might experience as an obstacle.
The attrition rates in this study (over 30% overall; see were somewhat lower than in other studies of electronically delivered SBIs, where attrition rates over time periods of 4-6 weeks ranged between 36.7 percent and 44.8 percent [bib_ref] Preventing high-risk drinking in youth in the workplace: a web-based normative feedback..., Doumas [/bib_ref] [bib_ref] Two fully automated web-based interventions for risky alcohol use: randomized controlled trial, Tensil [/bib_ref] [bib_ref] On-line randomized controlled trial of an internet based psychologically enhanced intervention for..., Wallace [/bib_ref]. This study was also subject to several limitations. First, we had no information on possible differences between student union members who entered the study and those who did not. Among those invited to participate, only 16.9 percent gave informed consent. One reason for this could be that a certain proportion of our e-mails did not reach the recipients due to spam e-mail filters or to changes in e-mail addresses. We are therefore unsure of how representative this sample is of the total student population. What is clear, however, is that prevalence rates of problematic drinking in this sample were consistent with earlier studies targeting university students in Sweden and the US [bib_ref] Alcohol involvement in Swedish university freshmen related to gender, age, serious relationship..., Andersson [/bib_ref]. Secondly, the two smartphone interventions differed significantly in graphic design and technical presentation. Moreover, the Promillekoll app had been publicly available for over 5 months before this study was launched. These circumstances made it difficult to conduct any meaningful comparison between the two apps, so we chose to compare each of them individually to the control group. Under ideal laboratory conditions, we would have added the planning and follow-up function that was part of PartyPlanner to the Promillekoll intervention, in order to facilitate participants' awareness of the extra pedagogical functions in the PartyPlanner app. In this case, the apps were produced by different designers, confounding design and format with content. Thirdly, the attrition rates in the two intervention groups differed significantly. Some of the participants who completed the follow-up left written comments on the PartyPlanner app. Their comments indicated that they disliked the technical web-based app solution, which required continuous internet connection. This may have caused time lags in app response that may have further deterred participants from using the app, and may have contributed to attrition. Our attrition analysis showed that completers and noncompleters in both the control and PartyPlanner groups differed on baseline characteristics measuring alcohol consumption, whereas Promillekoll participants did not. We are not sure why the Promillekoll app had a more equal distribution of baseline characteristics between the attriters and completers, but Promillekoll was released following a rigorous design process, and this may have led to a more pleasant user experience, which in turn led to higher retention. The Promillekoll app has also been extensively advertised in Sweden and participants may have been more curious to participate in its testing. The difference in attrition rates may also have contributed to our finding of significant differences for Promillekoll participants, since this sample may have been more representative in that men with higher levels of alcohol use-and possibly prone to engaging in competitive games-were retained in the sample. Highalcohol consumers in the PartyPlanner group, on the other hand, dropped out to a larger extent; these may also have been individuals with higher impulsivity, lower acceptance of frustration, and consequently, a lower tolerance for lag times. The results may have been tipped in favor of Party-Planner, given that fewer of the individuals with higher levels of alcohol consumption participated in follow-up.
A final limitation in the study is the lack of objective user data available for the apps. For the Promillekoll app, this was due to the fact that the app was programmed as a standalone app without data transmission on usage to any server. Our research group had no influence on the design and programming of this app. The PartyPlanner app, on the other hand, was developed by the research group and the possibility of accessing objective user data was included in the design. However, technical difficulties complicated the extraction of these data. We chose instead to rely on self-reported user data on whether participants had used the app, and on how many occasions. While these data may be subject to error, particularly given the unclear definition of "app use" as well as the number of app-use occasions, the extent of the error is approximately comparable. Had we used objective data for one app and self-report data for the other, we might have compromised the reliability of our comparisons to the control group even more.
A final ethical issue of note in this study is the fact that controls were not offered any specific intervention to address their risky alcohol use. Control group participants and intervention group participants received a recommendation to contact student health services both at registration and at follow-up if they felt concerned about their drinking. Also, at the end of the study, a considerable proportion of the students were above the levels for recommended weekly consumption, but they were not offered any further interventions or referral.
# Conclusions
Overall, participation in our study did not seem to affect drinking in any of the three study groups. However, the Promillekoll app seemed to be associated with a negative effect in the form of an increased number of drinking occasions over one week. Our conclusion from this study is that eBAC calculation in the app form is not effective for reducing alcohol consumption among university students. Future development of apps with this purpose may require additional input to supplement eBAC feedback.
Our secondary analysis suggests that there might be gender differences in how apps are used in the context of risky drinking among university students. The Promillekoll app produced by the Systembolaget had one possible negative effect for the men, but not for the women. The PartyPlanner app, with the additional functionality of planning ahead and comparing real drinking events with plans, did not seem to negatively affect men. However, participants in this trial arm had a higher dropout rate, consisting to a larger extent of male participants with higher alcohol consumption, in comparison to the Promillekoll and control groups. Further research is thus necessary to explore gender differences in the use of apps in this context. Such research should investigate which app features are associated with higher participant retention, as well as whether app design needs to take gender factors into account.
Future research should use a uniform design for apps with different intervention components in order to control for the confounding effects of differing designs. This would enable isolation of component effects from design and technical differences between apps. Finally, an important area of future research is offering further help to individuals drinking more than the recommended weekly levels who are identified through such research. Although we referred all individuals concerned about their drinking to student health services in this study, we suspect that many might be reluctant to approach such services. Some might not be aware of the harmful nature of their drinking, whereas others might experience approaching student services as stigmatizing. One possibility would be to offer more indepth automated interventions to individuals drinking more than the recommended levels in a separate, secondary study.
[fig] Figure 1: Consort diagram of the trial. Shaded areas were not included in analyses. [/fig]
[fig] 3: percent, compared to the Promillekoll (26.4%) and control (22.7%) groups [χ 2 (2, 1929) = 46.633, p < 0.001]. A subanalysis revealed that in the PartyPlanner group, all alcohol consumption-related baseline values were higher among the attrited individuals than among those who completed the follow-up; the same was true for all baseline values except for frequency and number of binge drinking occasions per week in the control group. For the Promillekoll group, there were no differences in baseline values between completers and the attrited group. Significantly more of the men (43.9%) in the Par-tyPlanner group did not complete follow-up in comparison to the women (35.3%) [χ 2 (1, 639) = 4.975, p = 0.026]; there were no gender differences for follow-up rates in the other two groups. [/fig]
[table] Table 1: Baseline characteristics of students with risky alcohol use in a randomized brief intervention app trial [/table]
[table] Table 4: Analyses comparing the two intervention groups to controls included only individuals who reported actually using the assigned app. Analyses were controlled for the number of times participants reported using the app, previous use of the publicly available Promillekoll app, and self-reports on having spoken to someone in the past 12 months about their alcohol use. [/table]
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Pathophysiology, Clinical Characteristics of Diabetic Cardiomyopathy: Therapeutic Potential of Natural Polyphenols
Diabetic cardiomyopathy (DCM) is an outcome of disturbances in metabolic activities through oxidative stress, local inflammation, and fibrosis, as well as a prime cause of fatality worldwide. Cardiovascular disorders in diabetic individuals have become a challenge in diagnosis and formulation of treatment prototype. It is necessary to have a better understanding of cellular pathophysiology that reveal the therapeutic targets and prevent the progression of cardiovascular diseases due to hyperglycemia. Critical changes in levels of collagen and integrin have been observed in the extracellular matrix of heart, which was responsible for cardiac remodeling in diabetic patients. This review explored the understanding of the mechanisms of how the phytochemicals provide cardioprotection under diabetes along with the caveats and provide future perspectives on these agents as prototypes for the development of drugs for managing DCM. Thus, here we summarized the effect of various plant extracts and natural polyphenols tested in preclinical and cell culture models of diabetic cardiomyopathy. Further, the potential use of selected polyphenols that improved the therapeutic efficacy against diabetic cardiomyopathy is also illustrated.
Diabetic cardiomyopathy (DCM) is an outcome of disturbances in metabolic activities through oxidative stress, local inflammation, and fibrosis, as well as a prime cause of fatality worldwide. Cardiovascular disorders in diabetic individuals have become a challenge in diagnosis and formulation of treatment prototype. It is necessary to have a better understanding of cellular pathophysiology that reveal the therapeutic targets and prevent the progression of cardiovascular diseases due to hyperglycemia. Critical changes in levels of collagen and integrin have been observed in the extracellular matrix of heart, which was responsible for cardiac remodeling in diabetic patients. This review explored the understanding of the mechanisms of how the phytochemicals provide cardioprotection under diabetes along with the caveats and provide future perspectives on these agents as prototypes for the development of drugs for managing DCM. Thus, here we summarized the effect of various plant extracts and natural polyphenols tested in preclinical and cell culture models of diabetic cardiomyopathy. Further, the potential use of selected polyphenols that improved the therapeutic efficacy against diabetic cardiomyopathy is also illustrated.
# Introduction
Diabetes, a group of metabolic diseases associated with damage and dysfunction of various organs, especially the heart, and lead to cardiovascular diseases. The current burden of diabetes has become a major threat to the health of populations, which reflects the cumulative effects of risk factors over the life span of people. Present western lifestyle and environmental factors promote the progression of these pathological conditions and are held responsible for rising rate of diabetic cardiomyopathies.
Persistent hyperglycemia causes the structural and molecular changes in cardiomyocytes, by increased production of advanced glycation end products (AGEs) in diabetes due to constant oxidative stress. AGEs accumulate in various tissues and can link with other proteins such as collagen type IV, laminin, and fibronectin, resulting in impaired cardiac function and enhanced myocardial stiffness. A significant increase in AGEs and its specific receptors (RAGEs) on cells trigger oxidative stress and activate protein kinase C, finally disrupting cellular and molecular functions. Oxidative stress induces the breaks in DNA, resulting in increased activity of poly ADP ribose polymerase (PARP) enzyme, which further promote the progression of various cardiovascular diseases.
The preponderance of diabetes will be accelerated by 50-60% in the population from 2015 to 2030, and also annual mortality rate attributing diabetes will be increased by 38% in the US population. Global healthcare expenditures were found to be higher to treat and prevent diabetes and its associated complications (∼376 billion US Dollars (USD) in 2010). This cost is projected to exceed ∼622 billion USD by 2030. However, the outcome of such expenditures is not satisfactory and people of low-income countries are still devoid of the treatments. Hence, an affordable therapy is required against diabetes, especially in the poorer socio-economic sections of country worldwide.
An alternative therapy by using plant polyphenols may have a great choice to end the cardiovascular complications developed by DCM. In this review, we discusses the pathophysiology and clinical features of DCM and the possible role of polyphenol in relation to the DCM therapy has been explored.
## Pathophysiology of diabetic cardiomyopathy
In the following sections, we have described the physiological mechanisms associated with progression of diabetic cardiomyopathy.
## Oxidative stress: critical contributor to diabetic cardiomyopathy
Free radicals are generated due to the continuous production of the oxygen, and considered as "reactive oxygen species" (ROS). Stress inducing agents stimulate the drastic release of these oxidants and create an imbalance in the equilibrium of ROS production and antioxidant capability.
Endothelial nitric oxide synthase (eNOS), a critical enzyme of endothelial cells, produces NO· and gets altered due to high glucose stress, which ultimately leads to vascular endothelial cell dysfunction. NO· further produces ONOO-, a cytotoxic free radical responsible for disturbing cardiovascular function. Oxidative stress is determined by the overproduction of ROS and RNS molecules. The unstable configuration leads to the breakdown of lipids, inactivation of enzymes, cell membranes, and DNA damage.
## Calcium homeostasis
Intracellular calcium (Ca 2+ ) is a significant marker of cardiac contraction. Hyperglycemic stress enhances Ca 2+ accumulation in cardiac myocytes, which imposes impairment in the ionotropic response in the heart. Ca 2+ influx activates its release after binding to troponin C and generates tension by activating the sliding of thin and thick filaments, which further resulting in cardiac contraction. Intranuclear Ca 2+ alteration may also change various kinases activities namely (extracellular-signalregulated kinase) ERK, (microtubule-associated protein kinase) (MAPK) and Janus kinase (JNK). MAPKs are also involved in the transcription of c-fos and c-jun, which activate phospholipase A 2, resulting in the plasma membrane and intracellular membranes permeabilization, further leading to cell death.
## Rennin-angiotensin system
In diabetic cardiomyopathy, rennin-angiotensin (RAAS) system is considered a life-saving system. RAAS is a complex pathway whose activation triggers a cascade of events leading to cardiovascular disease. Studies show that infusing Angiotensin-II (AngII) leads to stimulation of ACE/AngII/AT1R complex accelerating atherosclerosis and blocking of RAAS protect against cardiac damage. RAAS can be activated by hyperglycemia, leading to production of Ang-II. It has been known that the Ang-II can produce ROS through NADH/NADPH oxidase system. RAAS's role in NADH/NADPH bound oxidase is further supported by studies showing the effectiveness of ramipril (and ACE inhibitor) in preventing upregulation of p47phox, p22phox, and reduced NADH driven oxide production. This led to reduced fibrosis and hypertrophic gene expression. Blocking of Ang-II also showed reduced expression of p22phox, NADHoxidases and HG-induced p47phox. These studies show that the RAAS activity in diabetes supports an interaction between Ang-II and NADPH-oxidases in cardiomyocyte.
## Extracellular (ecm) remodeling in diabetic cardiomyopathy
The extracellular matrix is a complex meshwork of fibers comprised of proteins, polysaccharides, and provides structural as well as functional support to the surrounding cells, which are important for the cell-to-cell communication and adhesion. Alteration in the extracellular matrix components contributes to diabetic cardiomyopathy, which enhances stress in the diabetic heart which involves changes in mass, shape, and volume of the left ventricle, leading to ischemia, and pressure overload. Changes in physiological conditions due to stress stimulus can trigger various proteases activities such as serine proteases and matrix metalloproteinases (MMPs) that cause alteration in the expression of collagen, fibronectin, and ultimately leads to ECM remodeling.
Collagen fibrils are the fundamental blocks of extracellular matrix and give mechanical strength, stiffness, and toughness to the vasculature. The extracellular collagen matrix of the myocardium has a major function in maintaining cardiac organization. An excessive accumulation of fibrillar collagen in the myocardium was reported in hypertrophied heart. In absence of secondary risk factors such as hypertension or coronary artery disease, cardiac dysfunction in diabetic patients, increased collagen I, III, and IV deposition has been found to results in fibrosis and poor LV function. There is a delicate balance between continual degradation and synthesis of collagen in ECM. Specific collagen degrading MMPs enzymes as well as their inhibitors (tissue inhibitors of metalloproteinases, TIMPs), are essential in collagen remodeling.
## Matrix metalloproteinases: key enzymes for ecm modulation
MMPs have several conserved domains with different substrate specificity and inducibility. They play a major function in wound healing, tissue repair, and remodeling in various diseases. MMPs are of two types, membrane-bound and secretory. Membranetype MMPs (MT-MMPs) work in close proximity with the cell, whereas the secreted MMPs act within the matrix, away from the cells from which they are synthesized. MMP-2 (Gelatinase A) and MMP-9 (Gelatinase B) are the most abundant secreted proteases, which degrade gelatin. They are categorized in 6 main classes such as-gelatinases, collagenases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs.
MMPs are regulated in expression or activity and cellular inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs). The MMP-TIMP balance maintains the integrity of ECM by regulating the debasement rate of ECM proteins and tissue remodeling. Most of MMP inhibitors have wide-spectrum actions on other MMPs and could cause adverse effects. Usage of synthetic MMP inhibitors in experimental animal models against upregulated activities of MMPs are failed in clinical trials, except FDA approved doxycyclin. The synthesis and design of new generation biological and synthetic MMP inhibitors are required.
## Cardiac hypertrophy in diabetic cardiomyopathy
High glucose uptake initiates an imbalance in myocardial energetics and results in myocardial ischemia or hypertrophy. Whenever cells are exposed to high glucose stress, they enlarge and undergo hypertrophy (increase in size, not in number) to combat the excessive stress, resulting in an increase in myocytes length (eccentric hypertrophy), or myocytes width (concentric hypertrophy), which further enhances thickening of the septum and ventricular wall.
Cardiac hypertrophy, a phenomenon observed with many forms of human heart disease including diabetic cardiomyopathy, results in an increase in protein synthesis, addition of sarcomeres and fetal genes re-expression such as myosin heavy chain (β-MHC) and GATA-1 and activation of early response genes, such as c-jun, c-fos, and c-myc etc. In hypertrophic conditions, various signaling pathways such as mitogen-activated protein kinases, tyrosine kinase Src, GTPbinding protein Ras, protein kinase C, phosphoinositol 3-kinase are involved (29). Transforming growth factor β (TGF-β) mediates the production of transcription factors such as nuclear factor kappa B (NF-kB), small mothers against decapentaplegic (SMAD), signal transducer activating protein-1 (AP-1), and activator of transcription (STAT) that takes part in MMPs and TIMPs transcription leading to remodeling of the extracellular matrix.
Cardiac hypertrophy is prevalent in asymptomatic type 2 diabetes patients. The following fetal genes are used as an indicator/marker of cardiac hypertrophy under diabetic stress.
Sarcoplasmic Reticulum Ca 2+ ATPase 2 (Serca2) Sarcoplasmic reticulum Ca2+ ATPase 2 (Serca2), a candidate molecule for re-uptake of calcium into the sarcoplasmic reticulum, allows the muscle relaxation. Decreased Serca2 expression level was found in the diabetic heart and undergone diastolic dysfunction in cardiomyopathy. The SR Ca 2+ loading determines the Ca 2+ ion concentration available for next contraction dictating the rate of myocardial relaxation. Increases in Serca2 activity tend to enhance myocardial contraction and relaxation.
## Myofilament proteins
Myosin, actin, and titin filament proteins are highly expressed in the fetal heart than that of adult and major signs of cardiac hypertrophy. Myosin-binding protein C, interacts with actin, and changes the myosin cross-bridges (33) Myosin heavy chains (MHC), with integral ATPase activity, is one of the most underlying ways to find changes of MHC composition in the hypertrophied heart. Z-disc MLP-TCAP-titin complex defects can lead to cardiomyopathy and heart failure development.
## Peptide hormones
Atrial and brain natriuretic peptide (ANP and BNP) are small hormones, and their secretion occurs during cardiac stress. For acute heart failures, ANP and BNP can be clinically administered as therapeutic agents. For chronic cases, on the other hand, neprilysin (responsible for the degradation of ANP and BNP) (34) inhibitor is used. Recent studies reported that human proBNP, in bloodstream and its post-translational modification at the N-terminal region could lead to its higher levels in cardiac patients.
## Transcription factors and inflammatory signals
The main transcription factor, critically involved in hypertrophy is GATA-4, which was found to be highly expressed in the fetal myocardium. It triggers a wide group of heart-specific genes such as α and β-MHC (Myosin heavy chain), MEF-2 (Myocyte enhancer factor-2), SP-1 (Specificity protein-1), and NFκB (Nuclear factor-κappa B) associated with hypertrophy (36).
## Natural polyphenols: therapeutics of dcm
Natural products have multiple pharmacological actions against pathological conditions and the use of these products is safer than synthetic drugs. The use of plants as a source of natural polyphenols in various forms of traditional medicines from ancient time. Huynh et al. illustrates the pathway involving several cascades involves in the progression of diabetic cardiomyopathy. Based on the findings the key findings on disease pathways and treatment options such as targeting antioxidant-signaling pathways, we also proposed a figure that involves in the treatment strategy through various polyphenolics to target several genes and ameliorates the pathological conditions arises by cardiomyopathy.
Herbal remedies are gaining significant attention and that nearly 80% of the total world population uses conventional medicine, comprising 40,000-70,000 medicinal plants. In recent years, due to toxicological concerns with the synthetic substances in food and increasing awareness about herbal therapies, the use of natural substances are demanding. Herbal formulations are found to be cost-effective and having low side effects. Plant extracts have recently gained interest due to their antiglycoxidative activities, that defend cells against the free radical attack and have numerous biological consequences. The synthesis of different organic solvent extracts may differ in quality and concentration depending upon the difference in the polarity of the solvent used for extraction and the extracted polyphenols. Phenolic compounds such as gallic acid, caffeic acid, ferulic acid, trans-resveratrol, quercetin, fisetin have been shown to act as natural antioxidants by neutralizing free radicals.
We have summarized below some plant extracts and natural polyphenols, showing doses of polyphenols, experimental models studied, and key findings, which further suggest their beneficial effects against cardiovascular abnormalities. In vivo studies indicated the beneficial effects of some polyphenols in DCM such as methanolic extract of S. cumini seeds treated group showed normal morphological cardiac features compared to the diabetic control. This may be due to the presence of quercetin, ellagic acid, rutin, and gallic acid in the extract which contributes to reducing aspartate aminotransferase (AST), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) level up to the normal. The combined effect of A. sativum and voluntary exercise has worked as a powerful defense system, which decreases HbA1c and malondialdehyde (MDA) production in cardiomyocytes of diabetic models. B. oleracea also found to upregulate Nrf2 activation, a critical marker of DCM, which were found to be decreased in diabetic models. Decreasing MDA and AGEs formation levels was observed on the administration of H. sabdariffa (100 mg/Kg) in STZ treated rats, which also improved cardiac function by augmenting mitochondrial antioxidant defense. Administration of M. oleifera significantly reduced the lipid peroxidation products and increased the enzymatic as well as non-enzymatic antioxidants in the diabetic rat. Administration of A. augusta and A. marmelos could significantly reduce the levels of Interleukin (IL-6) IL-6, IL-1β, and (tumor necrosis factorα) TNF-α in cardiac tissues of diabetic rats, stimulates the antioxidants defense system by increasing catalase (CAT), superoxide dismutase (SOD) activities, and maintained the cardiac integrity. E. oleracea treatment along with exercise reduced leptin, IL-6, and TNF-α serum levels in diabetic models, which may improve the insulin sensitivity. P. pinnata significantly diminished the activity of cardiac enzymes such as LDH, CK-MB, AST compared with diabetic rats showing its cardioprotective effects. Resveratrol also promotes Nrf2 mediated cardiac protection. Treatment of catechins significantly increases SOD, CAT, and GSH activities up to control in diabetic rat hearts, however decreasing the higher levels of cardiac biomarkers CK-MB, AST, LDH, and troponin T, suggesting its cardioprotective effect.
Curcumin supplementation leads to lowering the level of blood glucose and proinflammatory cytokines in DCM models. Plant polyphenols have been studied extensively through in vitro cell models for the treatment of cardiovascular diseases. In cardiac H9C2 cells, gallic acid was found to suppress hypertrophy and fibrosis by regulating JNK2 signaling and Smad3 binding. In these cells, ferulic acid was also shown to protect cardiomyoblasts from high glucose induced oxidative stress by mediating Ca 2+ homeostasis. Similarly, epigallocatechin-3-gallate mediated cardioprotection was observed through Akt/GSK-3β/caveolin signaling. Quercetin, a flavonoid, inhibits AP-1, and activates the PPAR-γ pathways leading to protection against cardiac hypertrophy. Another agents such as, resveratrol and kaempferol have been shown to involve in Sirt-1 dependent pathways to provide protection against ER stress and reoxygenation injuriesleading to decrease in cardiomyocyte apoptosis. Administration of curcumin (10 µM) in cardiac cells, combat the glucose induced stress by reducing the overproduction of ROS and apoptosis through PI3K/Akt pathway.
Polyphenols are large phytochemicals and only a small fraction of them seem to be absorbed by the gastrointestinal tract in their consumed form. It is assumed that a large portion of these is first decomposed by the gut microbiome into lower molecule metabolites that are then absorbed in the gut. The polyphenols could work in two ways: firstly, the polyphenols could be broken down in the gut to produce several different metabolites which are absorbed and enter the system circulation. Secondly, polyphenols can directly affect the gut microbiota leading to health benefits in the patients. This could be through a change in microbiome composition, by facilitating the generation of short-chain fatty acidsand improving oxygen levels by reacting with ROS and improving the immune system in the gut, however, such mechanisms are unavailable to in vitro models, which could be a major limitation of these studies.
## Conclusion and future perspectives
Hyperglycemia is strongly correlated with the manifestation of cardiac malfunction and heart failure. Preclinical studies also revealed the beneficial effects of antioxidants, anti-inflammatory agents on cardiac dysfunction.
Although DCM has been found in both type-1 and type-2 diabetes, hyperglycemia induced cardiac fibrosis has been mainly observed in type-I DM hearts. Type-2 diabetes is primarily associated with cardiomyocyte hypertrophy and steatosis. Therefore, the studies showed in this paper are limited for type 1 diabetes.
In vitro studies involving high dosage of anti-oxidants have shown their protective effects. However, larger studies especially in vivo and clinical trials have shown variable results. Large clinical trials with anti-oxidant agents like vitamin C and E have not been able to provide clear evidence of their beneficial effects in diabetic patients. One of the reason antioxidant treatments are failed because the overall oxidation levels in cells are strongly regulated. Simply using anti-oxidants concentrates, as supplements may not work, as intended and new techniques need to be developed to improve the efficacy of anti-oxidant treatments. One such recent successful method is by reversing loss of enzyme functions such as treatment with drugs that prevent ROS-induced eNOS uncoupling. Another approach could be delivery of anti-oxidant enzymes using viral transfection gene therapy and targeted delivery at cellular or sub-cellular levels. Additionally, nanoparticles based drug delivery may lead to an effective treatment against such diabetic cardiomyopathies.
# Author contributions
NA and VR designed the manuscript. NA, DY, VR, and J-OJ wrote the manuscript. NA, DY, and J-OJ edited the manuscript. All authors contributed to the article and approved the submitted version. |
SAM and Severe Mitral Regurgitation Post–Acute Type A Aortic Dissection Surgery Treated With MitraClip
Post-operative systolic anterior motion associated with mitral regurgitation can be a challenging combination. We present the case of a 64-year-old male patient managed by MitraClip (Abbott Laboratories, Abbott Park, Illinois) implantation for systolic anterior motion and severe mitral regurgitation in the early post-operative period after aortic dissection surgery. This is the first description of MitraClip use post-aortic dissection. (Level of Difficulty: Intermediate.) (J Am Coll Cardiol Case Rep 2020;2:1582-6)
A lthough mitral valve (MV) systolic anterior motion (SAM) was initially described in patients with hypertrophic obstructive cardiomyopathy, it can also be present in patients with complex dynamic left ventricular anatomy.
This "dynamic SAM" can potentially occur after cardiac surgery and significantly affect perioperative management and the patient's prognosis (1).
## The mitraclip system (abbott laboratories, abbott
Park, Illinois) has been used to manage new onset SAM after MV repair [bib_ref] Firstin-man MitraClip implantation to treat late postoperative systolic anterior motion: rare cause..., Agricola [/bib_ref] or aortic valve (AV) interventions [bib_ref] MitraClip implantation for the treatment of new-onset systolic anterior motion of the..., Grasso [/bib_ref]. Clinical hemodynamic outcomes suggest that it could be a feasible and safe alternative in these settings [bib_ref] The use of MitraClip for symptomatic patients with hypertrophic obstructive cardiomyopathy, Thomas [/bib_ref].
We describe a rare case of SAM associated with severe mitral regurgitation (MR) complicating the early post-operative period after treatment of type A
## Learning objectives
Precise and prompt recognition of this potentially lethal association is vital to provide adequate clinical and interventional management.
MitraClip can be an effective and lifesaving alternative to manage SAM associated with severe MR in patients with high surgical risk or a contraindication to conventional cardiac surgery.
acute aortic dissection (AAD), which was successfully managed with a MitraClip intervention.
## History of presentation
A 64-year-old man was admitted to the emergency department with a sudden onset of retrosternal discomfort radiating to the back and superior abdomen associated with weakness in the right arm.
On emergency department arrival, vital signs were as This case shows that, in selected patients, Mitra-Clip use can be a feasible alternative to address refractory MR and SAM, with excellent safety profile and hemodynamic outcomes.
[fig] FIGURE 1: Pre-Operative TEE During Surgery for Acute Type A AAD (A) Moderate aortic valve regurgitation. (B) Moderate mitral regurgitation and nonsignificant systolic anterior motion. AAD ¼ acute aortic dissection; TEE ¼ transesophageal echocardiography. the first post-operative days, hemodynamic stabilization was not obtained, and high doses of inotropic and vasopressor agents were required. TEE was repeated and revealed severe MR, an anteriorly directed eccentric jet, P 1 to P 2 leaflet prolapse, and severe and persistent SAM (no LVOT obstruction, severe MR, not reversible with intravascular volume expansion and increase in the afterload) (Figures 2A and 2B, Videos 2 and 3). [/fig]
[fig] FIGURE 2 1: TEE Performed After AAD Surgery Images show (A) severe mitral regurgitation with an eccentric regurgitant jet directed anteriorly to the atrial roof, combined with (B) systolic anterior motion. Abbreviations as in Figure Tagliari systemic vascular resistance and persistent atrial fibrillation, may have led to left ventricular underfilling. This condition, aggravated by the presence of a hyperdynamic state resulting from inotropic agent and excessive sympathetic activation, has been identified as a potential SAMaggravating mechanism (6,7). Regarding the MR mechanism, it is noteworthy that, although MR mediated by SAM is traditionally characterized by a posteriorly directed jet on Doppler echocardiography, our patient had a predominantly anteriorly directed eccentric jet, which suggests the presence of an intrinsic MV disease. This statement is strengthened by the observation that, in patients with SAM and central or anterior MR jets, a significantly elongated posterior leaflet is usually present (8). A posterior leaflet basal portion bulging beyond the anterior leaflet may reduce the amount of posterior leaflet area effectively available to follow and coapt with the anterior leaflet toward the septum, thus explaining why MR is usually more significant when prolapse is present (9). Nonetheless, a more careful analysis of the available echocardiographic images could suggest the presence of chordal SAM associated with MV prolapse and increased left ventricular contractility, rather than true SAM involving the anterior leaflet body. When MR worsens following aorta surgery, investigators have proposed that because of the proximity between the MV and the AV, surgical intervention in the latter can damage the former or alter the normal dynamics of the aortomitral curtain (10). In our patient, the AV resuspension technique could have displaced the MV leaflets towards the aorta, thereby reducing the coaptation length and increasing the posterior leaflet prolapse. This surgically provoked MV anterior translocation may have aggravated the pre-existing MR and impaired clinical recovery. In the present case, transcatheter MV repair was considered the best option because of the patient's hemodynamic instability and absolute refractoriness to medical management, both of which created a condition of "too high risk" for open cardiac surgery reintervention. Despite the successful outcome obtained in this patient, it is important to keep in mind that conservative medical therapy is usually enough to stabilize patients with post-operative SAM. A 2-step approach, consisting of intravascular volume expansion simultaneously with any inotropic drug discontinuation (first step), followed by maneuvers to increase the afterload simultaneously and in the short term with a bolus administration of esmolol (1 mg/kg) (second step, applied when the first step fails to elicit a response), has been proposed. Following these steps, invasive management is indicated when the SAM does not disappear after conservative management (persistent SAM) (5). [/fig]
[fig] FIGURE 3: TEE After MitraClip Implantation Images show improvements in (A) systolic anterior motion and (B) mitral regurgitant jet. TEE ¼ transesophageal echocardiography. closure, the patient had no major complications related to the MitraClip procedure. At 1and 3-month follow-up, he had no cardiovascular symptoms. The 1-month echocardiographic image revealed mild AV regurgitation, mild MR, and 3 mm Hg mean MV gradient. CONCLUSIONS To the best of our knowledge, this is the first description of MitraClip implantation in the immediate post-operative period in a patient with type A AAD surgery performed to address SAM and severe MR. In this setting, less invasive percutaneous management can be considered as a first-line option instead of higher-risk redo surgery. ADDRESS FOR CORRESPONDENCE: Dr. Francesco Maisano, Cardiovascular Surgery Department, University Hospital of Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. E-mail: francesco.maisano@ usz.ch. KEY WORDS aorta, dissection, mitral valve, post-operative APPENDIX For supplemental videos, please see the online version of this paper. [/fig]
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Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through
# Introduction
Lentiviruses are a genus of retroviruses that include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), puma lentiviruses, and equine infectious anaemia virus (EIAV). Among these viruses, HIV is widely used and has become the standard for lentiviral vectors (LVs) to facilitate the delivery of genetic material (DNA or RNA) into target cells. HIV-1 derived LV is well known for its efficient and stable transduction in dividing and non-dividing cells. It integrates the desired transgene into the target cell genome using a viral integrase enzyme. In addition, LVs have the advantage of broad cell tropism and target-specific cell types through pseudotypes.
LVs can be stably integrated into the target cell genomes as proviruses. The three generations of LV-packaging systems are safer and are suited for the production with HEK293T cell line, while several studies reported increased vector production by utilising SV40 Tantigen. Vectors yielded at 48 and 72 h post-transfection can be harvested from the supernatant. Further purification and concentration protocols should be performed to produce debris-free LVs. Due to the integration of provirus at the random site of a target genome, . The HIV-1 virus contains three gene regions, gag, pol, and env, along with accessory genes , regulatory genes (tat, rev), and the 5′ and 3′ flanking long terminal repeats (LTR). The psi (Ψ) element is located at the 5′ end of the HIV-1 genome just upstream of the gag initiation codon.
## Gene
Size of Protein Function Accessory Proteins . The basic four-generation lentiviral vector plasmid system. The transgene vector is similar for both first-and second-generation lentiviral vectors, while the third-generation lentiviral vectors additionally have a central polypurine tract (cPPT) and woodchuck hepatis virus post-transcriptional response element (WPRE). SIN vectors also replace the U3 region of 3-LTR in both third-and fourthgeneration LVs. The packaging vectors differ for the four generations. The first-generation lentiviral vector has all of the accessory genes required for viral replication (vif, vpr, vpu, and nef ). In contrast, second-generation lentiviral vectors do not have these accessory proteins in their packaging vector. Third-generation LVs do not have the Tat regulatory protein. The fourth generation is differentiated by the split of the gag/pol and rev sequences into two different cassettes. All four systems contain VSV-G as an envelope vector.
## Non-integrating lentiviral vector (nilv) design
A vital component of the integration complex of LV is the viral integrase enzyme (IN) that catalyses viral DNA integration into the host genome. IN mediates the integration between vector and host DNA as the integration complex reaches the nucleus. However, this process carries predictable risks of harmful insertional mutagenesis, prompting an examination of alternatives to vector-mediated integration. To address this, NILVs were established. NILVs can stably express transgenes from the episomal DNA in non-dividing cells or transiently if the target cells divide both in vitro and in vivo. This also prevents the provirus formation while the vector DNA remains one of the primary episome types. Few approaches were revised in this generation of NILVs. NILVs were developed by mutating the integrase gene or by modifying the attachment sequences of the LTRs, and these mutations were divided into class I and II mutations according to their effects, as shown in. C-terminal domain Impairs binding to genomic DNAClass II mutations can be performed by exchanging the standard gag or pol packaging plasmid with an IN mutant strain. However, several studies reported that mutations in specific amino acids of IN resulted in impaired integration of lentiviruses. As class II mutations are mostly involved in altering the three protein domains in IN, namely the N-terminal domain, C-terminal domain, and IN core domain, they can affect the reverse transcription as well. Mutations introduced into IN not only affect the integration but also other relevant processes, especially by impairing the vital viral life cycle stages and causing pleiotropic effects that can make them unsuitable for vector development.
In contrast, class I mutations are limited in affecting the integrase multimerisation, linear episome processing, and DNA binding, as listed in. Mutations at a specific region will specifically target the integrase activity while keeping the other viral stages intact. The IN core domain has D64, D116, and E152, which are collectively known as a catalytic triad. Any alterations to these amino acids will inactivate the catalytic activity of IN and inhibit the integration while preserving the transgene expression. Additionally, mutations at W235, N120, and RRK (262-264) specifically block the genomic DNA binding. Alterations of H12 affect the IN multimerisation to generate NILVs. In addition, K264/K266/K273 mutations, also known as triad mutations, have been proven to impair both the DNA binding and strand transfer. Mutated 3 LTRs CA/TG dinucleotide gave rise to LTR att mutants and also make successful NILVs remain functional and efficient.
## Comparisons of nilvs with other non-integrating methods
Together with NILVs, various methods and options were also identified and studied for their efficiency in pre-clinical studies, as the elimination of transgene integrations is one of the major goals. Several integration-free approaches and their limitations are listed in, all of which have their own limitations. Among these methods, episomal plasmid and Sendai viral (SeV) vector are the methods that have highest efficiency and with only occasional integration. Therefore, the NILV approach was compared to episomal plasmid and SeV methods to examine its advantages and limitations before proceeding with NILVs as a better option in clinical applications. SeV vector, an RNA viral vector, is one of the most valuable tools in human iPSC generation. It is also well-known for its high transduction efficiency compared to other methods. Another recent study showed the ability of SeV vector to produce small regulatory RNAs with high transduction efficiency, durable expression, low cytotoxicity, and less risk of chromosomal insertion. Although the SeV vector is known as being "exgene-free", several researchers are still unsure of its safety in producing clinical-grade iPSC lines, as the avoidance of transgene integration passively depends on the cell passage. Adding to this, the expression levels of the genes may vary according to the infected cells, and this might affect the quality of the generated iPSC lines. Therefore, the long-term stability of the genome structure and epigenetic conditions must be thoroughly examined.
Although the SeV vector is considered safe due to its integration-free ability, these kinds of virus-based vectors have a size limitation in terms of their carriages compared to plasmid-based vectors, which theoretically have no restrictions in the size of their carriages. Thus, as an alternative option, researchers also studied Epstein-Barr virus (EBV) episomal plasmids, which have stable gene expression and long-term plasmid retention, especially in dividing cells. In addition, EBV has a cargo packaging capacity that overwhelms this vector compared to adeno-associated virus (AAV). However, EBV is not superior to the AAV vector because this virus vector does not integrate into the host genome and is non-pathogenic. AAV also has been shown to have limitations in a few gene therapy studies, such as its toxicity and unnecessary immunoreaction, especially in neuroscience research. Recently, one research group used a combined gene delivery method in which the EBV nuclear antigen (EBNA) and its OriP fragment with recombination sites were incorporated into the baculoviral vector. This fragment was then recombined in a host cell to create an episomal vector. This approach had high transgenic efficiency.
NILVs exhibit higher transfer efficiency in not only in the dividing but also the nondividing cells, attracting a wide range of researchers. Unlike LVs, NILVs do not present a risk of insertional mutagenesis. Moreover, NILVs are capable of transducing a broad range of cells or tissues with cargo packaging capacity compared to other vector systems. However, NILVs cannot introduce stable exogenous gene expression to the dividing cells, thereby limiting their application in clinical settings. Thus, adding modifications to the NILV design is one of the popular options. For example, Xu et al. added a minimal scaffold-matrix attachment region (S/MAR) sequence (SNIL) and successfully proved that this method manages to retain the episomal transgene expression in the dividing cells.
## Limitations of nilvs and possible solutions
As mentioned in the previous section, NILV has drawbacks that need to be corrected. Some major drawbacks can be fixed through simple modifications or by choosing NILVs wisely for appropriate studies. For example, NILV-transduced cells will harbour the circular non-replicating nuclear episomes but without the capability of replication, resulting in the loss of the NILV episomes during rapid division and limiting their use for long-term expression. Therefore, NILVs can be a great option for less-proliferating or slower-dividing cells such as mesenchymal stem cells (MSCs), but are not suitable for highly proliferating cancer cells.
Furthermore, NILVs also confer reduced transgene expression compared to their integrating versions. This can be amended by using strong enhancers or promoters that can produce sufficient transgene expression. On the other hand, one can also reduce the use of inhibitors to episomal transgene expression, by removing cis-acting genes, although this is still insignificantand requires further studies. Another approach to enhance the expression of integrase-defective LVs is by retaining the Vpr accessory protein via transcriptional activation of the HIV-1 LTR. However, studies found that Vpr induces global remodelling of the cellular proteome and could be responsible for the silencing of the unintegrated virus. Thus, one study suggested that Vpr itself can antagonise this silencing by degrading the SMC5-SMC6 complex localisation factor 2 (SLF2), which is responsible for the restriction of gene expression in the unintegrated viral genomes.
On the other hand, the residual integration of NILVs is still not accepted for certain clinical applications, although these integrating pro-viruses are non-canonical and would not necessarily display all of the characteristics of wild-type proviruses. Thus, NILVs are still the best option for quiescent cells, as critically summarised in one review paper. High transgene expression with reduced integration while maintaining low-level vector episomes should be the ideal goal for successful NILV development. Since the integrative lentivectors are useful for some applications, especially for achieving long-lasting persistence, avoiding the presence of integrated proviruses in the cells would strongly reduce the therapeutic effect. However, we cannot deny that the vector persistence in every transduced stem cell can potentially influence the safety profile in the clinical studies of the diseases.
## Clinical application of nilvs
LVs are among the most efficient gene transfer tools for dividing and non-dividing cells. However, insertional mutagenesis has been found in clinical trials with lentiviral vectors, prompting a detailed study of genotoxicity assays of all integrated vectors. Avoiding integration is the most direct approach to overcome this problem for many clinical applications. This can be facilitated through extensive studies of the integrating mechanisms of lentiviruses, as discussed earlier. NILVs have been studied for the treatment of infectious and genetic diseases, in addition to being great cell mediators for reprogramming. From our point of view, the transient expression of NILVs over stable expression is also highly preferable for vaccinations, cell type differentiation, site-directed integration, and persistent episomal expression. This is due to the progressive loss of the transgenes expressed from these non-integrated proviral forms.
## Vaccination
Transient expression of NILVs is required for vaccine development, as it shows sustained immune responses against various diseases in pre-clinical models, as shown in. Apart from cellular and humoral immunity, NILVs have also been proven successful for sustaining anti-tumour immunity. Dendritic cells (DC) are the most common targets for vaccines. They represent a safe and efficacious vaccination platform for the development of prophylactic vaccines. Many studies used dendritic cells as targets for malaria, thymoma, hepatitis B virus (HBV), and many more infectious agents that can be treated with NILVs. In a pre-clinical study, a single immunisation of NILVs encoded in a secreted form of the envelope of a virulent strain of West Nile virus (WNV) induced a strong B cell response, and a single immunisation was also sufficient to induce early and long-lasting protective immunity. On the other hand, one in vivo study involving the H5 influenza A virus (IAV) demonstrated that the monoclonal antibody (mAb) administered through NILVs did not persist for longer time points. However, this could be studied further to make it an effective strategy for rapid protection against infectious diseases in the future. Vaccinia virus, Zika virus, and human cytomegalovirushave also been investigated in pre-clinical studies to test their immune responses. NY-ESO-1 gene Dendritic cellsAdditionally, the injection of the DC-directed IDLV encoding OVA has shown promising immune responses in C57BL/6 mice and complete protection against a thymoma tumour expressing a delivered ovalbumin (OVA) antigen in mice. This shows that the IDLV system represents a promising and efficient vector platform for the future development of DC-based immunotherapy. Furthermore, cancer cell vaccines were also developed as an alternative approach to DC, since cancer cells express tumour antigens of interest and show robust improvements. Human immunodeficiency virus (HIV-1), human papillomavirus (HPV), and hepatitis C virus (HCV) were tested with cancer vaccines targeting the antigen-presenting cells. Developing a strong cellular immunity among HIV patients has been the aim of several clinical studies. Therefore, several studies have suggested working on SIV-based NILVs to evaluate the induction of transgene-specific immune responses against sensibly designed structural HIV antigens, as proven in BALB/c mice. This could be very efficient for studying the T cell immunogens for the development of longlasting and effective HIV vaccines. A study showed that NILVs injected intramuscularly express systemic CD8+ T cells and antibody responses to the secreted hepatitis B virus (HBV) surface antigen and have also been proven as a great tumour therapy.
As a cancer immunotherapy candidate, NILVs delivering the tumour antigen NY-ESO-1 to the human dendritic cells in vivo have shown promising clinical results by showing sustained CD8+ T cells within 14 days post-immunisation and anti-tumour responses. In 2020, this vaccine was improved with a heterologous boost by priming it with a recombinant protein, adenoviral vector, or self-replicating RNA boost. This resulted in increased efficacy of ZVex as a cancer vaccine. Improving the existing vaccines is a better option compared to developing new vaccines.
Another exciting study involved the application of NILVs to generate a vaccine candidate against coronavirus disease 2019 (COVID-19). Golden hamsters, which are naturally tolerant to SARS-CoV-2 replication and closely mimic human COVID-19 physiopathology, showed significant vaccination effectiveness and could limit lung deleterious damage by utilising the NILV approach. These findings showed that NILV-based intranasal vaccination against SARS-CoV-2 has a significant prophylactic effect and is a viable option against COVID-19.
## Cell-type differentiation
Regenerative medicine involving pluripotent stem cells has become an ideal goal for many researchers. Cellular reprogramming is another great application of NILVs via transient expression, as shown in. Additionally, iPSC production and differentiation into the lineage of interest is an alternative approach to the use of embryonic stem cells (ESCs). Furthermore, iPSCs can be an outstanding alternative to embryonic stem cells, although have several limitations and ethical concerns. Additionally, iPSCs also have similar pluripotency ability to human ESCs. As such, iPSC technology could be the desired application method for NILVs, as fewer genomic abnormalities are expected. Successful transformation of human somatic cells into iPSCs was first carried out in 2007 using a lentiviral system. Since then, multiple approaches were taken by other researchers worldwide in generating iPSCs with additional transcription factors, such as Oct4, Sox2, KLF4, and C-myc (OSKM), while concentrating on higher safety profiles in clinical settings. Certain transcription factors are known as oncogenes and possible side effects are foreseeable. Nonetheless, reprogramming without viral integration with plasmids or direct reprogramming protein delivery assays can solve this issue. Additionally, several more recent technologies and approaches have been suggested for producing iPSCs without transgene integration that can cause possible mutagenesis, such as non-integrating viral vectors, RNA virions, RNA replicons, non-integrating replicating episomal plasmids, minicircles, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, polycistron, and preintegration of inducible reprogramming factors. Polycistronic technology reduces the requirement for multiple viral vectors to deliver four different transcription factors, cutting this down to a single-stem cell cassette. A study by Awe et al. compared the reprogramming efficiency of the polycistronic stem cell cassette (STEMCCA) to other integration-free approaches. A similar polycistron approach was slightly modified using the Cre-Lox method. Another study was performed, whereby the integrated transgenes were excised from the reprogrammed genomes using the Cre-Lox method first and a single plasmid containing the four reprogramming factors was linked by the 2A sequence.
This type of advanced technology involving minimising genomic integrations can be used for potential human clinical trials with safer profiles. More studies are working on enhancing the iPSC reprogramming efficiency, such as by adding SV40 large T antigen (T) by 23-70-fold from human fibroblasts. A recent study conducted a DNA methylation landscape assessment for isogenic iPSCs to compare different reprogramming methods, which could also be a way of proving the safety of iPSC lines generated by reading any significant changes in DNA methylation profiles.
Various platforms can be used to generate iPSCs through non-integrating methods, such as by transient transfection, EBV episomal plasmids, synthetic mRNA, adenoviral vectors, microRNA mimics, minicircles, and SeV vectors. However, all of these methods have low re-programming efficiency, except the Sendai viral vector. NILV-based iPSCs can be used to study stem cell biology, as a cellular platform for pharmacological and toxicological studies, and can be considered as a possible source of autologous stem cells for use in regenerative medicine. Additionally, iPSCs have a great advantage in replacing human tissues or cells for disease modelling, drug screening (toxicity and efficacy), and cell-based therapy, especially for cardiovascular diseases.
However, most studies have used other non-integrated approaches, as discussed earlier, and only a few studies have used the NILV approach to produce iPSCs. In a previous study, purified integrase-deficient LV facilitated the generation of a population of purified hESC-derived hepatic progenitors that were devoid of integrated viral DNA. The ESCs could be differentiated into specific progenitor cells. For example, human embryonic stem cells (hESCs) encoding for green fluorescent protein (GFP) driven by the liver-specific apolipoprotein A-II (APOA-II) promoter can be differentiated into hepatic progenitors at day. Surprisingly, 99% of these APOA-II-GFP-positive cells expressed the hepatoblast markers, such as α-fetoprotein and cytokeratin-19, and were further cultured into more mature cells that resembled the hepatocytes. This approach can be used in cell therapy and in in vitro applications, such as drug screening. The reprogramming efficiency of NILV has not been well-explored or compared with the SeV approach in vitro. Therefore, evidence is needed in the future of the dynamic effects on genomic stability.
To the best of our knowledge, iPSCs in clinical trials mostly involve neurological and cardiovascular disease treatments (such as for Parkinson's disease) via iPSC-derived HSCs and cardiac cell injury via iPSC-derived cardiomyocytes. Japan started the world's first iPSC-based clinical study in 2013, whereby a macular degeneration patient received a transplant of iPSC-based retinal cells; however, the patient showed no improvement. Another study at Kyoto University focused on dopaminergic precursor cells that were differentiated from iPSCs injected into the brain of a patient with Parkinson's disease. This boosted the dopamine levels and improved the patient's symptoms.
## Site-directed integration
NILV is also used as a template for site-directed integration systems. A variety of systems are available for direct integration into genomic "safe sites" or for gene-specific correction to minimise the dysregulation of gene expression, as listed in . One approach involves combining a recombinase or transposase transiently with an NILV to facilitate integration at specific sites. The most recent example involved the use of the sleeping beauty (SB) transposon and transposase expression cassette. NILVs were able to facilitate transient transposase expression to the target cells. Several studies using NILVs have shown similar integration to the SB cassette without viral element integration, thereby reducing the insertional mutagenesis. An HIV-1/SB hybrid vector facilitated by the hyperactive SB100X transposase that allows efficient DNA transposition in primary human cells could be a valuable tool for therapeutic gene transfer, as it could be inserted into actively transcribed genomic regions. Another study explained that non-viral yeast Flpx9 recombinase produced by NILV can enter the circular viral recombination substrates and facilitate the site-directed genomic insertion. . A summary of the elements used in developing non-integrating lentiviral vectors (NILVs) for site-directed integration in pre-clinical studies. Different approaches for direct integration or site-specific modifications of safe genomic loci in different studies are listed with attached references. Moreover, NILVs can be designed to promote site-specific homologous recombination (HR). This includes NILVs combined with a rare cutting nuclease for targeted recombination at specific sites by HR. This will lead to successful targeted gene correction. For example, an NILV encoding a repair template was co-transported with an I-SceI nuclease expression vector to rescue a defective enhanced green fluorescent protein (EGFP) gene. Expression of the nuclease created a double-strand break (DSB) within the targeted locus and stimulated stable gene correction at the end of the process. Adding to this, calmegin (clgn) targets the neomycin-resistant transgene cassette that generates ES cells via transgene insertion. However, this method is unbeatable against the traditional electroporation method, which allows for more efficient gene transfer. Thus, better modifications and changes are the main concerns of many researchers. NILVs can also be used as templates for HR along with engineered zinc finger nucleases (ZFNs)and transcription activator-like effector nucleases (TALENs)to induce successful gene correction at the target gene locus with less off-target integration. Recently, the development of regularly clustered, interspaced, short palindromic repeat and CRISPR-associated protein 9 (CRISPR/Cas9) technology has made NILVs ideal tools. CRISPR/Cas9 and a guide RNA targeting the cytochrome b-245 heavy chain can correct the defects in hematopoietic cells. Another study also utilised CRISPR/Cas9-mediated, homology-directed repair via an ex vivo approach to conduct gene correction for recessive dystrophic epidermolysis bullosa.
## Nilv modification
## Gene therapy
From our viewpoint, the use of NILVs in gene transfer or gene therapy is a promising strategy for delivering therapeutic genes for genetic diseases such as cancer, macular degenerations, heart disease, diabetes, haemophilia, AIDS, and most blood-related disorders that is equal to the use of LVs. The LV-based gene therapies known as Zyntegloand Libmedlyhave been announced as treatments for β-thalassemia and metachromatic leukodystrophy (MLD), respectively, by the European Medicines Agency (EMA). However, this may to indicate a certain level of uncertainty related to future products, which may further delay the success and approval of LV-based therapies. Therefore, NILVs as alternatives with proven success in gene therapy can also be approved to meet the ultimate goal of treating genetic disorders without detrimental risks. Researchers have been replacing mutated and defective genes and making diseased cells more evident to the immune system. Gene transfer is a challenging process, as poorly studied unsuccessful steps can lead to adverse immune system reactions, incorrect cell targeting, infections caused by the virus, and tumour growth. For example, SCID-X1 patients who received HSCs transduced with murine leukaemia virus (MLV) developed abnormal T cell proliferation due to the insertion of a retroviral vector near the LMO2 proto-oncogene. In another study, a success-ful dopamine replacement gene therapy in patients with Parkinson's disease performed by targeting important genes for dopamine synthesis regulated the dopamine concentration, proving that gene therapy can be successful. Therefore, gene therapy involving NILVs also needs to be assessed in order to bring this method into clinical application, since it has been proven to be better and more successful than other gene delivery methods, as listed in. Hematopoietic cellsNILVs encoding for in vivo expression of GFP have been reported in organs such as the brain, liver, and spinal cord via stable gene transfer for longer periods (up to months) in several studies. In another study, hepatocytes that target NILVs with coagulation factor IX (FIX) in haemophilic mice induced an immune tolerance. A similar study using FIX transgenes carrying an R338L amino acid substitution associated with clotting hyperactivity and thrombophilia showed increased gene therapy efficacy but also less efficiency in hepatic transgene expression of NILVs. Recently, another study demonstrated that the NILV approach can overcome immune rejection and allow for the growth of transduced cells in an immunocompetent host by producing CRISPR-modified murine cell lines using mutated integrase vectors. In addition to the advantages of the CRISPR/Cas9 system, another study proved that a Cas9 protein delivery system with NILVs encoding both guide RNA and donor DNA resulted in efficient DNA breakage, one-time genome correction of the sickle cell disease (SCD) mutation, and long-term engraftment of HSCs. These studies explained the use of combined NILV approaches for a safer, long-lasting, and fruitful outcome in future research. Gene therapy has been given more importance compared to other clinical applications in treating many genetic disorders that are highly disruptive to human health. The latest research has also demonstrated a successful combination gene therapy strategy for HIV using a two-vector system design, which uses an integrating LV to transduce the cells sequentially and a non-integrating lentiviral LV to insert the conditional suicide gene, with knockout of CCR5, and transient expression of GFP to enrich the modified cells.
## Future directions and challenges
From the frame of reference of this review, the integration-free capability of NILV is highly desirable for gene therapy and could be the gold option for vaccination and cell immunisations studies with long-lasting immune responses. However, very limited or less studies have chosen NILVs as a gene delivery method to generate iPSCs, although this approach has potential. The earlier iPSC reprogramming work utilised the viral vector system to express the advantages of OKSM with a highly successful rate. This opened up new possibilities in regenerative medicine, especially for the development of diseasespecific models, including in drug toxicity studies. However, this viral vector also causes multiple potentially harmful integrations of the transgenes into the host genome, which lead to tumour formation. Although non-viral methods such as protein transduction, MiRNA expression, and mini circle vector expressionhave been introduced to eliminate this harmful effect, the efficiency of generating functional iPSCs using the viral system means it is still favoured. Therefore, the effort to refine and develop alternative approaches is being continued, including the use of non-integrating systems to deliver the reprogramming genes.
In addition, a study published a decade ago showed for the first time the successful reprogramming of blood cells using non-integrative SeV as an efficient integration-free gene delivery method. The high reprogramming efficiency without genomic integration of the SeV vector was also demonstrated commercially. Since iPSCs have not been generated using the NILV approach, the same concept can be expected to be applied using NILVs with a similar safety profile and with a slight possibility of transgene integration into the host in the future. This increases the hope of working more on NILVs by setting SeV vectors as an ideal example to generate successful high-clinical-grade iPSCs. In conclusion, research is still ongoing at every step of therapeutic application for NILVs in the hope of achieving better curative options in the future.
The major drawback of NILVs are their reduced transgene expression and gradual loss of episomal vectors, especially in dividing cells, which eventually reduce the therapeutic effect and mean it does not have long-lasting persistence. Some important improvements that can be further explored in NILV systems are the introduction of multiple mutation sites of the integrase sequence gene. This approach could help to increase the episomal gene expression, as reported in a previous study where different mutation sites of the integrase plasmid resulted in different gene expression levels. Another suggestion is to use a different type of promoter to drive the expression of the pluripotent genes, as the current promoter, the CMV promoter, is likely to be highly methylated during prolonged expression and might not be suitable or efficient for episomal expression due to the high replication rate of the transduced cells. Only targeted methylation-induced gene silencing could be reversed via the addition of 5-aza-2 -deoxycytidine. Alternatively, another option is to consider introducing a transduction domain at the 3 -5 exonuclease of the transcription gene, which could boost and enhance the gene expression, thereby increasing the reprogramming efficiency using NILVs.
Additionally, as NILVs have been used as the best option for non-invasive gene-based imaging only in non-dividing cells, highly proliferating cells should be considered for use with this approach in the future by merging OriP plasmid technology from EBV into NILV episomes that can retain the plasmids. Apart from the SNIL vector, incorporating the simian virus 40 (SV40) into NILVs could maintain the episomal DNA in dividing cells. One concern in merging or modifying the original vector to fix these limitations is the abnormal mutations and infections, which could multiply the existing limitations. Thus, careful epigenetic studies and DNA methylation profiles are needed prior to successfully applying the NILV approach in clinical settings.
In a nutshell, NILVs have emerged as an important tool in biomedical research as a therapeutic option. Future studies are needed with more advanced human clinical trials to achieve improvements and for optimisation in order to increase the safety and reduce illegitimate integration. The application of NILVs in the pre-clinical trials of recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation should be explored further to bring about even more advancements in the future. |
Interventions for preventing or treating malnutrition in homeless problem-drinkers: a systematic review
Background: Excessive drinking leads to poor absorption of nutrients and homeless problem-drinkers often have nutritionally inadequate diets. Depletion of nutrients such as vitamin B1 can lead to cognitive impairment, which can hinder efforts to reduce drinking or engage with services. This review aimed to assess effectiveness of interventions designed to prevent or treat malnutrition in homeless problem-drinkers.Methods: We systematically searched nine electronic databases and 13 grey literature sources for studies evaluating interventions to improve nutrition in homeless populations, without regional or language restrictions. Screening for inclusion was done in duplicate. One reviewer extracted data and assessed risk of bias, and another checked the extractions. Primary outcomes were nutrition status/deficiency, liver damage, and cognitive function. Secondary outcomes included abstinence, comorbidities, resource use, acceptability and engagement with intervention. Results were synthesised narratively.Results:We included 25 studies (2 Randomised Controlled Trials; 15 uncontrolled before and after; 7 surveys; 1 case-control). Nine studies evaluated educational and support interventions, five food provision, and three supplement provision. Eight studies evaluated a combination of these interventions. No two interventions were the same, and all studies were at high risk of bias. Nutritional status (intake/ deficiency) were reported in 11 studies and liver function in one. Fruit and vegetable intake improved with some education and support interventions (n = 4 studies) but not others (n = 2). Vitamin supplements appeared to improve vitamin deficiency levels in the blood (n = 2). Free or subsidised meals (n = 4) and food packs (n = 1) did not always fulfil dietary needs, but were usually considered acceptable by users. Some multicomponent interventions improved nutrition (n = 3) but acceptability varied (n = 3). No study reported cost effectiveness. Conclusions: The evidence for any one intervention for improving malnutrition in homeless problem-drinkers was based on single studies at high risk of bias. Various food and supplement provision interventions appear effective in changing nutritional status in single studies. Educational and multicomponent interventions show improved nutritional behaviour in some studies but not others. Further better quality evidence is required before these interventions can be recommended for implementation. Any future studies should seek the end user input in their design and conduct.
# Background
Problem-drinking is common among homeless people [bib_ref] The prevalence of mental disorders among the homeless in western countries: systematic..., Fazel [/bib_ref] [bib_ref] Substance misuse and related infectious diseases in a soup kitchen population, Magura [/bib_ref]. Homeless people are not just those sleeping rough on the street, but also include the 'hidden homeless' staying with friends or family, in hostels or bed and breakfasts, or in other vulnerable housing situations [bib_ref] The homelessness monitor: England, Fitzpatrick [/bib_ref]. Problem-drinking is a variably defined term and the concept can refer to alcohol abuse without physical dependenceor beyond 'safe' social drinking, or drinking above recommended levels and having problems in life as a result.
Problem-drinkers tend to obtain a large proportion of their energy intake from alcohol [bib_ref] Problems in nutritional status among homeless populations: an introduction, Coppenrath [/bib_ref] [bib_ref] Dietary inadequacies observed in homeless men visiting an emergency night shelter in..., Darmon [/bib_ref]. However, an alcohol-rich diet lacks important vitamins and minerals [bib_ref] Nutritional assessment of charitable meal programmes serving homeless people in Toronto, Tse [/bib_ref] and also reduces the absorption of nutrients by damaging the gut [bib_ref] Alcohol's role in gastrointestinal tract disorders, Bode [/bib_ref]. When left untreated, this can lead to impairment of cognitive, liver, and immune function [bib_ref] Differential depletion of carotenoids and tocopherol in liver disease, Leo [/bib_ref] [bib_ref] Alcohol's contribution to compromised immunity, Szabo [/bib_ref] [bib_ref] Severe infections are common in thiamine deficiency and may be related to..., Wijnia [/bib_ref]. Homeless people are at risk of being malnourished due to several factors, such as low income, limited knowledge and choice of food, and lack of cooking and storage facilities [bib_ref] Food choice and nutrient intake amongst homeless people, Sprake [/bib_ref]. The combined effect of homelessness and problem-drinking increases the risk of malnutrition [bib_ref] Nutrient intake and nutritional status in alcoholics, Salaspuro [/bib_ref].
Malnutrition costs around £19.billion per year to the public taxpayer in England and accounts for 33% of the hospital inpatient costs. In December 2016 the homelessness charity Shelter said a lower-end estimate of the number of homeless in England was 250,000 from official datasets [bib_ref] More than 250,000 are homeless in England -shelter, Richardson [/bib_ref]. The number of malnourished individuals in sheltered housing in England is estimated to be 22% higher than that in hospital inpatients. An approach to countering malnutrition is improving the nutritional quality of the food available to the population at risk. This could be achieved by educating people about healthy diet, distribution of nutritious meals or supplements, or advising the providers of food and healthcare how to tackle nutritional deficiencies.
Systematic reviews on interventions that either tackle homelessness or substance abuse in the homeless have been published [bib_ref] Effectiveness of interventions to improve the health and housing status of homeless..., Fitzpatrick-Lewis [/bib_ref] [bib_ref] A systematic review of interventions for homeless women, Speirs [/bib_ref] [bib_ref] Interventions to improve the health of the homeless -a systematic review, Hwang [/bib_ref] , but none have addressed nutrition. There are reviews addressing nutrition in housed problem-drinkers [bib_ref] Thiamine for prevention and treatment of Wernicke-Korsakoff syndrome in people who abuse..., Day [/bib_ref] [bib_ref] Alcohol consumption and body weight: a systematic review, Sayon-Orea [/bib_ref] , however, these will not necessarily be applicable to homeless problem-drinkers. Similarly, nutrition interventions that are in line with NICE guidelinesare considered cost effective for addressing malnutrition in the general population, but information on cost effective interventions for homeless drinkers is not available for decision makers.
This review aims to bring all these elements together and synthesise evidence on interventions for improving the nutritional status of homeless problem-drinkers [bib_ref] Interventions for preventing or treating malnutrition in problem drinkers who are homeless..., Thorley [/bib_ref].
# Methods
## Inclusion criteria
We included studies of any controlled or uncontrolled studies evaluating an intervention that aimed to improve the nutritional status, or macro-or micro-nutrient deficiencies in any problem-drinkers experiencing homelessness. We used the UK definition of homelessness in this review which includes: sleeping rough (outside); residing in temporary accommodation such as hostels, bed and breakfasts or night shelters; staying on a temporary basis with family or friends ('sofa surfers'); currently housed people who are at risk of being evicted; and currently housed people who cannot stay because they cannot afford to stay, the home is in a very poor condition or they are subject to violence, abuse or threats in the home. Problem-drinking is a commonly used term with no agreed definition. We therefore included all definitions of problem-drinking, as defined by included study authors [bib_ref] Interventions for preventing or treating malnutrition in problem drinkers who are homeless..., Thorley [/bib_ref].
We did not restrict inclusion of studies based on reported outcomes. The primary outcomes of interest for this review were: nutrition status or deficiencies; liver or bone marrow damage; and cognitive function. Secondary outcomes included: mortality; suicide; incidence of acute or chronic gastritis or pancreatitis; quality of life or wellbeing measures; and abstinence. We also collected process outcomes such as resource use, and engagement with or acceptability of interventions.
We excluded position papers; editorials; commentaries; qualitative studies; interventions solely aimed at improving the housing status of individuals or focused solely on reducing or stopping alcohol intake; institutionalised people; studies where entire communities are homeless (e.g. refugees or occupiers of slums or shanty towns). We also excluded studies on orphans or children in state care if not part of homeless families.
## Literature searches and study selection
A search of nine electronic databases and 13 grey literature sources was conducted. References of included studies were screened and authors were contacted to find any additional studies or data. The search was published in the protocol [bib_ref] Interventions for preventing or treating malnutrition in problem drinkers who are homeless..., Thorley [/bib_ref] and is up to date until 16th November 2016.
References identified in searches were screened in duplicate with disagreements resolved by discussion or a third reviewer. Identified relevant papers were read in full and assessed for inclusion in duplicate, with disagreements resolved by a third reviewer. Experts and homelessness charities were contacted to find unpublished studies and data.
## Data extraction
We extracted study details including aim of the study, country, participant characteristics, sample size; outcomes reported; and outcome data including treatment effect estimates, p-values, and confidence intervals. Data were extracted by one reviewer and checked by another. Discrepancies were resolved through discussion (with a third reviewer where necessary). Where data were unclear, we attempted to contact the authors for clarification.
## Risk of bias assessment
Risk of bias was assessed as part of data extraction. The Cochrane risk of bias toolwas used for the randomised controlled trials (RCTs) and the criteria listed by the Cochrane Effective Practice and Organisation of Care (EPOC) Group were used for other types of studies.
## Data synthesis
Meta-analysis was inappropriate because studies were heterogeneous with respect to their populations, interventions, and outcomes. Instead we carried out a narrative synthesis.
We analysed studies by intervention type: education/information and support; food provision; supplement or fortification; multicomponent interventions. The findings were summarized in tables for the main outcomes.
# Results
## Description of included studies
## Results of the search
Electronic searches resulted in 9189 citations. Twenty seven other references were identified in complimentary searches (contacting authors/ organisations, reference screening). In addition, we identified two further studies through contacts with subject expertsbut the authors did not provide sufficient information to assess eligibility to date. We included 25 studies reported in 37 articles / reports and excluded 257 papers (web appendix). The selection process and reasons for exclusion are shown in the PRISMA flowchart [fig_ref] Figure 1: PRISMA flow diagram of the review process [/fig_ref].
## Included studies
Of the 25 studies included, the majority (n = 12) were from the USA [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] [bib_ref] Providing WIC services to homeless families, Hamm [/bib_ref] [bib_ref] Life skill interventions with homeless youth, domestic violence victims and adults with..., Helfrich [/bib_ref] [bib_ref] Case management and access to services for homeless women, Heslin [/bib_ref] [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref] [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref] , and the rest were geographically diverse. [fig_ref] Table 1: Characteristics of included studiesKcal kilo calories, N number of participants analysed, NR... [/fig_ref] provides the characteristics of all included studies. Two studies [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] were RCTs, both from the USA; fifteen were uncontrolled before and after (UBA) studies ; seven were surveys [bib_ref] Case management and access to services for homeless women, Heslin [/bib_ref] [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref] [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] ; and one was a case-control study using a historical control sample [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref].
Studies varied widely in sample size from 5 participants [bib_ref] Challenges and opportunities for measuring the impact of a nutrition programme amongst..., Barbour [/bib_ref] to 128,365 [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] , with a median of 50. Most studies (n = 17) included less than 100 participants. The total number of participants included in this review is 131,054. Most studies (n = 9) did not report on type of homelessness. Six included shelter dwelling participants only while no study included just rough sleeping participants. Problem-drinking was not always defined. Only two studies provided a clear definition of problem-drinking as more than 80 g alcohol intake per session or per day [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] , while other studies used terms such as 'alcoholics' , 'alcohol dependency' , 'overt alcohol problem' , 'drinking at risky levels' , or 'seeking counselling for alcohol'. Proportion of problemdrinkers in the study population also varied (median 45%, IQR 26.5% -64%) across studies that reported this information.
Interventions assessed in included studies were grouped into four broad categories based on the different approaches to addressing malnutrition:
1. Education, information or support 2. Supplements (including vitamin injections or tablets, or fortified food products) 3. Food provision (including hot meals or food rations) 4. Multicomponent interventions, where studies combined more than one of the above approaches
Educational, information, and support interventions were the most common (n = 9) . These included motivational interviewing with nutrition information over the phone [bib_ref] Addressing smoking and other health risk behaviours using a novel telephone-delivered intervention..., Bonevski [/bib_ref] , a one-off lecture [bib_ref] Healthy hostels: a guide to promoting health and wellbeing among homeless people, Hinton [/bib_ref] , interactive group workshops [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Life skill interventions with homeless youth, domestic violence victims and adults with..., Helfrich [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] , and full curriculums on nutrition and diet [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref] [bib_ref] Challenges and opportunities for measuring the impact of a nutrition programme amongst..., Barbour [/bib_ref]. Three studies assessed the effect of supplements and fortification. These included oral or injectable multivitamin supplements [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] and a vitamin-fortified chocolate paste [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref]. Five studies tested food provision interventions [bib_ref] Food intake assessment of the 'bom prato' project users, Murakami [/bib_ref] [bib_ref] Impact of the social cafe meals program: a qualitative investigation, Allen [/bib_ref] [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref] ranging from prepared meals provided within organisations [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] and access to prepared meals at specified cafes [bib_ref] Food intake assessment of the 'bom prato' project users, Murakami [/bib_ref] [bib_ref] Impact of the social cafe meals program: a qualitative investigation, Allen [/bib_ref] to daily food packages given to clients at a tuberculosis clinic [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref]. Multicomponent interventions were assessed in eight studies . The combination of interventions included nutrition counselling on harm reduction (e.g. to eat before drinking) with naltrexone detoxing [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] , provision of education and kitchen facilities [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref] , provision of food along with kitchen facilities [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref] , education sessions with food and physical activity [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] , support sessions, recreational activities along with transport tickets, and free meals [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref] , Women, Infants and Children (WIC) programme [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] , and addition of group nutrition education, health checks, and food pack vouchers to the WIC programme [bib_ref] Providing WIC services to homeless families, Hamm [/bib_ref].
## Risk of bias in included studies
All studies were considered to be at a high risk of bias [fig_ref] Figure 2: Risk of bias in included studies [/fig_ref]. In the two RCTs the sequence generation and concealment of allocation were both rated 'unclear'. None of the studies reported attempts to adequately prevent knowledge of allocation to intervention (blinding) either for participants, providers or outcome assessors. Only two [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] out of the 25 studies adequately addressed incomplete outcome data in their analyses. The majority of the studies (n = 19) were also judged to be at high risk for the knowledge of allocated intervention to have affected data collection (based on Cochrane EPOC criteria).
## Findings
## Primary outcomes
Nutritional status Nutritional status measures were reported in 13 studies . Of these, three studies reported micronutrient deficiency in blood samples [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] and ten reported on nutritional intake .
Education, information or support interventions Educational sessions of varying intensity and duration were assessed in four studies [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref] [bib_ref] Challenges and opportunities for measuring the impact of a nutrition programme amongst..., Barbour [/bib_ref]. One involved motivational telephone interviewing [bib_ref] Addressing smoking and other health risk behaviours using a novel telephone-delivered intervention..., Bonevski [/bib_ref] , one was a single session on food hygiene and nutrition along with a cooking competition [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref] , and one involved life skill workshops [bib_ref] Life skill interventions with homeless youth, domestic violence victims and adults with..., Helfrich [/bib_ref]. One study assigned a case manager to provide information and support to optimise the uptake of the Supplemental Nutrition Program for WIC [bib_ref] Case management and access to services for homeless women, Heslin [/bib_ref]. Most of these were aimed at change in nutritional intake [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref] [bib_ref] Addressing smoking and other health risk behaviours using a novel telephone-delivered intervention..., Bonevski [/bib_ref] [bib_ref] Challenges and opportunities for measuring the impact of a nutrition programme amongst..., Barbour [/bib_ref]. The studies varied in design (one RCT, others uncontrolled before after studies) and outcome assessments (attempts to increase intake, frequency, or amount of intake). The effects were not consistent across studies, however, the majority indicate that education and support interventions could contribute to improved nutritional behaviour, i.e. eating healthier food [fig_ref] Table 2: Primary outcomes in included studies [/fig_ref].
## Supplement provision interventions
The oral [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] and injectablemultivitamin supplements were effective in lowering blood indictors of deficiency [fig_ref] Table 2: Primary outcomes in included studies [/fig_ref]. However, no longer term health or disease outcomes were measured.
Food provision interventions One study [bib_ref] Food intake assessment of the 'bom prato' project users, Murakami [/bib_ref] reported on a large-scale state supported intervention providing Primary outcomes of the review that were reported in the study. b This means that the study did not measure or report any of the primary outcomes of this review healthy meals at designated cafes and diners. The authors reported energy intake to be below recommended levels for clients eating there daily. One study providing food subsidy for meals at a local cafe [bib_ref] Impact of the social cafe meals program: a qualitative investigation, Allen [/bib_ref] reported that people ate more frequently, had weight gain and learnt food preparation skills and healthy eating habits, but quantitative data were not reported [fig_ref] Table 2: Primary outcomes in included studies [/fig_ref].
## Multicomponent interventions nutrition intake changes
were reported in three studies [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref]. All indicated a beneficial effect of the multicomponent interventions on healthier food intake [fig_ref] Table 2: Primary outcomes in included studies [/fig_ref].
Liver function Only one study [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] evaluating a multicomponent intervention (naltrexone detoxing and harm reduction counselling) reported liver function tests (mean aspartate transaminase and alanine transaminase levels) for the intervention group only, and found no difference between before and after measurements [fig_ref] Table 2: Primary outcomes in included studies [/fig_ref].
## Secondary outcomes
Change in drinking behaviour was reported in three studies [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref]. Several studies assessed some aspects of success of implementation of their intervention/ programme, such as the acceptability of the intervention [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] , or attendance and intervention/ programme completion [bib_ref] Providing WIC services to homeless families, Hamm [/bib_ref] [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] [bib_ref] Healthy hostels: a guide to promoting health and wellbeing among homeless people, Hinton [/bib_ref] [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref]. Cost of interventions was reported in four studies [bib_ref] Food intake assessment of the 'bom prato' project users, Murakami [/bib_ref] [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref]. However, no study reported cost effectiveness analyses.
Drinking behaviour Three studies, all assessing multicomponent interventions reported this outcome. Kadoura [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] reported a small non-significant decrease (Cohen's d = 0.15; p = 0.15) in mean alcohol consumption compared to baseline in the intervention group of homeless families. Stewart et al. [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref] reported 26% fewer participants drinking at the end compared to the midpoint assessment. Grazioli et al. [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] indicated the intervention (naltrexone + nutritional counselling) may lead to more drinking.
## Measures of implementation success
Eight studies reported some measure of implementation success (see Web appendix for details). Poor attendance (0-22%) was seen for an intervention using education sessions with a cooking competition at a shelter [bib_ref] Healthy hostels: a guide to promoting health and wellbeing among homeless people, Hinton [/bib_ref]. Higher treatment completion rates were seen in a tuberculosis clinic for homeless with a daily food pack provision [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref]. In taste testing carrot cake, beef burger, and apple crumble were liked the most in a study in the UK [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] , while a study in Spain [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] found that dairy, fruits and beans were favoured, however both reported a low preference for fish. There was high acceptability for the vitamin fortified chocolate spread packets [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref]. High attendance rates and perceived effectiveness were observed in a study that provided education, food provision and goal setting [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref]. In contrast, low acceptability and perceived effectiveness were seen in a study of naltrexone detoxing with counselling for better eating [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref]. Group nutrition education and health checks part of the WIC intervention were acceptable to homeless families. However, food voucher uptake and use was low [bib_ref] Providing WIC services to homeless families, Hamm [/bib_ref] and reasons for this low uptake of vouchers were: transiency, loss of the identification documents, loss of vouchers, lack of transport, and lack of time. One third of these families reported difficulty carrying a large amount of groceries, and 23% did not think the food met their needs fully.
Cost and resource use No studies reported cost effectiveness or provided enough data to assess cost effectiveness. Five studies reported cost and resource outcomes for the interventions tested [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref] [bib_ref] Food intake assessment of the 'bom prato' project users, Murakami [/bib_ref] [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref] [fig_ref] Table 3: Cost and resource use in included studiesUSD values [/fig_ref]. One other study [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref] recorded information on costs of meals provided but did not report this data in the paper. The table shows that although some of the cost information dates as far back as the 1980s and the comparisons are indirect, vitamin tablet supplementation could be cheaper than other interventions.
Other outcomes (detailed in web appendix) reported in included studies were life skills [bib_ref] Life skill interventions with homeless youth, domestic violence victims and adults with..., Helfrich [/bib_ref] [bib_ref] Challenges and opportunities for measuring the impact of a nutrition programme amongst..., Barbour [/bib_ref] , infant health and health visits by mothers [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref] [bib_ref] Addressing smoking and other health risk behaviours using a novel telephone-delivered intervention..., Bonevski [/bib_ref] , physical activity [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref] [bib_ref] Addressing smoking and other health risk behaviours using a novel telephone-delivered intervention..., Bonevski [/bib_ref] , access to shelter and food [bib_ref] Case management and access to services for homeless women, Heslin [/bib_ref] , and social outcomes such as loneliness and enjoyment [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref] [bib_ref] A support intervention to promote health and coping among homeless youths, Stewart [/bib_ref].
# Discussion
## Summary of findings
To our knowledge, this is the first review of the evidencebase on the effectiveness and costs of interventions for malnutrition in the homeless problem-drinking population. We included 25 studies assessing four broad categories of interventions. We found that in terms of nutritional status, educational interventions may increase fruit and vegetable intake, but this was not consistent across studies. A fortnightly multivitamin injection or daily multivitamin oral tablet could prevent vitamin deficiencies. A daily multivitamin fortified chocolate spread pack was acceptable but evidence of effectiveness was not reported. Free or subsidised meals or daily food packs also appeared acceptable but did not always fulfil adult energy needs. Three multicomponent intervention studies assessed nutritional status and all showed improved nutritional intake. In terms of implementation success or acceptability, provision of food or kitchen facilities were usually well received, but education in combination with detox or a cooking competition, or food vouchers that required money and time to be redeemed were not favoured. ASTaspartate transaminase, ALT alanine transaminase, B1 thiamine, B2 riboflavin, B3 niacin, B5 pantothenic acid,B6 pyridoxine, B7 biotin, B9 folic acid, B12 cobalamins, C ascorbic acid, g gram, gp group, kcal kilocalories, kJ kilojoules, L litre, MD mean difference, mg milligram, mmol millimoles, nmol nanomoles, μmol micromoles, N number of participants, NR not reported, NV no vitamin, pmol picomoles, P5P pyridoxal 5 phosphate, RCT randomised controlled trial, SD standard deviation, Tk transketolase, TDP thiamine diphosphate, TPP thiamine pyrophosphate, UBA uncontrolled before and after study, V vitamin
All studies were at high risk of bias. Many studies did not have control groups. Particular problems with interpreting data from uncontrolled studies are susceptibility to confounding (including seasonality) and regression to the mean. There were two RCTs and although these were at high risk of bias too, they are more reliable than the other studies because of randomization. Both reported higher intake of fruits and vegetables in the intervention group, one providing a 3 h workshop on resource management and diet [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] and the other providing newsletters, fruits, vegetables, and pedometers along with walking goals [bib_ref] Evaluation of a shelter-based diet and physical activity intervention for homeless adults, Kendzor [/bib_ref]. These interventions could be implemented in shelter settings similar to those in the respective studies.
It was not possible to estimate cost effectiveness as none of the studies reported cost-effectiveness analysis. Limited data on intervention costs from four studies on food or supplement provision interventions indicate the costs of daily oral vitamin supplements to be lower than that of meals. However, this information without the benefits associated with each is of limited use when choosing between competing interventions, especially when these were not directly or concurrently compared.
There were no assessments of cognitive function in the included studies. Although this may be difficult to measure accurately, it is an important outcome which can affect people's ability to optimally use healthcare and housing services, which may require learning and remembering new things [bib_ref] Cognitive impairments and the prevention of homelessness: research and practice review, Backer [/bib_ref]. Long term outcomes in health are also missing from the literature. These outcomes can be useful for decision makers in establishing whether the interventions provided the intended benefit. Nutritional outcomes reported across studies were illdefined and variably measured. There are known issues in this area of research associated with use of convenient measures, transiency of the population, and the extreme variation in food intake dependent on donations or the opening days/ h of soup-kitchens or similar facilities [bib_ref] Food, health and eating among single homeless and marginalized people in London, Evans [/bib_ref] [bib_ref] Nutrition and the homeless: the underestimated challenge, Seale [/bib_ref]. Future studies in this population should use more rigorous measures of nutritional change [bib_ref] Nutrition and the homeless: the underestimated challenge, Seale [/bib_ref].
Nine studies reported use of incentives (cash or gifts) to increase data collection and uptake of intervention. This, along with high dropouts, suggests that effectiveness may not be entirely attributable to the intervention.
# Strengths and limitations
Given that we expected limited evidence on the question, not using language restrictions and using an extensive grey literature search was a key strength of our review. This strategy was likely the reason we were able to identify a relatively large number of studies. We supplemented this exhaustive search with rigorous methods of inclusion and appraisal of the research identified. This makes our findings reliable. Another strength of this review lies in its inclusivity. This allowed for the unrestricted inclusion of and, consequently, the exploration of the range of interventions evaluated. The results of this review can therefore inform the implementation of these interventions to improve the health of homeless problem-drinkers in similar settings. No two studies of similar design assessed the same intervention and outcome and thus an expected limitation of this review was that results of the studies could not be combined in a meta-analysis. Thus the interpretation requires caution, especially considering the limitations in study quality. We therefore analysed interventions in broad categories and avoided subgrouping of results further.
## Applicability of evidence
There are still lessons to be learnt from this limited evidence base and implications for policy and research, although caution is required when implementing this evidence. Most interventions were set in shelters and inclusion often restricted to shelter dwellers. No study assessed an intervention for rough sleepers alone. This group often gets excluded from such research studies due to their transient location, yet they may have more complex needs [bib_ref] Food, health and eating among single homeless and marginalized people in London, Evans [/bib_ref] , so their inclusion in future research would be valuable. Although some shelters do, not all will accept rough sleepers who are heavy alcohol or drug users [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref] [bib_ref] Social and structural barriers to housing among street-involved youth who use illicit..., Krüsi [/bib_ref]. Rough sleepers with drinking or drug problems also sometimes avoid engaging with shelters [64]. Healthcare access points, such as general practices offering primary health care services to street drinkers may be a more inclusive setting to reach this subgroup [bib_ref] Food, health and eating among single homeless and marginalized people in London, Evans [/bib_ref]. Even when these are free at point of delivery, it could mean uneven reach where the most vulnerable homeless drinkers may be unaware of or unable to reach these services.
Although, education and training can raise awareness in and provide information to homeless people to make healthier food choices, it is harder for homeless people to make these healthy choices when they have no kitchen, cupboard, or fridge, and move residence frequently [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref]. In addition, vitamin B1 deficiency with heavy drinking over time leads to cognitive impairment [bib_ref] Thiamine deficiency: an update of Pathophysiologic mechanisms and future therapeutic considerations, Abdou [/bib_ref]. Once in this state, any counselling or health promotion efforts to bring about behaviour change would be less likely to work [bib_ref] Cognitive impairments in alcoholdependent subjects, Bernardin [/bib_ref]. Thiamine intake can often reverse this impairment [bib_ref] Thiamine for prevention and treatment of Wernicke-Korsakoff syndrome in people who abuse..., Day [/bib_ref]. Alcohol and/or drug support services often pursue harm reduction interventions (e.g. needle exchange) alongside interventions to reduce substance abuse. These services could also consider providing nutritional interventions for preventing or treating malnutrition in the homeless, heavy drinking population.
Our findings suggest that consideration for local taste preferences is important in meal provision services. However, only one study [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref] reported developing the content of the food rations according to local food tradition. Meal provision was an acceptable intervention although evidence suggested that it may not always fulfil energy needs. Meal services require a full kitchen and catering staff to serve meals every day. In addition, there is evidence that nutritional value of these meals may be constrained by resources and prioritising a satisfying meal over nutritional value by both providers and users [bib_ref] Improving the nutritional quality of charitable meals for homeless and vulnerable adults...., Pelham-Burn [/bib_ref]. No included study compared different types of food and/or supplement provision interventions and this should be assessed in future for comparative benefit and resources use.
Provision of kitchen facilities in two studies appeared to be effective in improving nutritional intake. It led to a reduction in vitamin deficiencies in one study in homeless families [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref]. The other study [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref] that made kitchens available to street-living homeless as well as the shelter participants reported that the participants enjoyed cooking, and the use of the kitchen facilities increased rapidly. This indicates that the lack of these facilities might be a key factor in malnutrition among homeless drinkers. It also suggests that experiential learning may be more effective than giving nutritional advice in this client group [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref].
It seems that an educational intervention with a cooking competition proposed by the shelter staff alone had low uptake [bib_ref] Healthy hostels: a guide to promoting health and wellbeing among homeless people, Hinton [/bib_ref] , but an intervention developed with service user input, involving education, kitchen facility and communal dining was popular [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref]. Involving the population in research can increase participation rates and user controlled research is encouraged in public health and social care research. Considering the difficulty in accessing homeless drinkers, their involvement in a project aimed at their health can improve uptake and measuring of the impact and continuity of service later. This would also give choice to a marginalised population.
Nine studies included only homeless women with children or homeless families [bib_ref] Lessons learned from the "spend less. Eat well. Feel better." program efficacy..., Derrickson [/bib_ref] [bib_ref] Providing WIC services to homeless families, Hamm [/bib_ref] [bib_ref] Case management and access to services for homeless women, Heslin [/bib_ref] [bib_ref] Nutrition education for homeless women -challenges and opportunities: a pilot study, Johnson [/bib_ref] [bib_ref] The brighter future for homeless families and their preschoolers' program in salt..., Kadoura [/bib_ref] [bib_ref] Maternal health behaviors and infant health outcomes among homeless mothers: U.S. special..., Richards [/bib_ref] [bib_ref] Striving for empowerment through nutrition education, Rusness [/bib_ref] [bib_ref] Nutrition knowledge and associated behavior changes in a holistic, short-term nutrition education..., Rustad [/bib_ref] [bib_ref] Nutritional and economic advantages for homeless families in shelters providing kitchen facilities..., Wiecha [/bib_ref]. These studies may therefore be less representative of the typical demographics of the homeless problemdrinking population in urban settings in Europe [bib_ref] Extent and profile of homelessness in European member states: a statistical update, Busch-Geertsema [/bib_ref] and North America [bib_ref] The 2016 annual homeless assessment report (AHAR) to congress. The U.S. Department..., Henry [/bib_ref] which is largely single male. Male homeless population was included in two studies exclusively [bib_ref] Vitamin status of homeless men, Darnton-Hill [/bib_ref] [bib_ref] A fortified street food to prevent nutritional deficiencies in homeless men in..., Darmon [/bib_ref] and five others had more than 80% men [bib_ref] Safer-drinking strategies used by chronically homeless individuals with alcohol dependence, Grazioli [/bib_ref] [bib_ref] Food acquisition practices of homeless adults: insights from a health promotion project, Tarasuk [/bib_ref] [bib_ref] Sensory and nutritional evaluation of a municipal service of community kitchen for..., Villena [/bib_ref] [bib_ref] Food incentives improve adherence to tuberculosis drug treatment among homeless patients in..., Garden [/bib_ref]. Gender differences have been seen in the homeless regarding use of health services [bib_ref] Gender differences among homeless persons: special services for women, Diblasio [/bib_ref]. Learning the composition and preferences of the target population before setting up an intervention and tailoring the intervention to the local demographics is therefore warranted. This will ensure optimal uptake and consequently the likelihood of intervention success.
The services for homeless problem drinking people are likely varied across the globe. In the USA, the United States Interagency Council on Homelessness (USICH) is responsible for reducing homelessness. It is an independent agency of the federal government designed to coordinate a response in partnership with state and local governments, and community groups. In the UK services to the homeless are provided by local councils under the guidance provided by the Department for Communities and Local Governmentsand by charities such as Crisis, Pathway, Salvation Army, and Shelter. This means that services and their structure vary across regions. In the UK, these services are also over stretched in the face of current political and economic situation and the housing crisis since 2008, which means fewer resources are available to address malnutrition for problem drinking subgroups of homeless people.
With limited available funds, the variations in services across settings and locations can serve as an alternative to a traditional comparative study. This can identify the most beneficial interventions for a specific outcome and/or setting. Thus in future, in addition to randomized designs, natural experiments such as those designed around a change in local or regional policy, a practice within a certain hospital or a charity organisation, can provide evidence that is more applicable in terms of effectiveness. These findings should then be reported in an accessible location and format to reduce publication bias.
# Conclusions
With the high risk, single study data on any of the studied interventions we cannot conclude which intervention may be most effective or cost effective for tackling malnutrition in homeless problem-drinkers. Nevertheless, several interventions appeared able to change nutrition related behaviour in a given setting and were acceptable. Decision makers need to carefully consider which of these interventions would translate well into their own setting and population for achieving a desired outcome. Including the target population in developing intervention content and delivery may optimise intervention success.
Better quality data and long terms outcomes such as malnutrition levels, health and disease status are also needed. Comparative cost and resource use should be part of any future intervention evaluation.
[fig] Figure 1: PRISMA flow diagram of the review process [/fig]
[fig] Figure 2: Risk of bias in included studies [/fig]
[table] Table 1: Characteristics of included studiesKcal kilo calories, N number of participants analysed, NR not reported, PRAMS Pregnancy Risk Assessment Monitoring System project for CDC, USA, RCT randomised controlled trial, UBA uncontrolled before after study, WIC The Special Supplemental Nutrition Program for Women, Infants, and Children in the USA. a [/table]
[table] Table 2: Primary outcomes in included studies [/table]
[table] Table 3: Cost and resource use in included studiesUSD values (In brackets) when the reported cost values were in other currencies calculated using historical exchange rates for the respective publication year's January [/table]
|
Background: The diagnosis of arterial hypertension based on measurements of blood pressure in the office has low accuracy.Objective: To evaluate the prevalence of masked hypertension (MH) and white-coat hypertension through home blood pressure monitoring (HBPM) in pre-hypertensive and stage 1 hypertensive patients.Method: Retrospective study, of which sample consisted of individuals with BP ≥ 120/80 mmHg and < 160/100 mmHg at the medical office without the use of antihypertensive medication and who underwent exams on the HBPM platform by telemedicine (TeleMRPA) between May 2017 and September 2018. The four-day MRPA protocol was used, with 24 measurements, using automated, validated, calibrated equipment with a memory function.Results:The sample consisted of 1,273 participants, of which 739 (58.1%) were women. The mean age was 52.4 ± 14.9 years, mean body mass index (BMI) 28.4 ± 5.1 kg/m 2 . The casual BP was higher than the HBPM in 7.6 mmHg for systolic blood pressure (SBP) and 5.2 mmHg for diastolic blood pressure (DBP), both with statistical significance (p < 0.001). There were 558 (43.8%) normotensive individuals; 291 (22.9%) with sustained hypertension; 145 (11.4%) with MH and 279 (21.9%) with white-coat hypertension (WCH), with a diagnostic error by casual BP in the total sample in 424 (33.3%) patients. In stage 1 hypertensive individuals, the prevalence of WCH was 48.9%; in prehypertensive patients, the prevalence of MH was 20.6%.Conclusion: MH and WCH have a high prevalence rate in the adult population; however, in prehypertensive or stage 1 hypertensive patients, the prevalence is higher. Out-of-office BP measurements in these subgroups should be performed whenever possible to prevent misdiagnosis. (Arq Bras Cardiol. 2019; 113(5):970-975)
# Introduction
The prevalence of arterial hypertension (AH) in the Brazilian adult population is high and varies according to the studied population and the method of assessment (31% to 35.8%).For this reason, the accurate diagnosis of the several scenarios related to blood pressure (BP) behavior is crucial for adequate stratification of cardiovascular risk, as well as for the definition of the best treatment strategies.In this context, considerations should be given to the possibility of normotension, masked hypertension (MH), white-coat hypertension (WCH), and sustained AH.MH is defined by the presence of normal BP in the office but high measurements outside of it; the WCH is defined by high values of BP in the office and normal values in the home measurements. It is noteworthy that MH, WCH and AH are highly prevalent and are related to an increase in cardiovascular morbidity and mortality.Therefore, they should be investigated and diagnosed, and for this purpose, it is essential to use methods capable of monitoring BP outside the doctor's office environment.Ambulatory (ABPM) or home (HBPM) BP monitoring can be used for home BP measurement, with the latter offering the possibility of providing the necessary information for the appropriate diagnosis with greater comfort and better cost-benefit ratio.Also, in relation to HBPM, the inclusion of the habitual BP measurements into the patient's routine has shown an increase in compliance with drug treatment. This benefit seems to be even greater when using telemedicine platforms.An adequate identification of MH and WCH is so important that the main guidelines of AH recommend the use of ABPM or HBPM in the diagnostic investigation whenever possible, emphasizing their use at the initial BP alterations.The present study is the first national study to evaluate the prevalence of MH and white coat hypertension through the HBPM in prehypertensive and stage 1 hypertensive patients.
## Original article
# Method
This study was submitted and approved by the Human Research Ethics Committee of Hospital das Clínicas da Universidade Federal de Goiás under CAEE number 99691018.7.0000.5078. This is a retrospective study that evaluated the data of all patients who underwent exams on the TeleMRPA platform (www. telemrpa.com) from May 2017 to September 2018. Of the total, those who underwent the examination for diagnostic purposes and were not using antihypertensive drugs were selected.
Inclusion criteria were age older than 18 years; individuals assessed on the TeleMRPA platform, without the use of antihypertensive drugs and who had, by the casual measure (mean of two measurements) performed at the clinic on the first day of the protocol, SBP and DBP that met the criteria for the diagnosis of prehypertension (PH) -SBP ≥ 120 mmHg and/or DBP ≥ 80 mmHg and SBP < 140 mmHg and DBP < 90 mmHg; or stage 1 AH -SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg and SBP < 160 mmHg and DBP < 100 mmHg, according to the Brazilian Guideline of Arterial Hypertension (DBHA, Diretriz Brasileira de Hipertensão Arterial).The TeleMRPA platform was developed as a telemedicine monitoring tool, with characteristics that allow the analysis and filtering of the database according to the scientific questions to be investigated. There was a concern to develop and improve the mathematical algorithm aiming to allow the high quality of the analyzable data, either for the interpretation of the exam or for the development of research projects. In this context, the database protects the identification data of the patient and clinics or health units. Prior to the inclusion of data into the platform, the co-investigators were trained regarding the scientific evidence and methodology of the HBPM, as well as for the use of Omron automatic devices for BP measurements.
The protocol used to obtain home measurements follows the recommendation of the Brazilian guidelines for HBPM,which advises two measures to be carried out on the first day at the office or clinic (these measures are not used for the analysis of the mean home measurements) and six measurements a day on four consecutive days (three in the morning and three in the evening), with a total of 24 measures to calculate the mean. This mean value is considered normal when lower than 135/85 mmHg.It is recommended, on the first day, when the patient receives the device at the health unit, that the patient be taught the correct handling of the BP measuring device, as well as the technique for adequate and reliable measurement. This recommendation follows the DBHA guidelines.Subsequently, the patient (or caregiver/companion) is instructed to measure the BP twice a day, following the abovementioned protocol.
Patient data, as well as BP values, were included into the TeleMRPA platform and analyzed for the following variables:
Socio-demographic data: gender, age, sample distribution and BP behavior by geographic regions;
Anthropometric data: BMI using the Quetelet formula (BMI = weight in kg/height in meters 2 ).
Blood pressure: mean BP at the clinic (first day), mean of home BP measures (second to fifth days) and mean BP in morning and evening, mean pulse pressure and BP variability.
## Database and statistical analysis
The database was created using the Excel® (Microsoft) software with data imported from the TeleMRPA platform; the numerical codes were typed by three researchers with subsequent cross-checking to identify and correct typing errors.
Continuous variables were presented as mean and standard deviation and categorical variables as absolute and relative frequencies. The Kolmogorov-Smirnov test was used to verify the distribution of the continuous variables. The paired t-test was used to compare BP measurements between the casual measurements and HBPM. To compare the frequencies of masked AH between stage 1 and stage 2 prehypertensive patients, the chi-square test was used, which was also used to compare the diagnoses of prehypertension and stage 1 hypertension between the casual BP measure and the HBPM. The significance level was set at p < 0.05. The Stata® software, version 14.0 was used for the analysis.
# Results
The initial sample consisted of 4,350 individuals who underwent HBPM from May 2017 to September 2018 in nine Brazilian states. Of these, 1,273 participants with a clinical diagnosis of prehypertension or stage 1 AH and without use of medicationswere selected, of which 853 (67.0%) were from the Northeast region, 43 (3.4%) from the North region, 10 (0.8%) from the Midwest region, 307 (24.1%) from the Southeast region and 60 (4.7%) from the South region. Mean age was 52.4 ± 14.9 years and the mean BMI was 28.4 ± 5.1 kg/m 2 . As for gender, 739 (58.1%) were women.
The mean values of casual BP were 133.2 ± 11 mmHg and 84.1 ± 8 mmHg, and for HBPM, the mean values were 125.5 ± 11.7 mmHg and 78.9 ± 8 mmHg for SBP and DBP, respectively. The mean number of valid measures was 22.96.
When comparing the means of the casual BP with the HBPM, higher values were found for casual BP in 7.6 mmHg for SBP and 5.2 mmHg for DBP, both with statistical significance (p < 0.001)When analyzing only the prehypertensive group, there were 145 individuals (20.6%) who actually had masked hypertension, and if we separate those individuals with SBP ≥ 130 mmHg and < 140 mmHg , and/or DBP ≥ 85 mmHg and < 90 mmHg (n = 364), the prevalence of MH increases to 27.8%.
In the stage 1 hypertensive group, 279 individuals (48.9%) with WCH were identified.
# Discussion
The present study confirmed that the BP measurements obtained through HBPM is very useful in the diagnosis of AH phenotypes and allowed the reclassification of 33.3% of the assessed individuals, thus adding important information to the AH is a highly prevalent disease in the adult population; in a worldwide survey of 1,128,635 individuals, the prevalence was 34.9%, most of them with stage 1 hypertension. It is also known that the prevalence of PH is at the same level, reinforcing the need for a correct diagnosis, so that the most appropriate conduct can be implemented.It is also well established that BP monitoring methods outside the office, when compared to the casual measure, have a higher diagnostic accuracy and show a better prediction of cardiovascular risk.In the studied sample, when we compared the means of casual BP with HBPM, we found significantly lower means in HBPM, with statistically significant differences (p < 0.001) for both SBP and DBP.
## Original article
Based on this scientific evidence, to avoid diagnostic error, the most recent AH guidelines have strongly recommended the use of ABPM or HBPM for diagnostic evaluation, especially in individuals with initial BP alterations.When only those individuals with diagnostic criteria for PH or stage 1 AH were assessed, in which the chance of casual measures that induce misdiagnosis is higher, we found a prevalence of true normotension and hypertension of only 66.7%, that is, in 33,3% of the cases, the diagnosis would have been wrong, if we considered only the casual measure. These data coincide with other publications that found similar error rates.The risk of WCH in stage 1 hypertensive patients is even more important, or MH in prehypertensive patients because, contrary to what was believed, both WCH and MH are associated with higher cardiovascular mortality and, in the specific case of MH, this mortality is even higher than that of patients with sustained hypertension.It is worth mentioning that this prevalence is high in adults, but it is also significant in adolescents.In the sample of the present study, among the prehypertensive patients (n = 703), we found a prevalence of MH of 20.6%; this value is even higher when we analyze the highest BP strata in this group. In PH patients with SBP ≥130 mmHg and/or DBP ≥ 85 mmHg, this prevalence is 27.8%, significantly higher (p < 0.001) than PH patients with BP levels lower than 130/85 mmHg. Our findings are in agreement with the trend seen at the last guidelines and statements, 1,2,5,18-21 which dedicate special attention to prehypertensive individuals, since some of these individuals will show a significant increase in cardiovascular outcomes. It is also justifiable recommending the American and European guidelines regarding the performance of ABPM or HBPM whenever possible when casual BP is ≥ 130/85 mmHg.Finally, when we evaluated the individuals diagnosed as stage 1 hypertension (n = 570), we found a prevalence of WCH of 48.9%, which could result in misdiagnosis and use of inadequate therapeutic strategies in almost half of these individuals.
## Original article
Most of this sample consisted of individuals from the Northeast region of the country (67.7%); this may be considered a limitation; however, as the analysis refers to AH phenotypes, it seems to us that the geographic region distribution would have little or no effect on the results.
The Telemedicine MRPA (TeleMRPA) platform is an extremely useful, low-cost, easy-to-perform and wide-ranging modality, allowing a systematic evaluation of home-based BP measures, in accordance with the DBHA recommendations. In addition to the individual evaluation in clinical practice, the accumulation of information on this platform represents an important database for analyses on the diagnosis and treatment of AH in several regions of Brazil, both in public and private institutions.
# Conclusion
The casual BP measurement is a method used for AH screening, but it is necessary to take into account that home measurements are more accurate for the diagnosis.
We found high prevalence rates of MH and white-coat hypertension in individuals diagnosed with prehypertension or stage-1 hypertension. |
Anti-Yo positive and late-onset paraneoplastic cerebellar degeneration associated with ovarian carcinoma
Rationale: Paraneoplastic cerebellar degeneration (PCD) is a rare nonmetastatic neurological complication often associated with ovarian, breast, and other gynecologic cancers. Anti-Yo is one of the antionconeural antibodies found in patients with PCD. It primarily emerges before a malignancy is detected.Patient concerns: In this report, we describe an unusual case involving a patient who exhibited anti-Yo-positive PCD 1 year after being diagnosed with ovarian cancer.Diagnoses: Histopathology of the resected tissues and Antineuronal antibody testing.Interventions: The patient was treated with intravenous immunoglobulin (IVIG, 1 g/d) for 1 week and a large-dose of methylprednisolone (0.4 g/kg/d) for 5 days. At the same time, underlying complications were prevented actively, and the peripheral nerves were protected.Outcomes: Although most patients with anti-Yo-positive PCD do not improve after treatment, our patient significantly improved after receiving active and effective treatment.Abbreviations: CSF = cerebrospinal fluid, CT = computed tomography, MRI = magnetic resonance imaging, PCD = paraneoplastic cerebellar degeneration, PNS = paraneoplastic neurological syndromes.
# Introduction
Paraneoplastic neurological syndromes (PNS) are a rare group of syndromes that occur in patients with cancer and are not caused by the presence of metastases or the direct infiltration of tumors into the nervous system. Malignant tumors associated with PNS are mainly found in ovarian cancer, breast cancer, [bib_ref] Immunological markers in neurological disorders, Karim [/bib_ref] small cell lung cancer, [bib_ref] Small cell lung cancer presenting with paraneoplastic limbic encephalitis, Bowyer [/bib_ref] and so on. Paraneoplastic cerebellar degeneration (PCD) is a rare and unusual nonmetastatic neurologic complication, which is a remote effect of cancer. [bib_ref] Ovarian cancer complicated by cerebellar degeneration: a paraneoplastic syndrome, Hall [/bib_ref] According to previous findings, it is well known that a malignant disease can lead to antibody formation, causing secondary clinical effects. PCD with ovarian cancer is 1 example. Clinically, it is characterized by acute or subacute onset with progressive pancerebellar dysfunction, including asymmetry of truncal and limbs, gait ataxia, dysarthria, and nystagmus (mostly vertical). [bib_ref] Paraneoplastic cerebellar degeneration: clinical-immunological correlations, Anderson [/bib_ref] Several specific antionconeural antibodies have been found in serum and cerebrospinal fluid (CSF) in some patients with PCD, depending on the underlying tumor. [bib_ref] Paraneoplastic disorders: antineuronal antibodies and therapeutic options, Dropcho [/bib_ref] Anti-Yoassociated PCD occurs almost exclusively in middle-aged women with ovarian cancer. [bib_ref] Paraneoplastic cerebellar degeneration. I. A clinical analysis of 55 anti-Yo antibodypositive patients, Peterson [/bib_ref] Several reports have shown that response to treatment in PCD is not satisfactory, especially in anti-Yo-positive PCD patients. In this report, we present an anti-Yo (+) ovarian cancer patient with subacute-onset PCD, whose symptoms significantly improved after treatment.
## Case report
This study was conducted in accordance with the declaration of Helsinki. This study was conducted with approval from the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College. Written informed consent was obtained from all participants.
A previously healthy 65-year-old married female farmer, mother of 5 children, was admitted to a local hospital for progressive abdominal distention for 3 months since September 2012. She had been menopausal for more than 10 years. As an ovarian tumor marker, her CA-125 level was 270 U/mL (normal range: 0-35 U/mL), while other serum tumor markers (CA19-9, CA15-3, CEA, and leukemia cell marker) were within normal limits. Pelvic color Doppler ultrasound examination revealed a cystic solid and irregularly fixed mass in the left ovary. The patient underwent exploratory laparotomy, which revealed a 10 Â 15 cm left ovarian tumor with an irregular surface. She underwent total abdominal hysterectomy, bilateral salpingo-oophorectomy, lymphadenectomy, and omentectomy. Final histopathology of the resected tissues revealed stage III serous ovarian carcinoma [fig_ref] Figure 1: Histopathology of the resected tissues reveals stage III serous ovarian carcinoma [/fig_ref]. The papillary structure disappeared, and the tumor cells showed a large mass, the parenchyma adenocarcinoma. The cell atypia was large, the mitotic figures were few, and the interstitial was very few. She was treated with paclitaxel and cisplatin for 7 cycles and exhibited a complete response to chemotherapy. She remained disease-free and had no recurrence.
In September 2013, the patient presented with imbalance, episodic vertigo, nausea, and vomiting without any apparent reason. Her brain computed tomography (CT), which was performed in a local hospital, was normal. Thus, the patient and her family ignored her symptoms. Nevertheless, these symptoms were progressively exacerbating. Slurred speech and vertical nystagmus gradually appeared. In February 2014, she was admitted to our department with severe ataxia of the trunk, limbs and gait, along with slurred speech, dysmetria, and pathological nystagmus, which had developed over the preceding month. Neurological examination: The patient suffered from ataxia, imbalance and gait disturbances, speech disorder (dysarthria), and vertical nystagmus. There were Romberg sign (+), finger-nose test (+), and heel-knee test (+). There was no obvious abnormality in sensory system. She had normal defecation and urination, but poor diet and sleep.
Mild neutrophilic granulocytosis was found (72.9%, normal: 50%-70%) in the blood analysis. Biochemical and urine analysis revealed no abnormalities. Furthermore, emission-CT, electroencephalogram, CT of the brain, and magnetic resonance imaging (MRI) of the brain with gadolinium enhancement examinations were unremarkable. The patient only received MRI of the brain, which could exclude cancerous metastasis of central nervous system. According to the follow-up of the patient, the possibility of cancer metastasis could be ruled out. Based on these results, stroke, infection, toxic cerebellar lesions, cerebellar tumors, and hereditary cerebellar degeneration were excluded from the possible diagnoses. Her CA-125 level was 20 U/mL. CT scans of the abdomen and pelvis were normal. There were no definitive evidences for the recurrence of ovarian cancer. CSF examination did not reveal any abnormality. Antineuronal antibody testing (anti-Yo, anti-Ri, anti-Hu, anti-CV2, antiamphiphysin, and anti-Ma2/TA) was positive only for anti-Yo [fig_ref] Figure 2: Antineuronal antibody testing [/fig_ref]. These findings were consistent with the diagnosis of PCD.
The patient was treated with intravenous immunoglobulin (IVIG, 1 g/d) for 1 week and a large-dose of methylprednisolone (0.4 g/kg/d) for 5 days. At the same time, underlying complications were prevented actively, and the peripheral nerves were protected. Audiovestibular testing clearly indicated improvement after treatment. She was independently mobile in bed, and could sit and stand with supervision. Moreover, her speech was more intelligible. Besides, seeing to fast forward and side face, the patient had vertical nystagmus when she was admitted to our hospital, while the nystagmus disappeared after treatment. Audiovestibular testing included Romberg sign. The patient was not stable, no matter whether eyes open or closed, but she could stand steady after treatment. Finger-nose test and heel-knee test were negative after treatment. This empirical treatment with IVIG and methylprednisolone may be effective and worthy of being recommended. To date, she has been alive for 19 months without any evidence of recurrence.
# Discussion
At present, the pathogenesis of PCD is not completely understood. However, it has been considered to be associated with antibody and T-cell responses against the expression of shared epitopes in the nervous system and the tumors. [bib_ref] Innate and adaptive autoimmunity directed to the central nervous system, Bhat [/bib_ref] In recent years, 6 specific antionconeural antibodies have been found in serum and CSF of some patients with PNS such as anti-Hu [antineuronal nuclear antidodies (ANNA)-1], anti-Yo (purkinje cell antibody type 1), anti-Ri (ANNA-2), anti-CV2, antiamphiphysin, and anti-Ma2/TA. [bib_ref] Immunological markers in neurological disorders, Karim [/bib_ref] [bib_ref] An unusual presentation of anti-Hu-associated paraneoplastic limbic encephalitis, Langer [/bib_ref] Furthermore, it has been believed that anti-Yo-associated PCD occurs almost exclusively in women and is most likely associated with ovarian, breast, and other gynecologic cancers, although exceptions have been seen. There were many previous studies correlating PCD and cancer. [bib_ref] Paraneoplastic neurological syndromes associated with ovarian tumors, Zaborowski [/bib_ref] [bib_ref] Paraneoplastic cerebellar degeneration with anti-Yo antibodies associated with metastatic uveal melanoma, Valpione [/bib_ref] [bib_ref] Paraneoplastic cerebellar degeneration caused by ovarian clear-cell carcinoma, Negishi [/bib_ref] The fact that this patient responded to the treatment for ovarian cancer would strengthen the association between gynecological malignant tumors and paraneoplastic syndrome. PCD occurs at any stage of the course of cancer. This case has proven that if acute or subacute cerebellar disease occurs in middleaged woman with ovarian cancer and progresses without any signs of intracranial hypertension, we should consider the PCD; but only when cerebellar stroke, infection, toxic cerebellar lesion, cerebellar tumors, or hereditary cerebellar degeneration have been excluded based on careful physical examination and imaging findings. Early definite diagnosis is conducive to timely treatment.
A subset of patients with anti-Yo PCD shows improvement. The natural tendency of the disease to plateau around 6 months after onset makes it difficult to determine whether treatments were effective. Isolated cases of PCD related with anti-Yo only respond favorably to the immunomodulatory therapy. This is one of the interesting aspects that can offer this case. In general, PCD predates the cancer diagnosis. However, in approximately 30% of patients, the ataxic symptoms occur when the cancer is in remission.
However, the specific pathological mechanism of PCD was still not fully understood. Some research showed that dysregulation of calcium homeostasis by anti-Yo antibodies may be the initial mechanism of attack on purkinje's cells. The authors suggest a pathway where PCs are first silenced by interruption of calcium signaling by internalized anti-Yo antibodies, and then cleared by cytotoxic T cells and microglia. PKCc (the catalytic subunit of PKC), a calcium-dependent kinase, Cav2.1, a voltage-gated calcium channel, and the calcium-dependent protease calpain-2 were upregulated, which would increase intracellular calcium levels, potentially triggering cell death pathways, which need to be further explored. [bib_ref] Paraneoplastic cerebellar degeneration with anti-Yo antibodies-a review, Venkatraman [/bib_ref] For such patients, the removal of the primary tumor is the mainstay of treatment, along with plasma exchange, IVIG, as well as immunosuppressive agents (cyclophosphamide), antitumor drugs (rituximab), or corticosteroids, which can be administered after surgery. Based on the results of this case, a combination of IVIG and methylprednisolone can be recommended.
It has been known that approximately 50% of the damage to a patient's nervous system manifests before the cancer is diagnosed. Before the primary lesion is found, the detection of related antibodies could provide basis for early diagnosis and has significance for diagnosing the tumor type. However, no antibodies have been identified in approximately 40% of patients. [bib_ref] Case records of the Massachusetts General Hospital. Case 4-2007. A 56-year-old woman..., Dalmau [/bib_ref] Further studies are required to characterize mechanisms leading to neuronal death in PNS.
Therefore, it can be seen that on one hand, it is vital to investigate the mechanism of PCD in patients with ovarian cancer in the pathogenesis of immunology. On the other hand, female patients who present with symptoms of PCD should be thoroughly screened for gynecological malignancy. Therefore, female patients who present with symptoms of ovarian cancer should be provided timely and effective treatment to prevent PCD. Early awareness of PCD and timely treatment is important for patients to have a good prognosis.
The potential causal relationship between ovarian cancer and PCD needs to be investigated. Therefore, in addition to the need of neurologists for continuous learning, surgeons working in this field also need to study and gain an interdisciplinary understand-ing of these so-called marginal diseases. Early treatment with maximal preservation of central nervous system function and improvements in antitumor immunity are the main treatment goals for the future. Further studies that would eventually enable physicians to prevent or cure this disease are therefore desirable.
# Conclusion
PCD with anti-Yo (+) could exhibit after ovarian cancer, whose symptoms could be significantly improved after treatment by IVIG and a large-dose methylprednisolone. This would contribute to the early detection and treatment of PCD for ovarian cancer patients, which is important for patients to have a good prognosis.
[fig] Figure 1: Histopathology of the resected tissues reveals stage III serous ovarian carcinoma. The papillary structure disappeared, and the tumor cells showed a large mass, the parenchyma adenocarcinoma. The cell atypia was large, the mitotic figures were few, and the interstitial was very few. [/fig]
[fig] Figure 2: Antineuronal antibody testing (anti-Yo, anti-Ri, anti-Hu, anti-CV2, antiamphiphysin, and anti-Ma2/TA). The testing was positive only for anti- [/fig]
|
Phenotype Standardization for Drug Induced Kidney Disease
Drug induced kidney disease is a frequent cause of renal dysfunction; however, there are no standards to identify and characterize the spectrum of these disorders. We convened a panel of international, adult and pediatric, nephrologists and pharmacists to develop standardized phenotypes for drug induced kidney disease as part of the phenotype standardization project initiated by the International Serious Adverse Events Consortium. We propose four phenotypes of drug induced kidney disease based on clinical presentation: acute kidney injury, glomerular, tubular and nephrolithiasis, along with primary and secondary clinical criteria to support the phenotype definition, and a time course based on the KDIGO/AKIN definitions of acute kidney injury, acute kidney disease and chronic kidney disease. Establishing causality in drug induced kidney disease is challenging and requires knowledge of the biological plausibility for the specific drug, mechanism of injury, time course and assessment of competing risk factors. These phenotypes provide a consistent framework for clinicians, investigators, industry and regulatory agencies to evaluate drug nephrotoxicity across various settings. We believe that this is first step to recognizing drug induced kidney disease and developing strategies to prevent and manage this condition.
# Introduction
Drug induced kidney disease (DIKD) accounts for approximately 19-26% of cases of acute kidney injury (AKI) in hospitalized patients [bib_ref] Spectrum of acute renal failure in the intensive care unit: the PICARD..., Mehta [/bib_ref]. There are no standards to identify drug induced nephrotoxicity and as a result -DIKD is often unrecognized. In recent years, the International Serious Adverse Event Consortium (iSAEC) has initiated a phenotype standardization project for drug induced adverse events [bib_ref] Case definition and phenotype standardization in drug-induced liver injury, Aithal [/bib_ref]. In conjunction with the iSAEC, we have developed consensus definitions for DIKD, taking into account its wide spectrum and the need for balancing practicality with reliability of the classifications across different settings.
## Consensus process
With the support of the iSAEC, we organized a series of eight teleconferences followed by two face-to-face meeting of international, adult and pediatric, nephrologists and pharmacists. The panel developed phenotypic criteria using a modified Delphi process to allow identification of patients across 4 categories representing the spectrum of DIKD, for subject recruitment into a genetic study of DIKD (DIRECT). The panel was divided into subgroups and researched specific phenotypes. Criteria were summarized and presented to the larger group for consensus. Criteria were considered in the context of using electronic medical records to screen for patients with DIKD in both hospitalized and ambulatory settings.
Panelists were asked to consider the known mechanisms of nephrotoxicity, time course of drug exposure and the setting as discussed in more detail below. For the acute kidney injury (AKI) phenotype, established definitions were considered as the starting point and adapted for DIKD (e.g. AKIN/KDIGO criteria for AKI) [bib_ref] KDIGO Clinical Practice Guideline for Acute Kidney Injury, Group [/bib_ref].
## Description of phenotype
We propose that DIKD presents in one of four phenotypes: AKI, glomerular disorder, tubular disorder, or nephrolithiasis/crystalluria. The clinical presentation of each phenotype is based on a change in biomarkers and other evidence: Scr (AKI), proteinuria or hematuria (glomerular), electrolyte abnormalities (tubular), ultrasound findings (nephrolithiasis). To standardize the initial phenotype, we developed primary and secondary criteria. We suggest that at least one primary criterion must be met for all drugs suspected of causing DIKD .
## Mechanisms
Adverse drug reactions can be classified into type A and B reactions. Type A reactions are dose-dependent toxicities that are predictable based on the known pharmacology of the drug and alleviated by reducing drug exposure (i.e. dose reduction) or withdrawal of the drug (e.g. aminoglycoside toxicity). Type B reactions are unpredictable based on the known pharmacology of the drug. Toxicity is not dose-dependent and usually requires drug withdrawal for resolution (e.g. acute interstitial nephritis from proton pump inhibitors).
Often, the same drug may present as different DIKD phenotypes. For instance, NSAIDS can result in AKI due to hemodynamic changes or acute interstitial nephritis (AIN), or nephrotic range proteinuria from glomerular injury. The risk factors that predispose individuals to develop an adverse reaction from an individual drug are unknown in most cases. Genetic risk factors are emerging for the development of serious drug induced adverse reactions [bib_ref] HLA-A*3101 and carbamazepine-induced hypersensitivity reactions in Europeans, Mccormack [/bib_ref]. In type A reactions, genetic variation in drug elimination may determine overall drug exposure and pharmacological effect. For example, alterations in the expression of organic anion transporters (OAT) in the kidney could lead to increased intracellular concentrations of certain antimicrobials with increased toxicity to the renal tubules. The mechanisms underlying type B reactions are more complex and variable than type A reactions. In many instances of organ-directed toxicity, drug-induced disease may mimic other diseases. For example, hydralazine associated glomerulonephritis (GN) is immune mediated, may mimic a lupus or ANCA positive GN and may be categorized as a type B reaction. Based on our current understanding we classified common drugs associated with DIKD in relation to Type A and B reactions .
## Time course
Several factors affect the presentation of DIKD, including the drug exposure duration, time course for biomarker change, identification of renal abnormalities and the duration of the DIKD event. Drugs vary widely in the presentation of nephrotoxicity within each mechanistic type with some causing acute injury (examples: aminoglycosides (Type A) and cephalosporins (Type B)) while others are associated with a slower insidious insult (example: lithium (Type A)). Recognition of DIKD depends on the frequency with which the diagnostic tests are obtained and reviewed and will differ based on the setting (discussed further below). The interplay of factors including the mechanism of toxicity, duration of drug exposure and frequency of biomarker testing influence the recognition, management and outcomes of DIKD. Based on these observations we propose categorizing DIKD into three broad subsets reflecting the time course of events. These categories build on conceptual models proposed by KDIGO for AKI (considered if the injury develops within 7 days) and CKD (persistence of injury for >90 days). Injury to the kidney beyond 7 days but less than 90 days reflects sub-acute injury similar conceptually to acute kidney disease proposed in KDIGO guidelines. The development of DIKD can similarly be divided into acute (1-7 days), sub-acute (8-90 days) and chronic (>90 days) post drug exposure . We propose utilizing this framework as a practical approach for applying the primary criteria for all 4 phenotypes of DIKD as discussed further below. Based on this conceptual model, for each phenotype, thresholds could be established to detect DIKD, define its severity and ascertain recovery.
## Setting
Nephrotoxicity is common in both hospitalized and ambulatory care settings, but its reported frequency varies based on several factors. Hospitalized patients are generally sicker, have a higher risk of exposure to nephrotoxins, contrast agents and procedures, and are more frequently monitored than ambulatory care patients. Recognizing DIKD in ambulatory care is more difficult, more likely to be missed and not reported. Additionally, establishing causality is more difficult when biomarker values are lacking and history of drug exposure is incomplete. This is particularly important for the AKI phenotype, where our current definitions evaluate changes in Scr over a set period of time in relation to a specific reference. Often, this reference creatinine is unavailable. Chronic forms of DIKD (e.g. tubular disorders, nephrolithiasis and glomerular disorders) are similarly more likely to be recognized in clinic settings but it may be more difficult to establish causality to a specific drug.
In order to account for these factors, we propose that DIKD cases meet the minimal criteria, as follows:
1. The drug exposure must be at least 24 hours preceding the event.
## 2.
Reasonable evidence for biological plausibility for the causal drug, based on known mechanism of drug effect; metabolism and immunogenicity.
## 3.
Complete data (including the medication history, biomarker concentrations, comorbid diseases, concurrent risk factors) is required to account for concomitant risks and exposures to other nephrotoxic agents.
## 4.
The strength of the relationship between the attributable drug and phenotype should be based on drug exposure duration, extent of primary and secondary criteria met and the time course of the injury.
## Acute kidney injury phenotype key features
The AKI phenotype was based on KDIGO criteria with modifications to account for the presence of underlying CKD, time course and setting [bib_ref] KDIGO Clinical Practice Guideline for Acute Kidney Injury, Group [/bib_ref]. Since changes in serum creatinine are the hallmark of this phenotype, it includes acute tubular necrosis and acute interstitial nephritis. Although hemodynamic alterations are recognized for specific drugs e.g. angiotensin converting enzyme inhibitors (ACE), angiotensin receptor blockers (ARB), and non-steroidal anti-inflammatory drugs (NSAIDS), there are currently no consensus definitions of hemodynamic injury. Since transient changes in creatinine (usually Stage 1 AKI) can occur from other factors e.g. dehydration and hypotension in the setting of drug exposure and resolve when these factors are corrected, it is often difficult to distinguish primary drug induced effects from other factors. Recent studies from cardiac surgery patients exposed to ACE/ARB suggest that these changes are generally mild (Stage 1 criteria), resolve with dose reduction or withdrawal, and may not represent a clinically significant injury [bib_ref] Adjudication of etiology of acute kidney injury: experience from the TRIBE-AKI multi-center..., Koyner [/bib_ref]. Consequently, in order to increase specificity, we did not include hemodynamic changes as distinct criteria and we proposed that the primary criteria must meet a minimum of KDIGO Stage 2, to be considered a potential DIKD event . The overall severity of AKI is to be based on KIDGO staging criteria.
To further characterize the AKI phenotype based on initial presentation, we propose the secondary criteria shown in . Secondary criteria are used to further distinguish phenotypes (i.e. positive gallium scan for AIN), permit stratification and analysis within prespecified subgroups (e.g. oliguric vs non-oliguric presentations). The AKI phenotype itself comprises several mechanisms. For instance, renal functional change can reflect a direct nephrotoxic effect (i.e. ATN from aminoglycoside or cisplatin), or an idiosyncratic effect (i.e. AIN from a proton pump inhibitor). In both instances, changes in Scr and urine output would define the phenotype but secondary criteria such as urine and peripheral eosinophilia and a positive gallium scan could provide additional classification . We suggest that whenever feasible, kidney biopsy data would be used to confirm the underlying mechanisms (e.g. AIN vs ATN) and characterize the phenotype. A patient case is presented in , demonstrating the application of the AKI criteria for DIKD. In this AKI case, the patient presents with a significant rise in serum creatinine secondary to drug injury. In this case, there is a reference serum creatinine prior to drug initiation and repeated during the course of treatment establishing a clear timeline of injury. In addition to renal injury, the patient has a pruritic rash suggesting this may be a type B reaction. Based on the timeline of drug exposure and plausible mechanisms of toxicity, it is likely she has vancomycin induced AKI but the sub-classification of acute tubular necrosis versus acute interstitial nephritis is difficult to establish without histologic evidence. Subsequently, a kidney biopsy confirms acute tubulointerstitial injury and the treatment plan includes changing antibiotics and a course of steroids. Emerging biomarkers of kidney damage (e.g. NGAL, KIM-1< IL18) combined with functional markers (e.g. serum creatinine and urine output) may permit further delineation of these events including transient hemodynamic alterations and could aid in defining the AKI phenotype.
## Influence of ckd
To permit a clear assessment of underlying CKD status, and to establish a standardized approach to determine the onset and duration of AKI, we standardized the definitions of "baseline" and "reference" Scr values (Appendix 1). We recognized that in some instances, patients may present with an elevated Scr without a preceding reference value. In these instances, we propose accepting an absolute or relative decline in Scr equivalent to a Stage 1 over 48 hours (following drug dose change) or 7 days (drug discontinuation), respectively. In these instances, the decline in Scr would need to meet the criteria within 2 weeks of stopping the drug to ensure specificity. Some forms of AKI may take longer to resolve after discontinuation of the drug (in the case of AIN) and would be categorized as a sub-acute injury. The development of AKI in the setting of pre-existing CKD requires a similar level of change in Scr or urine output; however, patients must meet criteria for CKD (Appendix 1).
## Effect of time course and setting
Since many drugs manifest biomarker changes outside the time frame of the acute time period, we propose a sub-acute phenotype that requires a similar severity of Scr change as in the AKI phenotype but permits a Scr elevation within 4 weeks from initiation of drug and in the setting of continued drug exposure or within a maximum of two weeks of drug discontinuation. For patients where a decline in Scr was to be considered as evidence of kidney injury, the criteria would need to be met within 90 days of a change in drug dosing or discontinuation. This approach permits classification and tracking of injuries for duration and outcomes.
## Glomerular phenotype
Although several drugs have been associated with the development of glomerular injury, this is a relatively infrequent form of DIKD. Significant proteinuria, hematuria and associated urinary sediment abnormalities are the key hallmarks of this phenotype; however, this must be distinguished from a primary (e.g. idiopathic minimal change disease) or secondary (e.g. diabetes) glomerular process. Consequently, we proposed that the phenotype requires a kidney biopsy during continued drug exposure or within 4 weeks after stopping the drug, showing specific features previously associated with drug toxicity (e.g. collapsing focal segmental glomerulosclerosis with pamidronate). We recognized that often the biopsy features would be confounded by other factors (e.g. concurrent diseases) and would need to be consistent with the drug exposure period. We selected a urine protein to creatinine (UPCR) and urine albumin to creatinine (UACR) ratios > 0.8 or a 24 hour protein excretion > 1 gram per day as evidence for significant proteinuria [bib_ref] Hematuria and proteinuria in a mass school urine screening test, Park [/bib_ref]. We additionally proposed that a urinalysis with greater than 50 red blood cells per high powered field or dysmorphic red blood cells (such as acanthocytes) or RBC casts as evidence for significant hematuria and glomerular involvement. These definitions reflect clinical situations where most clinicians would consider a kidney biopsy. We recognized that UPCR and UACR are not equivalent however for practical purposes we suggest that either test be used in the absence of a timed collection to identify patients with suspected glomerular lesions. We opted for more specificity in defining this syndrome given its rarity in contrast to idiopathic glomerular disorders and other causes of asymptomatic proteinuria. presents a glomerular disorder case highlighting the application of the above criteria with confirmatory evidence of DIKD on renal biopsy.
## Tubular disorder
Drug induced tubular disorders have been described with several medications that are handled through tubular transport mechanisms and it is possible that mutations in renal transporters could give rise to tubular toxicity. Several different mechanisms have been implicated depending on the site of drug handling, drug exposure and duration of treatment.
In most instances, these are dose-related and usually seen with chronic, continued exposure. Several patterns have been described ranging from isolated abnormalities (e.g. phosphate leak) to more generalized lesions contributing to a proximal renal tubular acidosis (RTA) or an acquired Fanconi's syndrome. Recognizing the wide spectrum of tubular dysfunction, we proposed classifying this phenotype to include abnormalities in urinary losses of phosphate, glucose, magnesium, potassium, and tubular proteins or water handling. These would be associated with secondary changes in serum electrolytes, bicarbonate, and pH . A key issue is to distinguish DIKD from congenital defects, other diseases (e.g. sarcoid) or toxin mediated tubular dysfunction. In the case of tubular disorders, the primary criteria alone may be used for electronic surveillance and detection of possible injury, but secondary criteria are essential to confirm diagnosis and improve specificity.
## Europe pmc funders author manuscripts
## Europe pmc funders author manuscripts
## Nephrolithiasis
Medications may precipitate into crystals depending on their urinary solubility. The precipitation of medications spans the spectrum of asymptomatic, isolated crystalluria to obstructive stones. Crystalluria may also lead to AIN. This has been well described with anti-retrovirals such as indinavir which commonly causes isolated crystalluria but less commonly obstructive nephropathy. Drug induced renal calculi have also been described with sulfa antibiotics and triamterene. Additionally, nephrolithiasis can be associated with RTA syndromes related to tubular disorders. Imaging is often the only method to detect nephrolithiasis but may not be available in all cases. However, given the high incidence of nephrolithiasis in the general population, it is important to demonstrate the temporal relationship to the drug and analyze the stone composition, if available.
## Combination phenotypes
Although the phenotypes have distinct features, a patient may develop more than one phenotype. For example, drug induced crystalluria and nephrolithiasis could lead to AKI from obstruction or AIN. We considered that these combination phenotypes are possible. In such cases, each of the phenotypes will need to be evaluated independently, to establish a relationship to the drug exposure.
## Causality assessment and adjudication
Causality assessment tools such as the Naranjo scale have been used to attribute drug adverse reactions and have been modified to improve sensitivity for specific types of adverse reactions [bib_ref] A method for estimating the probability of adverse drug reactions, Naranjo [/bib_ref]. Causality assessment tools for DIKD have not been developed or reported. Challenges in causality assessment include multi-drug exposures and concurrent AKI risks. For instance, the risk of DIKD from antibiotics in the setting of sepsis would be enhanced by hypotensive episodes and exposure to contrast agents. In these situations, we propose that each drug be evaluated individually with respect to its possible contribution to the phenotype and underlying risk factors assessed. With multi-drug exposure, each causal agent should be rank classified (i.e. primary, secondary) based on the temporal relationship, magnitude and duration of effect, and knowledge of the underlying mechanism.
## Anticipated uses and limitations
There is no current systematic way of identifying DIKD given the variability in the presentation with each individual drug. Phenotype standardization provides framework guidance to pharmaceutical industry for drug development, to regulatory agencies for safety surveillance, physicians and patients for recognition. The proposed classification requires validation but could be utilized by regulatory agencies to standardize the documentation of kidney toxicity in clinical trials. If these criteria are validated in clinical trials, physicians would have a uniform method to describe and record adverse drug events and to inform patients of the potential risk and consequences of specific drug toxicities. Researchers would build on these initial phenotypes with new tools e.g. damage biomarkers to further characterize the component toxicities. Electronic medical records (EMR's) could be trained to identify the 4 broad phenotypes and build alert systems for pharmacists and physicians to recognize drug nephrotoxicity and develop quality metrics to prevent drug nephrotoxicity. This approach has been successfully implemented by recent studies in pediatrics and adults demonstrating the incidence of drug toxicity and the efficacy of an alert system to correct it [bib_ref] Acute kidney injury and increasing nephrotoxic-medication exposure in noncritically-ill children, Moffett [/bib_ref]. The phenotype standardization is anticipated to improve recognition of DIKD. Once these criteria have been validated, they can also be used for quality measurement. This will enhance the description of the epidemiology of DIKD, as was demonstrated by Selby and colleagues with the implementation of KDIGO criteria for screening and recognition of AKI [bib_ref] Use of electronic results reporting to diagnose and monitor AKI in hospitalized..., Selby [/bib_ref]. We recognize that the phenotype categorization is broad. However, a hierarchical categorization with primary and secondary criteria brings forward commonalities of the injuries to allow for enhanced recognition. The structured secondary criteria further distinguish the injury. For example, patients recognized as exhibiting tubular dysfunction can be further categorized as disordered water handling or acid base disorders. Broad categorization addresses the multi-mechanisms of injury since definition is based on biomarker presentation. This facilitates the standard detection and alerting of injury when a recognized nephrotoxic drug is being administered. We recognize these proposed phenotypes do not include every possible mechanism of DIKD. We have deliberately excluded hemodynamic injury because there is no consensus definition on hemodynamic changes and transient AKI. The current KDIGO definitions require adequate consideration and correction of pre-renal factors affecting recognition. In the absence of these standardized definitions, we have opted for greater specificity. We recognize this may lead to misclassification, if the effect is mild, transient and limited. We anticipate over time, with emerging biomarkers for pre-renal conditions, the identification of hemodynamic alterations secondary to DIKD will improve, leading to refinement of the AKI phenotype. In addition, if the phenotype is too sensitive in definition, the risk of prematurely stopping a drug for patient care or halting drug development for a candidate drug increases.
We recognize several limitations of the proposed framework. The AKI phenotype encompasses different pathologic injuries and the absence of specific mechanistic biomarkers makes the differentiation of AKI clinically challenging. We have proposed the KDIGO criteria as a unifying definition to identify patients and using the secondary criteria to provide further specificity of the nature, site and extent of injury. We anticipate that emerging biomarkers of kidney damage can identify site specificity and coupled with functional assessments, we can further refine the phenotype. The proposed approach is one of practicality and raises the need for biomarkers to distinguish injury. In addition, the proposed biomarker cut-offs were chosen for specificity, however, these cut-offs should not replace clinical judgment as there may be patients who develop DIKD but do not meet these thresholds for biomarker changes. For example, a patient who develops an increase in serum creatinine due to aminoglycosides but does not meet the criteria for Stage 2 AKI could still be diagnosed with DIKD based on the physician's assessment. The current phenotypes do not address the multi-mechanism injury as an entity which may have a different prognosis from single mechanism injuries. Additionally, as mentioned previously, these definitions do not ascertain causality and often the patient may be exposed to multiple drugs. Recognizing the phenotype is limited by how often biomarker measurements are taken so there is inherently uncertainty on the exact time course of DIKD. The practicality for utilizing the primary and secondary criteria for EMR screening is yet to be determined. Although in broad categorization of DIKD loses some granularity, it enhances the feasibility of EMR detection strategies.
# Conclusions
We have utilized a consensus based approach to establish 4 specific phenotypes to characterize DIKD based on existing knowledge of disease mechanisms, time course and setting. We acknowledge the inherent limitations of a consensus approach and the absence of any prospective validation. We recognize that the phenotypes would be subject to further revision based on their performance in prospective studies. However, we are confident that these phenotypes provide a consistent framework for clinicians, investigators and industry and regulatory agencies to evaluate drug toxicity across various settings. We believe that this is first step to recognizing DIKD and developing strategies to prevent and manage DIKD.
# Appendix 1: definitions
Acute kidney injury (AKI): is a process that causes an abrupt reduction in kidney function, and will be defined by meeting any of the following criteria [bib_ref] KDIGO Clinical Practice Guideline for Acute Kidney Injury, Group [/bib_ref] : - Urine sodium < 10 mEq/L AIN = acute interstitial nephritis, ATN = acute tubular necrosis, DM = diabetes mellitus, FeNa= fractional excretion of sodium, FePO4 = fractional excretion of phosphorus, GN = glomerulonephritis, HPF = high powered field, LDH = lactate dehydrogenase, RBC= red blood cell, SIADH= syndrome of inappropriate antidiuretic hormone, UPC = urine protein to creatinine ratio, UACR= urine albumin to creatinine ratio, WBC = white blood cell.
[formula] i [/formula]
1 Hemodynamic changes may contribute to ATN, however, in the absence of any specific features are not considered individual criteria for the AKI phenotype.
2 SIADH does not reflect direct tubular damage but rather the impact of a drug on ADH secretion and subsequent impaired water handling.
Kidney Int. Author manuscript; available in PMC 2016 February 18. |
Asymptotically local synchronization in interdependent networks with unidirectional interlinks
Synchronization in complex networks has been investigated for decades. Due to the particularity of the interlinks between networks, the synchronization in interdependent networks has received increasing interest. Since the interlinks are not always symmetric in interdependent networks, we focus on the synchronization in unidirectional interdependent networks to study the control scheme. The mathematical model is put forward and some factors are taken into consideration, such as different coupling functions and strengths. Firstly, the feasibility of the control scheme is proved theoretically by using Lyapunov stability theory and verified by simulations. Then, we find that the synchronization could be maintained in one sub-network by utilizing our control scheme while the nodes in the other subnetwork are in chaos. The result indicates that the influence of interlinks can be decreased and the proposed scheme can guarantee the synchronization in one sub-network at least. Moreover, we also discuss the robust of our control scheme against the cascading failure. The scheme is verified by simulations to be effective while the disturbances occur. synchronization [9, 10], projective synchronization [11], general synchronization [12, 13] and lag synchronization [14, 15]. Meanwhile, a number of control schemes, include adaptive control [16], decentralized control [17], impulse control [18], and pinning control[19], are provided. In the existing literature, the synchronization in multilayer networks has received increasing interest. The quantity and distribution of the controllers are analyzed to achieve lag synchronization[14]. In Ref.[15], the realizations of different kinds of synchronization with the consideration of different dynamics for each node between general complex networks are discussed. It is found that synchronization could be achieved when driving-response networks have identical connections[20]. To investigate synchronization on complex networks of networks, the attack and robustness of the pinning scheme are analyzed[21].However, far too little attention has been paid to the synchronization in interdependent networks. The synchronization behavior and the synchronicity in the interdependent systems are discussed in Ref.[22]and[23]. The mathematical models of interdependent networks are proposed and a variety of control schemes are provided to achieve synchronization[24][25][26]. These studies to date have tended to focus on interdependent networks with bidirectional interlinks, but it is found that the interdependencies are not always mutual or symmetric[27][28][29]. For instance, a computer hub might work with electricity supplied by a power substation but does not necessarily provide information control to it. Similarly, the operation of a gas station must depend on electricity supplied by a power plant, but the power plant does not need the support of a gas station.Moreover, the coupling function of the nodes in different sub-networks is usually regarded as the same one and one variable is always used instead of a matrix to describe the intercoupling between sub-networks[22,24,26]. These are not appropriate. The couplings are complicated. In[30], the dynamics of the local synchronization is studied in adaptively coupled neuronal network, in which the coupling between two neurons is determined dynamically by the states of the neurons. As for the interdependent networks, both the coupling functions and strengths must be different in different sub-networks at least. And note that the interdependency does not only exist between nodes i in one sub-network but also in the other sub-network. This indicates that the intercoupling matrix is very important and should not be ignored.The aim of this paper is to explore the realization of synchronization in interdependent networks with unidirectional interlinks. The major contributions of our work are as follows. First, we propose the model of a unidirectional interdependent network composed of two sub-networks. This is different from previous works. Second, different coupling functions and strengths are considered in the proposed model, and an intercoupling matrix is introduced. These are in line with the fact. Third, the design of controller is more simply, and the feasibility of the control scheme is proved theoretically by using Lyapunov stability theory and verified by simulations. Finally, by utilizing our control scheme, the influence of the interdependencies is decreased, and the synchronization could be maintained in one sub-network while the nodes in the other sub-network are in chaos.The rest of this paper is organized in the following way. In section II, the model of unidirectional interdependent networks is proposed, and some preliminaries are given. The control scheme is presented and proved theoretically in section III. The simulations are run and the results are discussed in section IV. The conclusion is given in section V.Model presentation and preliminaryConsidering the interdependent networks composed of two sub-networks X and Y. Each subnetwork is consisting of nodes which n-dimension nonlinear systems and coupled to eachPLOS ONEAsymptotically local synchronization in interdependent networks with unidirectional interlinks
# Introduction
Real-world is composed of large numbers of complex networks. The states of the nodes and couplings change continuously or discretely in a single network and multilayer complex networks [bib_ref] Multilayer networks, Kivelä [/bib_ref] [bib_ref] The structure and dynamics of multilayer networks, Boccaletti [/bib_ref]. Buldyrev et al. [bib_ref] Catastrophic cascade of failures in interdependent networks, Buldyrev [/bib_ref] present the concept of interdependent networks in 2010 and discuss the particularity of the interdependent links. The interdependent relations can be found in many real-world network systems, such as social networks in which the same actors are shared [bib_ref] Multirelational organization of large-scale social networks in an online world, Szell [/bib_ref] , plant-communication networks in which the computers work with the support from the power plant and conversely deliver control messages to them [bib_ref] Catastrophic cascade of failures in interdependent networks, Buldyrev [/bib_ref] , transportation networks in which the same locations are shared by airplanes, buses, and trains. Nowadays, interdependent networks has become one of the hot topics in the field of complex networks [bib_ref] Percolation in real interdependent networks, Radicchi [/bib_ref] [bib_ref] Networks formed from interdependent networks, Gao [/bib_ref].
The synchronization in complex networks has been concerned for decades. Several synchronization methods have been proposed, such as cluster synchronization [bib_ref] Symmetry-and input-cluster synchronization in networks, Siddique [/bib_ref] other, and each node is an n-dimension system. Sub-network Y unidirectionally depends on sub-network X with one-to-one mode. Then the dynamic equations of two sub-networks could be described as follows
[formula] _ x i ¼ f ðx i Þ þ a X N j¼1 a x ij Hðx j Þ i ¼ 1; 2; . . . ; Nð1Þ_ y i ¼ gðy i Þ þ b X N j¼1 a y ij Kðy j Þ þ g X N j¼1 c ij ðHðx j Þ À Kðy i ÞÞ i ¼ 1; 2; . . . ; Nð2Þ [/formula]
where, for node i, x i 2 R n ðy i 2 R n ) is the state vector in sub-network X(Y);f ðx i Þ : R n ! R n ðgðy i Þ : R n ! R n ) is a smooth nonlinear vector function; H : R n ! R n ðK : R n ! R n ) is a smooth nonlinear coupling function in sub-network X(Y); α(β) is the coupling strength in sub-network X(Y); γ is the intercoupling strength from subnetwork Y to sub-network X; A x ¼ ða x ij Þ 2 R n�n ðA y ¼ ða y ij Þ 2 R n�n Þ is the coupling matrix of sub-network X(Y), and if a connection exists between node i and node j(
[formula] i 6 ¼ j), then a x ij ða y ij Þ ¼ 1, otherwise a x ij ða y ij Þ ¼ 0; the diagonal elements a x ii ða y ii Þ satisfy dissipative condition a x ii ¼ À P N j¼1;j6 ¼i a x ij ða y ii ¼ À P N j¼1; [/formula]
j6 ¼i a y ij Þ; C ¼ ðc ij Þ 2 R n�n is the intercoupling matrix from subnetwork Y to sub-network X, that is, if a interdependency exists from node i in sub-network Y to node j in sub-network X, then c ij = 1, otherwise c ij = 0. Remark 1. We consider that the coupling functions are different in different sub-networks and α, β, γ are not equal. These agree with the fact in the real-world.
Remark 2. In this paper, the intercoupling matrix C is not symmetric for unidirectional interdependency and the construction of C follows the law that the node with a high degree in sub-network Y will preferentially depend on the node with a high degree in sub-network X. It is more universal in actual interdependent networks.
To achieve local synchronization, we add controllers u x i and u y i into two sub-networks respectively. Then Eqs (1) and (2) can be rewritten as
[formula] _ x i ¼ f ðx i Þ þ a X N j¼1 a x ij Hðx j Þ þ u x i i ¼ 1; 2; . . . ; Nð3Þ_ y i ¼ gðy i Þ þ b X N j¼1 a y ij Kðy j Þ þ g X N j¼1 c ij ðHðx j Þ À Kðy i ÞÞ þ u y i i ¼ 1; 2; . . . ; Nð4Þ [/formula]
Considering the interdependent networks composed of (3) and (4), there are two isolate nodes and the state vectors of them are s x ðtÞ 2 R n and s y ðtÞ 2 R n . s x (t) and s y (t) are utilized as reference trajectories for each sub-network, and satisfy
[formula] kx i À s x ðtÞk ¼ 0; i ¼ 1; 2; . . . ; Nð7Þlim t!1 ky i À s y ðtÞk ¼ 0; i ¼ 1; 2; . . . ; Nð8Þ [/formula]
Remark 4. The states of two isolate nodes are totally different and used as reference trajectories for sub-network X and Y, respectively. So, the asymptotical synchronization in sub-network X is not the same as the one in sub-network Y. We call the asymptotical synchronization in the interdependent networks as asymptotical local synchronization.
In order to design appropriate u x i and u y i , we need the following assumptions and lemma. Assumption 1. Let F(t) = Df(s x (t)) = [f ij (t)] n×n be Jacobian matrix of function f(s x (t)) on s x (t). F ¼ ðf ij Þ n�n 2 R n�n , and f ij is the maximum value of f ij ðtÞ ðt 2 RÞ; Let G(t) = Dg(s y (t)) = [g ij (t)] n×n be Jacobian matrix of function g(s y (t)) on s y (t). G ¼ ðg ij Þ n�n 2 R n�n , and g ij is the maximum value of g ij ðtÞ ðt 2 RÞ.
[formula] Assumption 2. Let B(t) = DH(s x (t)) = [b ij (t)] n×n be Jacobian matrix of function H(s x (t)) on s x (t). B ¼ ðb ij Þ n�n 2 R n�n , and b ij is the maximum value of b ij ðtÞ ðt 2 RÞ; Let D(t) = DK(s y (t)) = [k ij (t)] n×n be Jacobian matrix of function K(s y (t)) on s y (t). D ¼ ðk ij Þ n�n 2 R n�n , and k ij is the maximum value of k ij ðtÞ ðt 2 RÞ. Lemma 1. [31] For any matrices X; Y 2 R n�m , if A T ¼ A > 0; A 2 R n�n , then X T Y +Y T X � X T AX + Y T A −1 Y. [/formula]
# Main results
According to [bib_ref] Networks formed from interdependent networks, Gao [/bib_ref] and (8), local synchronization error vectors are defined as
[formula] e x i ¼ x i À s x ðtÞð9Þe y i ¼ y i À s y ðtÞð10Þ [/formula]
According to (3)-(6), local synchronization error systems can be derived
[formula] _ e x i ¼¼ f ðx i Þ À f ðs x ðtÞÞ þ a X N j¼1 a x ij Hðx j Þ þ u x i ð11Þ _ e y i ¼ gðy i Þ À gðs y ðtÞÞ þ b X N j¼1 a y ij Kðy j Þ þ g X N j¼1 c ij ðHðx j Þ À Kðy i ÞÞ þ u y ið12Þ [/formula]
## Theorem 1
For the interdependent networks composed of sub-network (3) and (4), if assumptions 1-2 hold, then asymptotical local synchronization could be achieved via controllers
[formula] u x i ¼ À a X N j¼1 a x ij Hðs x ðtÞÞ þ d x i e x ið13Þu y i ¼ À b X N j¼1 a y ij Kðs y ðtÞÞ À g X N j¼1 c ij Hðs x ðtÞÞ þ g X N j¼1 c ij Kðs y ðtÞÞ þ d y i e y i ð14Þ _ d x i ¼ À k x i e xT i e x i ð15Þ _ d y i ¼ À k y i e yT i e y ið16Þ [/formula]
where d x i and d y i are adaptive laws, k x i and k y i are feedback gains, [bib_ref] Lag synchronization of complex networks via pinning control, Guo [/bib_ref] into error system (11) and [bib_ref] Generalized synchronization of complex networks, Shang [/bib_ref] respectively and use linearization method.
[formula] k x i > 0; k y i > 0, i = 1,2,. . .,N. Proof. Introduce (13),_ e x i ¼¼ FðtÞe x i þ a X N j¼1 a x ij BðtÞe x j þ d x i e x i ð17Þ _ e y i ¼ GðtÞe y i þ β X N j¼1 a y ij DðtÞe y j þ γ X N j¼1 c ij BðtÞe x j À γ X N j¼1 c ij DðtÞe y i þ d y i e y ið18Þ [/formula]
Let l F max be the maximum eigenvalue of matrix (
[formula] F T + F), l G max be the maximum eigenvalue of matrix (G T + G), l B max be the maximum eigenvalue of matrix BB T , l D max be the maximum eigen- value of matrix DD T . Letã x 1 ¼ max 1�i�N ja x ij j;ã x 2 ¼ max 1�i�N ja x ji j;ã y 1 ¼ max 1�i�N ja y ij j;ã y 2 ¼ max 1�i�N ja y ji j;c 1 ¼ max 1�i�N jc ij j;c 2 ¼ max 1�i�N jc ji j: Choose the candidate Lyapunov function V t ð Þ ¼ X N i¼1 e xT i e x i þ X N i¼1 e yT i e y i þ X N i¼1 ðd x i þ d x � Þ 2 k x i þ X N i¼1 ðd y i þ d y � Þ 2 k y ið19Þ [/formula]
where d x � ; d y � are constant, and satisfy d x
[formula] � > ðl F max þ aNã x 1 l B max þ aNã x 2 þ gNc 2 Þ=2; d y � > ðl G max þ bNã y 1 l D max þ gNcl B max þ bNã y 2 Þ=2. [/formula]
Then, with Eqs (15) and (16), the derivative of V(t) along error system (17) and (18) are obtained as
[formula] _ V t ð Þ ¼ X N i¼1 ð_ e xT i e x i þ e xT i _ e x i Þ þ X N i¼1 ð_ e yT i e y i þ e yT i _ e y i Þ þ 2 X N i¼1 d x i þ d x � k x i _ d x i þ 2 X N i¼1 d y i þ d y � k y i _ d y i ¼ X N i¼1 � e xT i F T ðtÞe x i þ a X N j¼1 a x ij e xT j B T ðtÞe x i þ e xT i FðtÞe x i þ a X N j¼1 a x ij e xT i BðtÞe x j þ e yT i G T ðtÞe y i þ b X N j¼1 a y ij e yT j D T ðtÞe y i þ g X N j¼1 c ij e xT j B T ðtÞe y i À g X N j¼1 c ij e yT i D T ðtÞe y i þ e yT i GðtÞe y i þ b X N j¼1 a y ij e yT i DðtÞe y j þ g X N j¼1 c ij e yT i BðtÞe x j À g X N j¼1 c ij e yT i DðtÞe y i À 2d x � e xT i e x i À 2d y � e yT i e y i �ð20Þ [/formula]
According to lemma 1, we can get the results as follows:
[formula] a X N j¼1 a x ij e xT j B T ðtÞe x i þ a X N j¼1 a x ij e xT i BðtÞe x j � a X N j¼1 ja x ji je xT i e x i þ a X N j¼1 ja x ij je xT i BðtÞB T ðtÞe x i ð21Þ b X N j¼1 a y ij e yT j D T ðtÞe y i þ b X N j¼1 a y ij e yT i DðtÞe y j � b X N j¼1 ja y ji je yT i e y i þ b X N j¼1 ja y ij je yT i DðtÞD T ðtÞe y i ð22Þ g X N j¼1 c ij e xT j B T ðtÞe y i þ g X N j¼1 c ij e yT i BðtÞe x j � g X N j¼1 jc ji je xT i e x i þ g X N j¼1 jc ij je yT i BðtÞB T ðtÞe y ið23Þ [/formula]
Introduce (21)-into [bib_ref] Synchronization between two coupled complex networks, Li [/bib_ref] , and with assumptions 1-2 _ V ðtÞ can be rewritten as
[formula] _ V ðtÞ � X N i¼1 � e xT i F T ðtÞe x i þ a X N j¼1 ja x ji je xT i e x i þ e xT i FðtÞe x i þ a X N j¼1 ja x ij je xT i BðtÞB T ðtÞe x i þ e yT i G T ðtÞe y i þ b X N j¼1 ja y ji je yT i e y i þ g X N j¼1 jc ji je xT i e x i À g X N j¼1 c ij e yT i D T ðtÞe y i þ e yT i GðtÞe y i þ b X N j¼1 ja y ij je yT i DðtÞD T ðtÞe y i þ g X N j¼1 jc ij je yT i BðtÞB T ðtÞe y i À g X N j¼1 c ij e yT i DðtÞe y i À 2d x � e xT i e x i À 2d y � e yT i e y i � � X N i¼1 e xT i ðF T þ F þ a X N j¼1 ja x ij jBB T þ a X N j¼1 ja x ji j þ g X N j¼1 jc ji j À 2d x � Þe x i þ X N i¼1 e yT i � G T þ G þ b X N j¼1 ja y ij jDD T þ g X N j¼1 jc ij jBB T þ b X N j¼1 ja y ji j À g X N j¼1 c ij ðD T þ DÞ À 2d y � � e y i < ðl F max þ aNã x 1 l B max þ aNã x 2 þ gNc 2 À 2d x � Þ X N i¼1 e xT i e x i þ ðl G max þ bNã y 1 l D max þ gNcl B max þ bNã y 2 À 2d y � Þ X N i¼1 e yT i e y ið24Þ [/formula]
Note that d x
[formula] � > ðl F max þ aNã x 1 l B max þ aNã x 2 þ gNc 2 Þ=2 and d y � > ðl G max þ bNã y 1 l D max þ gNcl B max þ bNã y 2 Þ=2, so _ V ðtÞ < 0. The proof is completed. [/formula]
## Simulation examples
To verify the theoretical analysis, we construct the interdependent networks composed of two undirected sub-networks. Sub-network X is constructed as WS small world network (N = 10, K = 2, P = 0.5) and sub-network Y is constructed as BA scale free network (N = 10, m 0 = 3, m = 2). The characteristics of sub-network X(Y) are as follows: the average path length is 1.6 (1.62), the clustering coefficient is 0.39(0.76) and the average degree is 4(3.4). The nodes in sub-network Y unidirectionally depend on the nodes in sub-network X with one-to-one mode. The construction of interlinks follows the law that the node with a high degree in subnetwork Y will preferentially depend on the node with a high degree in sub-network X. The coupling matrices A x , A y and the intercoupling matrix C are as below: Each node in sub-network X is a Lorenz system
[formula] _ x i1 ¼ À a 1 x i1 þ a 1 x i2 _ x i2 ¼ b 1 x i1 À x i2 À x i1 x i3 _ x i3 ¼ x i1 x i2 À c 1 x i3 8 > > > < > > > : i ¼ 1; 2; � � � ; 10ð28Þ [/formula]
where a 1 = 10, b 1 = 28, c 1 = 8/3, and Lorenz system is in chaos. Each node in sub-network Y is a Rössler system [bib_ref] Phase synchronization on scale-free networks with community structure, Zhou [/bib_ref]. All of our simulations are run in MATLAB R2016a and the time step is 0.01.
[formula] _ y i1 ¼ À y i2 À y i3 _ y i2 ¼ y i1 þ a 2 y i2 _ y i3 ¼ b 2 þ y i3 ðy i1 À c 2 8 > > > < > > > : i ¼ 1; 2; � � � ; 10ð29Þ(0) = [i, −i, i] T , y i (0) = [0.3i, 0.3i, −i] T , d x i ð0Þ ¼ 0:1i; d y i ð0Þ ¼ 0:2i; k x i ¼ 0:1i; k y i ¼ i, i = 1,2,� � �, [/formula]
## Example 1
Add controllers (13)-(16) into interdependent networks (3) and (4) according to Theorem 1. The states of error systems are shown in Figs 1 and 2. We can find that the states of error systems tend to zero quickly. That is to say, the proposed control scheme works, and asymptotically local synchronization in each sub-network is achieved respectively.
When asymptotically local synchronization was achieved in each sub-network, the values of adaptive laws tend to be stable. The trajectories of adaptive laws are shown in Figs 3 and 4.
## Plos one
Sub-network Y unidirectionally depends on sub-network X. If all controllers in sub-network Y were out of order, would asymptotically local synchronization in sub-network X still be achieved? The nodes in sub-network Y have no influence on the nodes in sub-network X as the interdependencies are unidirectional. So, the synchronization in sub-network X should be maintained intuitively. We run the simulations and the results are shown in Figs 5 and 6.
The results shown in that the synchronization in sub-network X is still achieved while the controllers in sub-network Y do not work. Meanwhile, [fig_ref] Fig 6: States of error system between the nodes and the isolate node in... [/fig_ref] shows that the states of error system in sub-network Y are oscillatory as expected.
## Example 3
Here we suppose all controllers in sub-network X do not work. This is just opposite to example 2. Simulations are run and the results are given in Figs 7 and 8. we can see that the states of error system in sub-network X fall into oscillation. That is to say, the states of the nodes in sub-network X are in chaos. Because of unidirectional interdependency, this would have a great influence on the nodes in sub-network Y and should result in loss of the synchronization in sub-network Y. But on the contrary, the results in [fig_ref] Fig 8: States of error system between the nodes and the isolate node in... [/fig_ref] indicate that the synchronization in sub-network Y is still achieved. This means that the influence is decreased by using the proposed control scheme. The synchronization can be guaranteed in sub-network Y, even though the nodes in sub-network X are in chaos.
## Example 4
In the interdependent networks, the failure of one or more nodes in one sub-network will result in the cascading failure of the corresponding nodes in another sub-network due to the existence of the interdependency. So it is worth observing whether the synchronization implemented by our controllers is robust against the disturbances.
In our work, the interdependency is unidirectional and one-to-one mode. For simplicity, the connectivity of each sub-network is not consideration and two cases, the nodes in sub-network X or Y are out of work early, are studied in the simulations. Firstly, some nodes in subnetwork X or Y are chosen randomly to be out of work. Then the connections between the invalid nodes and their neighbor are deleted. Secondly, if the failed nodes belong to sub-network X, the cascading failure starts, i.e., the corresponding nodes in sub-network Y will also be invalid due to the interdependency. But if the failed nodes belong to sub-network Y, the cascading failure will not start in sub-network X for the interdependency is unidirectional. Finally, the cascading failure results in the deletion of the connections between the failed nodes and their neighbor in sub-network Y.
It is found in our simulations that whether different nodes or different numbers of nodes are chosen early in sub-network X or Y, the results are similar. So only two simulation results are given in Figs 9 and 10 by choosing node 4 and 7 in sub-network X to be out of work early, and in Figs 11 and 12 by choosing node 1 and 6 in sub-network Y early.
From Figs 9-12, it is shown that the synchronization by using our controllers could be retained when some nodes in sub-network X or Y are out of work. Whether the cascading failure happens or not, our control scheme is still effective, which shows the robustness of the proposed method.
# Conclusion
In this article, we design adaptive controllers to achieve asymptotically local synchronization in unidirectional interdependent networks. In the proposed model, different coupling strengths, different coupling functions, intercoupling strength, and intercoupling matrix are considered to agree with the fact in real-world. The feasibility of the control scheme is proved theoretically by using Lyapunov stability theory and verified by simulations in MATLAB. The numerical results show that asymptotically local synchronization in unidirectional interdependent networks can be achieved quickly via the adaptive controllers. Furthermore, we find that the synchronization in one sub-network can be achieved by using our control scheme, even if the failure of the controllers exists in the other sub-network. This indicates that the influence can be decreased to a certain extent. Also, the effectiveness of our control scheme is verified while the cascading failure occurs, i.e., the synchronization of the remaining nodes in each sub-network can be retained.
Our work enriches the research contents of the synchronization in the interdependent networks. Further investigations on the synchronization in interdependent networks are needed to promote the deeper research of complex networks.
## Supporting information
## S1 appendix. a hurwitz matrix a is given which satisfies the condition.
(DOCX)
[fig] where a 2: = 0.1, b 2 = 0.1, c 2 = 14, and Rössler system is in chaos. The coupling strength α = β = 0.01, and the intercoupling strength γ = 0.03. the coupling functions H(x i ) = [sinx i1 , cosx i2 , x i3 ] T , K(y i ) = [tany i1 , y i2 , y i3 ] T , i = 1,2,� � �,10. The other initial values are as below: s x (0) = [0, −20, −5] T , s y (0) = [10,0,0] T , x i [/fig]
[fig] Fig 1: States of error system between the nodes and the isolate node in sub-network X (Eq (3)).https://doi.org/10.1371/journal.pone.0267909.g001 [/fig]
[fig] Fig 2: States of error system between the nodes and the isolate node in sub-network Y (Eq (4)).https://doi.org/10.1371/journal.pone.0267909.g002 [/fig]
[fig] Fig 3: Trajectory of adaptive laws of sub-network X (Eq (3)).https://doi.org/10.1371/journal.pone.0267909.g003 [/fig]
[fig] Fig 4: Trajectory of adaptive laws of sub-network Y (Eq (4)).https://doi.org/10.1371/journal.pone.0267909.g004Fig 5. States of error system between the nodes and the isolate node in sub-network X (Eq (3)). https://doi.org/10.1371/journal.pone.0267909.g005 [/fig]
[fig] Fig 6: States of error system between the nodes and the isolate node in sub-network Y (Eq (2)).https://doi.org/10.1371/journal.pone.0267909.g006PLOS ONEFig 7. States of error system between the nodes and the isolate node in sub-network X (Eq (1)).https://doi.org/10.1371/journal.pone.0267909.g007 [/fig]
[fig] Fig 8: States of error system between the nodes and the isolate node in sub-network Y (Eq (4)).https://doi.org/10.1371/journal.pone.0267909.g008 [/fig]
[fig] Fig 9: States of error system between the remaining nodes and the isolate node in sub-network X while node 4 and 7 are chosen to be out of work. https://doi.org/10.1371/journal.pone.0267909.g009 Fig 10. States of error system between the remaining nodes and the isolate node in sub-network Y with the failed node 4 and 9 for the cascading failure. https://doi.org/10.1371/journal.pone.0267909.g010 Fig 11. States of error system between the nodes and the isolate node in sub-network X which is immune to the cascading failure. https://doi.org/10.1371/journal.pone.0267909.g011 [/fig]
[table] , phase: PLOS ONEPLOS ONE | https://doi.org/10.1371/journal.pone. [/table]
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Long-Distance Movement of mRNAs in Plants
Long-distance transport of information molecules in the vascular tissues could play an important role in regulating plant growth and enabling plants to cope with adverse environments. Various molecules, including hormones, proteins, small peptides and small RNAs have been detected in the vascular system and proved to have systemic signaling functions. Sporadic studies have shown that a number of mRNAs produced in the mature leaves leave their origin cells and move to distal tissues to exert important physiological functions. In the last 3-5 years, multiple heterograft systems have been developed to demonstrate that a large quantity of mRNAs are mobile in plants. Further comparison of the mobile mRNAs identified from these systems showed that the identities of these mRNAs are very diverse. Although species-specific mRNAs may regulate the unique physiological characteristic of the plant, mRNAs with conserved functions across multiple species are worth more effort in identifying universal physiological mechanisms existing in the plant kingdom.
# Introduction
Higher plants have evolved a communication system that enables the coordination of developmental cues and environmental inputs. The local communication is achieved between different cellular compartments inside a cell or adjacent cells by symplasmic transmit through plasmodesmata, while inter-organ communication is realized by long-distance signaling that takes place in the vasculature. Phloem, one of the major components in the vasculature system, has long been recognized as a tissue that transports carbohydrates and amino acids. In recent years, it has been found that phloem harbors a diverse population of components, e.g., mRNAs, small RNAs, proteins, small peptides and hormones.
While scientists have had a relatively comprehensive understanding of the biological functions of many phloem located, long-distance mobile molecules, little is known on the importance and breadth of mRNAs in participating in the systemic signaling and regulation of plant physiology. mRNAs have been traditionally viewed as local intermediate components between the genomic DNAs and the proteins in a cell. However, this conventional view of mRNAs has been challenged because a handful of studies have shown that mRNAs produced in mature leaves can leave their original cells and move to distal tissues via the phloem to exert plant physiology functions. For example, the long-distance movement of AtIAA18, AtIAA28 and AtTCTP1 mRNA is indispensable for the root development in Arabidopsis; although FT (Flowering Locus T) protein is demonstrated to be the systemic florigenic signal, long-distance movement of AtFT mRNA from mature leaf to shoot apex has also been suggested to be involved in flowering initiation in Arabidopsis; similarly, the mRNAs of a few FT homologs, NsSP3D, NsSP5G, and NsSP11A, are required in the flowering induction in Nicotiana Sylvestris; in potatoes, a number of mRNAs, i.e., StBEL5, StBEL11, StBEL29 and StPOTH1, have been found to be able to move from mature leaves to stolon for tuberization initiation and storage root development; in tomatoes, the long-distance movement of PFP-LeT6 and SlPS mRNAs is essential for tomato leaf developmentand resistance to the necrotrophic fungus Botrytis cinereal, respectively. These pioneer studies conducted in the last two decades demonstrated the importance of long-distance movement of mRNAs in physiological regulation; however, how common this phenomenon is for other mRNAs remains an intriguing question until the recent development of a few systems that enabled the discoveries of large-scale mobile mRNAs in plants.
## Identification of large-scale movement of mrnas using heterograft methods
Three major sampling methods, i.e., stylectomy, EDTA facilitated, and cucurbit bleeding, can be used to study mobile mRNAs in the phloem. However, the lengthy sampling procedure associated with the stylectomy method, the possibility of contaminations derived from the damage of EDTA to plant tissues, and the non-phloem origin of most of the cucurbit "phloem" sap make these methods less ideal in dissecting phloem mobile molecules. In the last 3-5 years, important breakthroughs have been made in the area of mRNA movement using other alternative approaches. One of the most prominent discoveries was derived from a system in which parasitic plants and their hosts were used. It was found that a total of 9518 mRNAs (45% of the whole genome) from the host Arabidopsis moved into the parasitic Cuscuta; meanwhile, 8665 mRNAs from the parasitic Cuscuta moved into the Arabidopsis. On the contrary, when parasitic Cuscuta was attached to the tomato host, only 347 tomato mRNAs moved into Cuscuta, and 288 Cuscuta mRNAs moved into the tomato. In addition to the host parasitic system, a few heterograft systems, in which one species or genotype is grafted onto the other species or genotype, were used to demonstrate the existence of the large scale, vasculature-mediated movement of mRNAs between different organs, as shown in. For example,determined that 2006 mobile mRNAs moved from the shoot to the root or vice versa, in which two ecotypes of Arabidopsis were used as either scion or rootstock;identified 138 Arabidopsis mobile transcripts in the scion in a Nicotiana benthamiana/Arabidopsis heterograft system;reported that 3333 mRNAs were transmittable within a heterograft system consisting of two grape varieties; revealed that 3546 mRNAs moved from the cucumber mature leaves into watermelon sink tissues; Wang et al. (2020) identified 2386 mobile mRNAs in a watermelon/bottle gourd heterograft system; we (2018) detected 1063 shoot-to-root mobile mRNAs in which the N. benthamiana plant was used as scion and the tomato was used as rootstock. Comparisons of the mobile mRNAs from these systems showed that the identities of the mRNAs are highly species specific. This indicates that a better understanding of the basic physiological processes, e.g., cellular origin, destination, related to mRNAs is needed before large-scale functional characterization of these mRNAs at the molecular level are pursued.
## Cell origin of mobile mrnas
The aforementioned studies have ambiguously demonstrated that certain mRNAs produced in the leaf can move to other distal organs via phloem. Due to the complicated cell ultrastructure and diverse cell types in leaf, it is legitimate to assume that not all leaf cells contribute equally in the biosynthesis and transport of mRNAs. Evidence from our studyand Thieme et al.demonstrated that the mobile mRNAs detected in the heterografts were over represented in previously identified phloem mRNAs derived from other methods. Via a computational analysis, Calderwood et al.also concluded that mRNAs located in the phloem companion cells had a higher possibility to move.
Another interesting discovery was from Yang et al.who found that among the most abundant 33 leaf mRNAs identified from the grapevine grafting system, approximately half (17) of them moved over a long distance. This finding implied that the abundance of mRNAs in leaves has a high correlation with movement. However, in our N. benthamiana/tomato heterografting system, we found that the abundance of mRNAs in leaves had no correlation with the mobility. In our system, none of the 100 most abundant mRNAs in the leaf moved to the rootstock. What is the explanation of the big difference between our discovery and the one from Yang et al. We believe that the anatomical structural difference of leaf cells between herbaceous (our system) and woody species (system in the Yang study) may play a role in the generation of this distinction, as shown in.
In herbaceous species, there are very limited plasmodesmata connections between the phloem cells, which are involved in long-distance transport and signaling, and the other surrounding leaf cells, such as bundle sheath and mesophyll cells. On the contrary, in woody species, the phloem and its surrounding leaf cells are connected with a high abundance of plasmodesmata. At present, it has been widely accepted that such structural characteristics in herbaceous and woody plants are associated with specific transport mechanisms for small molecules, such as carbohydrates. In herbaceous species, sugars produced in mesophyll cells during photosynthesis cannot directly move into the phloem through plasmodesmata, due to the low abundance of plasmodesmata. Sugars have to be exported to the apoplast of the phloem and then taken up by sugar transporters localized on the plasma membrane of phloem companion cells (CC). In woody plants, this apoplast step is not needed because sugars produced in mesophyll cells can be directly transported to the phloem via the abundant plasmodesmata. The discrepancy between our observation and that from Yang et al. indicated that the aforementioned theory related to sugar movement may also apply to mRNAs. In most plants, phloem cells (including companion cells and sieve elements (SE)) account for only 1 to 3% of all the leaf cells. Therefore, highly expressed mRNA populations identified by RNA-seq or quantitative reverse PCR in leaves are more likely to be expressed in all leaf cells. In woody plants (such as grapevine), these high-abundance mRNAs, regardless of their cell types, are likely to move because of the rich plasmodesmata among all the cells in leaf, as shown in. Therefore, a strong correlation between the abundance and movement is more obvious. However, the scenario may be completely different in a system involved in herbaceous species (such as our N. benthamiana/tomato heterograft system). The high-abundance mRNAs, unless located in phloem, are not prone to move due to the low density of plasmodesmata between phloem and the surrounding cells, shown in. Therefore, a positive correlation between the mRNA abundance and mobility is less likely to appear. In agreement with this hypothesis, a study suggested that the long-distance movement of AtGAI mRNA was compromised when it was ectopically expressed outside of the phloem in Arabidopsis. In herbaceous species, there are very limited plasmodesmata connections between the phloem cells, which are involved in long-distance transport and signaling, and the other surrounding leaf cells, such as bundle sheath and mesophyll cells. On the contrary, in woody species, the phloem and its surrounding leaf cells are connected with a high abundance of plasmodesmata. At present, it has been widely accepted that such structural characteristics in herbaceous and woody plants are associated with specific transport mechanisms for small molecules, such as carbohydrates. In herbaceous species, sugars produced in mesophyll cells during photosynthesis cannot directly move into the phloem through plasmodesmata, due to the low abundance of plasmodesmata. Sugars have to be exported to the apoplast of the phloem and then taken up by sugar transporters localized on the plasma membrane of phloem companion cells (CC). In woody plants, this apoplast step is not needed because sugars produced in mesophyll cells can be directly transported to the phloem via the abundant plasmodesmata. The discrepancy between our observation and that from Yang et al. indicated that the aforementioned theory related to sugar movement may also apply
## Destination of mobile mrnas in the root
When leaf-produced mRNAs arrive in root via phloem, they or the translated proteins derived from these mRNAs need to be unloaded from the sieve tube to other cells to exert physiological functions. The root system in plants is complex and consists of multiple types of cells, e.g., phloem, xylem, cortex, endodermis, epidermis and pericycle.. Similar to the limited exploration on the cellular origin of the mobile transcripts, very few efforts have been focused on the identification of the recipient cells in roots. A previous study on StBEL5 mRNA indicated that the root has a mechanism to distinguish the shoot-born mRNAs because the mRNA transcript is only enriched in stolon. This tissue-specific distribution of the StBEL5 mRNA in underground tissue is important for the initiation of tuberization and tuber development. The enrichment mechanism is not clear but two possibilities exist. StBEL5 mRNAs could be specifically unloaded to stolon as suggested by Banerjee et al., or the transcripts were unloaded to all root cells but a cell distinctive mRNA degradation mechanism is involved that leads to the low abundance of mRNAs in cells with high mRNA turnover activities.
Recently, Ross-Elliott et al.established a mathematical model and suggested that phloem unloads solutes through the symplasmic plasmodesmata connection into the adjacent phloem pore pericycle (PPP), by a combination of mass flow and diffusion in Arabidopsis roots. While small solutes such as sugars are exported without restriction, macromolecules (e.g., proteins) are restricted in the PPP due to the size-dependent filtration. Experimental evidence supporting the mathematical model came from a study conducted by Yang et al.. It was found that YFP-TCTP mRNA can be translated into its corresponding YFP-TCTP protein after they arrive in the root. However, YFP-TCTP proteins were mainly observed in PPP but totally absent in other root cells such as xylem pore pericycle (XPP). It is important to realize that the specific localization of YFP-TCTP protein in PPP cells does not restrict its physiological effect to be solely in these recipient cells because the movement of AtTCTP from the shoot to the root promoted the initiation of lateral root primordia, a tissue different from PPP. A previous study has shown that LR primordia of Arabidopsis arises in the XPP, a cell that does not directly receive the TCTP protein. The underlined mechanism is not clear but one possibility is that the arrival of the TCTP proteins in PPP cells stimulates a cascade of molecular processes in which certain downstream components further move from PPP to XPP to initiate the primordia formation.
## Factors conferring mobility
All the heterografting systems demonstrated that only a portion of the transcribed mRNAs in the leaf can move to the root. This indicates that certain factors associated with these mRNAs are related to their mobility. As discussed above, it was suggested that mRNAs with high abundance in the phloem companion cells are more prone to move. If abundance in the companion cells is the only factor conferring mobility, then transcripts, including those that are not mobile, should move if their abundances are increased in the companion cells. Our research showed that this is not the case. AtAMT1;2 and AtCHL1 are two transcripts that were identified to be immobile in all the published heterograft systems. When these two mRNAs were individually overexpressed in the companion cells driven by a companion cell-specific promoter in potato, the lack of detection of these mRNAs in the root from the heterografts in which the transgenic plants were used as scion and wild type as rootstock, suggested that increasing abundance solely was not enough to promote the mobility of these two transcripts. It should be noted that our result does not negate the importance of mRNA abundance in companion cell in conferring mobility. Instead, it was suggested that other factors were also involved in regulating mRNA movement.
In addition to abundance, another factor that may participate in the regulation of mobility is the plasmodesmata between the companion cells and the sieve element. The size exclusion limit (SEL) of these plasmodesmata in the collection phloem of leaf is reported to be~67 kDa in Arabidopsis. Small molecules, such as sugars and amino acids, can move freely through these plasmodesmata but the movement of macromolecules larger than the SEL may be restricted. For example, when GFP is overexpressed in the companion cells, the protein of GFP can enter the SE for long-distance movement, while the mRNA of GFP cannot. Although the lack of endogenous RNA-binding protein for GFP transcript may be one reason related to this observation, the large size of GFP mRNAs (230 kDa), which is beyond the SEL of the plasmodesmata, could restrict the GFP mRNAs in the companion cells.
Multiple studies have indicated the importance of specific sequence motifs in mRNAs in conferring mobility. Similar to the lack of mobility of GFP mRNAs, GUS, with a molecular mass at 652kDa, was not mobile when it was overexpressed by the 35S promoter. However, the mobility of GUS can be achieved if a TLS (tRNA-like sequence) motif is fused with GUS. An analysis of the mobile mRNAs from most of the published systems showed that only 10-15% of these transcripts harbor TLS. This indicates that other motifs may also be involved in transcript mobility. Banerjee et al.demonstrated that both 5' and 3' untranslated regions of the mRNAs of StBEL5 were involved in mediating long-distance transport from shoot to stolon. A cis-acting element required for RNA mobility was mapped to the coding region of AtFT mRNAs. The sequence of AtGAI at coding and 3' untranslated regions constitutes the motifs necessary for RNA movement. In addition, Yang et al. showed that the 5-methylcytosine (m5C) modification of mobile mRNAs plays a crucial role in facilitating their transport.
It has been suggested that the movement of RNAs is facilitated by RNA-binding proteins (RBPs) and specific motifs located in different mRNAs can be recognized by certain RBPs. For example, the 3' UTR of StBEL5 harbors a poly-pyrimidine sequence element that specifically interacts with polypyrimidine tract-binding proteins (PTBs) in potatoes. It is known that the RNA-RBP complex not only facilitates movement, but it also protects the RNA from degradation. In our study, we found that some of the leaf-born mRNAs had a higher abundance in roots, but most of them were degraded during their shoot-to-root movement. It remains interesting to explore whether the degradation of the mobile mRNAs was due to the lack of RBPs to them. Other potential routes of mRNA transport to enter the phloem, e.g., via vesicles, have also been proposed.
## Physiological functions of the mobile mrnas
Various studies have demonstrated that the long-distance movement of mRNAs may be associated with important physiological processes, such as root development, flowering, tuberization and leaf development. Notaguchi et al.found that AtIAA18, AtIAA28 transcripts were synthesized in the vascular of mature leaves and their movement to the root regulated lateral root development. Yang et al.determined that the 5-methylcytosine (m5C) modification of AtTCTP1 mRNA was required for its transport and the movement of m5C-modified TCTP1 mRNA was essential for root growth. Branco and Maslealso revealed that the long-distance transported AtTCTP1 mRNA specifically stimulates the emergence of the lateral root along the primary root pericycle, while the root elongation is partially controlled by the local constitutive TCTP1 expression. The AtFT mRNA functions as a systemic floral signaling to promote vegetative-to-reproductive transition in Arabidopsis. The FT belongs to phosphatidylethanolamine-binding domain protein (PEBP) family. Further study suggested that a number of members in the PEPB family, such as NsCET1, NsSP9D, NsSP3D, NsSP5G, NsSP11A, and NsSP2G, are phloem mobile. Potato tuberization is also controlled by mobile mRNAs. The potato StBEL5 mRNA was demonstrated to be a long-distance signal that is expressed in the phloem cells of leaves and transmitted into roots and stolons to initiate tuberization. The overexpression of StBEL5 mRNA using a leaf-specific promoter helped overcome the inhibitory effects of long days on tuber formation and enhanced the tuber yield. Phloem-mobile StPOTH1 mRNA functions synergistically with StBEL5, while StBEL11 and StBEL29 functions antagonistically to StBEL5. The long-distance movement of mRNAs also controls leaf development. Kim et al.reported that the chimeric PFP-LeT6 fusion mRNA was transported from the mutant rootstock to the heterografted wild-type scion and caused the leaf's morphological changes in the scion. AtGAI, CmGAIP and CmNACP were found to be mobile and regulated the shoot apex development in Arabidopsisand pumpkin. A study has also shown that CmPP16 functions as an RNA-binding protein that carries various mRNA molecules from companion cells to the sieve element. The long-distance movement of tomato SlPS mRNAs in Arabidopsis was essential for resistance to the necrotrophic fungus.
To our knowledge, the aforementioned list of mRNAs were the major ones for which in-depth functional characterizations have been conducted. However, in recent years, hundreds of mRNAs have been identified to be mobile from the Arabidopsis, grapevine, cucumber and Solanaceae grafting systems, respectively. It is legitimate to assume that mRNAs with conserved biological functions should exist in most, if not all, of these systems. However, we compared the mobile mRNAs and found only one "core" mRNA shared by all the systems. This indicated that either most of the mobile mRNAs have no functions or they play species-specific functions in plant physiology. In addition to the N. benthamiana/tomato heterografting system, we also developed another system in which canola was the scion and Arabidopsis was the rootstock. The extremely low number, i.e., twenty-three, and the high variation of mobile mRNAs identified from this system further indicated that more understanding is needed from the perspective of these mobile mRNAs as a population before in-depth molecular functional characterizations of individually selected mobile mRNA was pursued.
If some of the mobile mRNAs do not have specific physiological functions, why do plants generate and transport such a large population of mRNAs? Two mechanisms, i.e., selective and non-selective, have been proposed to be used by plants to transport their mRNAs. For mRNAs with important physiological roles in the distal tissues, the generation and movement should be selective and tightly regulated. For example, a mRNA has to harbor a specific motif or be of a certain length; however, for mRNAs that are produced in the leaf, particularly companion cells, at high abundance but without essential features for long-distance movement, they may still enter the phloem sieve tube translocation stream. These mRNAs may be completely or partially degraded during their movement. To test this possibility, we designed a N. benthamiana/tomato heterograft system in which the stem of the tomato rootstock was 2.5 m long. We discovered that a total of 1,096 N. benthamiana mRNAs passed the graft joint, but 854 of them disappeared during their movement from shoot to root. It is reasonable to assume that the movement of mRNAs undergoing degradation in the phloem is not selective and regulated; therefore, it is less likely that there are physiological functions associated with them. An alternative way to interpret this phenomenon is that plants use the degradation mechanism to remove excess cellular mRNAs. In addition, Melino et al. suggested that the turnover of RNAs and the catabolism of nucleotides may supplement the internal nitrogen pool and support the growth of the plant.
## Methods to identify mobile mrnas
Previous studies using EDTA-facilitated exudation or cucurbit exudation identified large numbers of mRNAs in the phloem translocation stream. However, the authenticity of these identified mRNAs was often questioned. The recent adoption of the multiple heterograft systems significantly improved the authenticity and increased the numbers of identified mobile mRNAs. Nonetheless, attention must be paid when using heterografts for mobile mRNA identification. Each of these steps, e.g., finding the two compatible species, sampling, the preparation of libraries and the next-generation sequencing, and data mining, could lead to false discoveries.
The two species used in the heterograft should be elaborately selected. In general, these criteria should be satisfied: a) the two species should be phylogenetically close enough so they can be grafted; b) the genome sequences for the two species should be distant enough so the mRNAs can be unambiguously assigned to one species or another. For example, in the Arabidopsis Col/Arabidopsis Ped heterograft system, it was predicted that 72% of mobile mRNAs were not able to be identified due to the high genome-sequence similarities between the two ecotypes. On the contrary, in the N. benthamiana/Arabidopsis system, the highly different genome sequences between the two species was an advantage in the identification of mobile mRNAs; however, the two species involved belong to different families. This may lead to strong physiological distortion and some of the identified mRNAs may be related to the non-native physiological alterations. Indeed, a number of the mobile mRNAs identified from this system are related to stress responses.
One of the essentials shared by all these heterografts is the adoption of the RNA-Seq for the exhausted identification of mRNAs transmitted between the two plants. Two major analytical methods were developed to facilitate the analysis. Thieme et al.and Yang et al.used SNPs in their systems in which different genotypes of the same species were used; while other investigators directly compared genome sequences for the identification because species with more distant relationships were used in their grafting systems. Due to the close relationship and similar genome sequences of the two genotype/species in the heterografts, it is extremely important to apply strict informatics procedures so false discovery can be avoided. First, RNA-Seq libraries prepared from the source and the recipient tissues should be loaded into different lanes for sequencing. This will eliminate the possibility of crosstalk between samples even if various adapters are used to index samples during library construction. Second, a homograft in which both the scion and rootstock are the recipient species in the heterograft system should be established. This homograft is used as a control to eliminate false positives derived from the heterografts.
## Future perspective
Thousands of mRNAs have been identified to be mobile from the various heterograft systems. The functional characterization of individual mRNAs should be pursued in the near future. However, it is equally important to elucidate the general mechanisms related to the movement of these mobile mRNAs. For example, do herbaceous and woody plants differ in the origin of mobile mRNAs? To address this question, a mobile mRNA fragment, e.g., GFP+TLS, can be overexpressed either in the phloem or outside of the phloem in herbaceous and woody plants. The transgenic plants can be grafted on the non-transgenic rootstock and the abundance of GFP transcripts can be measured in the rootstock. A high abundance of the GFP will indicate the successful export of this transcript from the overexpressed cells. When sugars arrive in roots, they need to be unloaded from phloem to other root cells for metabolism. Do the shoot-to-root mobile mRNAs need to be unloaded, and if so, what are the recipient cells? In the heterografting system, usually~30 m reads on the whole root tissue were sequenced for mobile mRNA discovery. The depth of single-cell sequencing can now reach as high as 15-30 m reads for each cell type and it can be used to locate cell-specific shoot born-mRNAs. A third question that is interesting to ask is the integrity of the mobile mRNAs. Our N. benthamiana/tomato heterograft system with a 2.5 m long stem let us discover that degradation occurs during the movement of mRNAs. However, partially degraded mRNAs could still be caught by RNA-Seq and assigned to be mobile. Therefore, it remains extremely important to study the integrity of a mRNA before any in-depth functional characterization is pursued. Other intriguing questions that remain to be answered include the identification of factors conferring the mobility and proteins that bind to the mobile mRNAs. The last, but not the least interesting and challenging question, is the differentiation of the effect between the mobile mRNAs and their corresponding proteins in the recipient organs.
## Conflicts of interest:
The authors declare no conflict of interest. |
The influence of time on the sensitivity of SARS-CoV-2 serological testing
Sensitive serological testing is essential to estimate the proportion of the population exposed or infected with SARS-CoV-2, to guide booster vaccination and to select patients for treatment with anti-SARS-CoV-2 antibodies. The performance of serological tests is usually evaluated at 14-21 days post infection. This approach fails to take account of the important effect of time on test performance after infection or exposure has occurred. We performed parallel serological testing using 4 widely used assays (a multiplexed SARS-CoV-2 Nucleoprotein (N), Spike (S) and Receptor Binding Domain assay from Meso Scale Discovery (MSD), the Roche Elecsys-Nucleoprotein (Roche-N) and Spike (Roche-S) assays and the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples collected as part of the Co-STARs study (www. clini caltr ials. gov, NCT04380896) up to 200 days following infection. Our findings demonstrate the considerable effect of time since symptom onset on the diagnostic sensitivity of different assays. Using a time-to-event analysis, we demonstrated that 50% of the Abbott nucleoprotein assays will give a negative result after 175 days (median survival time 95% CI 168-185 days), compared to the better performance over time of the Roche Elecsys nucleoprotein assay (93% survival probability at 200 days, 95% CI 88-97%). Assays targeting the spike protein showed a lower decline over the follow-up period, both for the MSD spike assay (97% survival probability at 200 days, 95% CI 95-99%) and the Roche Elecsys spike assay (95% survival probability at 200 days, 95% CI 93-97%). The best performing quantitative Roche Elecsys Spike assay showed no evidence of waning Spike antibody titers over the 200-day time course of the study. We have shown that compared to other assays evaluated, the Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche Elecsys Spike Assay and the MSD assay maintained a high sensitivity for the 200-day duration of the study. These limitations of the Abbott assay should be considered when quantifying the immune correlates of protection or the need for SARS-CoV-2 antibody therapy. The high levels of maintained detectable neutralizing spike antibody titers identified by the quantitative Roche Elecsys assay is encouraging and provides further evidence in support of long-lasting SARS-CoV-2 protection following natural infection.Following natural infection or vaccination, sensitive measurement of SARS-CoV-2 serological status is important to identify immune correlates of protection from future waves of the pandemic, evaluate those in need of booster OPEN
www.nature.com/scientificreports/ vaccination and identify candidates for SARS-CoV-2 antibody therapy. The rapid response to the COVID-19 pandemic has led to the development of a wide range of serological tests suitable for evaluating SARS-CoV-2 exposure, infection or vaccination status [bib_ref] Diagnostic accuracy of serological tests for covid-19: Systematic review and meta-analysis, Lisboa Bastos [/bib_ref] [bib_ref] Molecular and serological tests for COVID-19. A comparative review of SARS-CoV-2 coronavirus..., Kubina [/bib_ref] [bib_ref] Testing for SARS-CoV-2 (COVID-19): A systematic review and clinical guide to molecular..., La Marca [/bib_ref]. Typically, these tests are approved for use by the regulatory authorities based on their performance against a panel of reference sera including positive and negative controls at either 14-or 21-days post infection [bib_ref] Serodiagnostics for severe acute respiratory syndrome-related coronavirus 2: A narrative review, Cheng [/bib_ref]. Public Health England reported a 93.9% sensitivity for the Abbott SARS-CoV-2 IgG Nucleoprotein assay 5 and 100% for the Roche Elecsys Nucleoprotein assay at ≥ 14 days post infection. This led to widespread adoption of these tests across NHS laboratories for testing at population level. Other studies have confirmed this test performance at 14-21 days post infection [bib_ref] Comparison of SARS-CoV-2 serological tests with different antigen targets, Coste [/bib_ref] [bib_ref] Performance characteristics of five immunoassays for SARS-CoV-2: A head-to-head benchmark comparison, Ainsworth [/bib_ref]. Population level serological studies have also based their conclusions-vital to guide national policy-on the basis of these tests 9 without considering how time since infection influences the performance of the test. The problem with this approach is that it does not take into account SARS-CoV-2 humoral dynamics and changes in avidity over time [bib_ref] Long-term persistence of spike protein antibody and predictive modeling of antibody dynamics..., Grandjean [/bib_ref] [bib_ref] The challenge of avidity determination in SARS-CoV-2 serology, Bauer [/bib_ref]. Although serological tests with limited diagnostic range may demonstrate excellent sensitivity shortly after infection, it is unclear how they will perform with time following infection or vaccination.
In order to address this question, we applied 4 widely used serological assays in parallel to serial samples from the Co-STARs studyin which staff testing seropositive to SARS-CoV-2 were followed for up to 200 days following infection. We compared the proportion of samples that remained seropositive over time using a survival analysis and determined the decay rate of the nucleoprotein (N) antibody and the spike (S) antibody for each test using a previously published mathematical model fitted to the data.
# Materials and methods
Study setting and design. Serological testing was performed on stored serum samples collected as part of the Co-STARs study (www. clini caltr ials. gov, NCT04380896), approved by the UK National Health Service Health Research Authority and run at Great Ormond Street Hospital between April 29th and November 2020 in accordance with the relevant guidelines [bib_ref] Long-term persistence of spike protein antibody and predictive modeling of antibody dynamics..., Grandjean [/bib_ref]. Briefly, Co-STARs was a 1-year single-centre prospective cohort study of antibody responses to COVID-19 infection in healthcare workers. Serum samples were taken from the 3657 participants at baseline and underwent a screening ELISA using the EDI assay. Repeated monthly serum samples were then taken from those with a seropositive baseline screening test for up to 250 days after the date of infection. Written informed consent was obtained from all participants. Those samples identified as seropositive with available symptom start date had further confirmatory testing with the quantitative three antigen MSD assay. Study participants. The majority of hospital staff were eligible for the Co-STARS study. Only those participants with significant immunosuppression, those that had received blood products within 6 months of recruitment and those that had active and ongoing symptoms of SARS-CoV-2 infection (within the last 21 days) were excluded. Only samples from individuals with at least one positive test from any platform were included in the analysis. Moreover, individuals without a known symptom start date were removed.
## Data collection.
As part of the Co-STARs study all participants undertook a detailed standardised online questionnaire at study entry. This included the date of onset of COVID-19 symptoms, and any SARS-CoV-2 diagnostic test results.
Comparison of serological assays. Samples taken as part of the Co-STARS studywhich had an accompanying symptom start date available for analysis were initially screened for seropositivity by the EDI assay or by any of the three antigens of the Meso Scale Discovery (MSD) assay. The selected samples each underwent testing with 4 serological assays: (1) The Roche Elecsys Anti-SARS-CoV-2 electrochemiluminescence immunoassay (ECLIA) assay detects the nucleocapsid (N) antigen (Roche-N); (2) the Roche Elecsys Anti-SARS-CoV-2 S electrochemiluminescence immunoassay (ECLIA) assay detects the spike (S) antigen (Roche-S); (3) the Abbott Nucleoprotein Chemiluminescent Microparticle Immunoassay (CLMIA) assay detects the nucleocapsid (N) antigen (Abbott-N); (4). All tests were performed as per manufacturer's specifications.The four antigen Meso Scale Discovery (MSD) assay was undertaken at the WHO Pneumococcal Supranational Reference Laboratory at the UCL Institute of Child Health. Only 3 antigens were reported from the MSD assay (the Spike, the Nucleoprotein and the RBD) as the baseline test performance of the N-terminal domain (NTD) antibody response was insufficient for further evaluation as previously reported [bib_ref] Evaluation of a novel multiplexed assay for determining IgG levels and functional..., Johnson [/bib_ref]. The Roche-N and Roche-S assays were undertaken by the Laboratory Medicine Service of Swansea Bay University Health Board, Morriston Hospital, Swansea. The Abbott-N assay was undertaken by Public Health Wales Microbiology at Cardiff and Vale University Hospital. All samples were stored and transported between laboratories at − 80 °C and only removed for aliquoting prior to testing to avoid unnecessary freeze-thaw cycles.
Statistical analysis and modelling. In order to evaluate the relative proportion of seropositive tests in the parallel serological assays over time, a time-to-event analysis was performed using the time from symptom onset and the first negative test for each assay after a first positive test as the event of interest using the R package survival. Only tests taken > 14 days after symptom onset were considered in the analysisNo tests were performed between 14-and 21-days post symptoms, and thus using 14 or 21 days post symptom onset as threshold did not affect our results. A participant was defined as seropositive when at least one of the 4 tests undertaken was seropositive. If the other tests that were run in parallel never became seropositive, the time-to-event was set to the earliest test taken for that individual. If a participant never became seronegative during the follow-up period, a right-censored observation was added at the time of the last serological test. www.nature.com/scientificreports/ Additionally, the decay rate after 21 days since symptom onset was estimated using a Bayesian generalized linear mixed model as implemented in the R package MCMCglmm 16 , where time from symptom onset was included as a fixed effect and study participants as a random effect. Therefore, a unique slope for the regression was estimated for the entire population, while the intercept was allowed to vary between the study participants. The decay rate was estimated from the slope of the linear model.
To assess the overall diagnostic capability of the Abbott-N assay, a receiver operating characteristic curve (ROC) analysis was performed using the pROC package within R 17 . The MSD-N and Roche-N assays were used as the gold standard for the comparison.
Ethical approval and consent. The study had national Integrated Research Application System (IRAS) approval and all participants in the study provided informed consent.
# Results
A total of 950 samples from 329 participants seropositive by any assay after 14 days underwent testing with the Roche-N, Roche-S, the MSD and the Abbott-N assay. The majority of the participants (98%, 321/329) had a positive result by two or more assays. Antibody decay with time. Plotting the raw log transformed antibody titers over time since symptom onset demonstrated that antibody dynamics were dependent on the assay undertaken. The production of spike antibodies was demonstrated to be maintained at high levels up to 200 days when evaluated by the MSD and the quantitative Roche -S assay. All nucleoprotein antibody assays demonstrated decay of the nucleoprotein antibody over time. This was most pronounced in the Abbott-N assay and much less so in the Roche -N assay which demonstrated slow waning of the nucleoprotein antibody.
Assay sensitivity with time post symptom onset. The existing published test performance for all assays undertaken is provided in . The sensitivity of all assays (at least 14 days from symptom onset) at 50, 100 and 150 days is provided in [fig_ref] Table 2: Sensitivity of compared assays at 50, 100 and 150 days from symptom... [/fig_ref]. All assays demonstrated a reasonable sensitivity at 50 days following infection [fig_ref] Figure 2: Comparison of seropositivity and antibody dynamics between serological tests [/fig_ref]. As time passed following infection, the Abbott-N assay rapidly became seronegative [fig_ref] Figure 2: Comparison of seropositivity and antibody dynamics between serological tests [/fig_ref] , with a median survival time inferred at 175 days (95% CI 168-185 days), whereas the survival probability at 150 days was inferred to be 95% for the Roche-N (95% CI 0.92-0.97), and 91% for the MSD-N assay (95% CI 0.87-0.94). The Roche-S and MSD-S assays remained seropositive for the duration of the study. The MSD-RBD assay showed some evidence of waning seropositivity over time (90% Survival probability at 150 days, 95% CI 0.88-0.94).
A total of 45% (159/329) of the individuals had a negative result using the Abbott-N assay during the course of the study. For the MSD test, 16% (52/329) of participants had a negative test for the N antigen, 11% (36/329) . Log transformed serial serological antibody titer data plotted by time from symptom onset. Antibody dynamics are dependent on the assay used with the sensitive Roche-S and MSD-S assay demonstrating maintenance of the spike protein antibody while the nucleoprotein antibody is shown to wane with the MSD and Abbott-N assays but to a lesser extent with the Roche-N assay. Mathematical model fits to estimate antibody decay. To estimate the decay rate for each antibody and assay studied, a generalized linear mixed model was fitted to the trajectory of antibody decay after 21 days from symptom onset, where the decay rate was estimated as the slope of the antibody titer through time. Under the most sensitive and quantitative Roche -S assay the spike antibody demonstrated no decay at all and rather a slow rate of increased titers over time from symptom onset (0.0031, 95% CI 0.0018-0.0044, [fig_ref] Figure 2: Comparison of seropositivity and antibody dynamics between serological tests [/fig_ref]. In accordance with the raw observed data, all nucleoprotein antibodies under the mathematical model decayed. This was most pronounced in the Abbott-N assay (− 0.022, 95% CI − 0.023 to − 0.02) and least pronounced in the Roche -N assay (− 0.0025, 95% CI − 0.0039 to − 0.0012, [fig_ref] Figure 2: Comparison of seropositivity and antibody dynamics between serological tests [/fig_ref] , . The lower performance of the Abbott-N assay can be explained by a lower detection of titer values as their concentration wanes over time. When compared to the quantitative MSD-N, 26% (222/860) of all positive samples by the MSD-N were negative for the Abbott-N test [fig_ref] Figure 3: Comparison of antibody titers between the Abbott-N assay and the MSD-N assay [/fig_ref]. A total of 75% of samples (137/ 183) positive by the MSD-N with an MSD arbitrary titer value lower than 403 were negative for the Abbott-N assay. Using the currently manufacturer recommended threshold of 1.4 arbitrary units, the Abbott-N test was characterized by a high specificity of 0.96 and a sensitivity of 0.74 using all our test results after 14 days. Using a ROC curve [fig_ref] Figure 3: Comparison of antibody titers between the Abbott-N assay and the MSD-N assay [/fig_ref] , the optimal cut-off that maximises both specificity and sensitivity was estimated to be 0.845 arbitrary units.
# Discussion
Sensitive measurement of SARS-CoV-2 seropositivity is key to evaluate who has been infected or exposed to SARS-CoV-2, to determine the correlates of protection from future disease, stratify those that need booster vaccination and target the use of anti-SARS-CoV-2 antibodies to those that are seronegative. To our knowledge no other study has evaluated the sensitivity of multiple diagnostic tests in parallel on longitudinally collected serological samples. This study demonstrates that as time elapses after infection, the sensitivity of serological . Decay rate for each serological assay (log arbitrary units per day) estimated in a generalized linear mixed model. www.nature.com/scientificreports/ testing varies widely depending on the test used. Although serological tests may be demonstrated to perform well 14-21 days after infection, this initial test performance often diminishes as time passes. In order to evaluate whether or not the population maintains SARS-CoV-2 antibodies it is vital that we utilize serological tests that remain sensitive over time. Initial published baseline test performance reports concluded that the Abbott-N assay was a high-performance test and a key tool in SARS-CoV-2 surveillance [bib_ref] Performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in, Bryan [/bib_ref]. Our data demonstrate that as time passes following infection the sensitivity of this assay declines rapidly until at < 6 months following infection it is no more than 50% sensitive. Our findings support the concerns raised by others regarding the poor performance of some nucleoprotein based assays [bib_ref] Testing for responses to the wrong SARS-CoV-2 antigen, Rosadas [/bib_ref] [bib_ref] SARS-CoV-2 seroprevalence survey estimates are affected by anti-nucleocapsid antibody decline, Bolotin [/bib_ref].
In contrast, the Roche assays, particularly the Roche Elecsys Anti-SARS-CoV-2 Spike assay maintained high sensitivity for the 200-day duration of the study. Although there remains no single correlate of sterilizing or protective immunity following SARS-CoV-2 infection or vaccination, it is clear that natural infection and the presence of neutralizing spike antibodies decreases the possibility of re-infection and the severity of disease upon re-exposure to currently circulating strains [bib_ref] SARS-CoV-2 infection rates of antibody-positive compared with antibody-negative health-care workers in England:..., Hall [/bib_ref]. Our finding that spike antibodies remained at high titers 200 days after infection adds to our previous study on this topic [bib_ref] Long-term persistence of spike protein antibody and predictive modeling of antibody dynamics..., Grandjean [/bib_ref] and provides further evidence in support of long-lasting protection against severe disease from currently circulating strains. Fitting mathematical models to the raw data of the Roche spike assay demonstrated that spike antibody titers did not decay but rather increased slightly over the duration of the study. The Roche nucleoprotein assay also maintained sensitivity for the duration of the study with a low rate of decay. Although this assay is semi-quantitative, our findings suggest that this could be used to sensitively identify those that have been vaccinated from those that have been both vaccinated and infected.
Many studies have evaluated the impact of time on test sensitivity over the first 3 weeks following symptom onset [bib_ref] Evaluation of SARS-CoV-2 serology assays reveals a range of test performance, Whitman [/bib_ref] [bib_ref] Performance of SARS-CoV-2 serology tests: Are they good enough, Piec [/bib_ref] [bib_ref] Meta-analysis of diagnostic performance of serology tests for COVID-19: Impact of assay..., Wang [/bib_ref]. However, we found no other study that had examined the sensitivity of antibody testing on parallel longitudinal samples collected between 1 and 6 months after infection or exposure. Assays with a higher titer cut-off for detection may perform well in the initial period after infection, but fail to detect seropositivity as antibody levels wane over time. We show that the Abbott-N test failed to detect 75% of samples positive for the MSD-N with a titer value lower than 403, which makes the Abbott-N assay less suitable for seroprevalence studies. Using a ROC curve and the MSD-N and Roche-N assays as the gold standard, we showed that a lower threshold of 0.845 instead of 1.4 arbitrary units may be more suitable to optimize the sensitivity and specificity. Even though different thresholds may be relevant depending on whether sensitivity or specificity needs to be prioritized, our findings suggest that the high Abbott-N test threshold results in a high number of false negatives. These findings are concordant with previous reports showing a range of high uncertainty between 0.49 and 1.4 [bib_ref] Performance verification of the Abbott SARS-CoV-2 test for qualitative detection of IgG..., Castro [/bib_ref]. Barzin et al.used Abbott-N testing alone to determine SARS-CoV-2 seroprevalence in 2,973 asymptomatic out-patients in North Carolina estimating a seroprevalence of 0.8%. Similarly, Wilkins et al. [bib_ref] Seroprevalence and correlates of SARS-CoV-2 antibodies in health care workers in Chicago, Wilkins [/bib_ref] used Abbott-N on 6510 healthcare workers up to 150 days after symptom onset and estimated a seroprevalence of 4.8%. Our findings suggest that previously published surveys of SARS-CoV-2 seroprevalence such as these could have significantly underestimated the true prevalence of SARS-CoV-2 humoral immunity.
Memory T-cell interferon gamma release or proliferation assays in response to SARS-CoV-2 antigens provide an alternative means of assessing prior exposure to infection. However, these assays are limited by cross reactive immunity to the seasonal coronaviruses decreasing specificity [bib_ref] SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected..., Le Bert [/bib_ref] [bib_ref] Cross-reactive memory T cells and herd immunity to SARS-CoV-2, Lipsitch [/bib_ref].
Although all serological tests used in the study demonstrated a high initial specificity, one limitation is that only 38% of participants had a confirmatory SARS-CoV-2 PCR result. Our data may therefore be influenced by an unknown proportion of falsely positive serological tests. However, at entry to the study, all seropositive participants had both a screening EDI nucleoprotein assay and an MSD assay performed which limited the chances of a falsely positive result due to a single erroneous test. Not all samples were processed at the same time; the Roche and Abbott-N assays were processed 3 months after the MSD assays. Despite this, we believe that sample storage and freeze-thawing cycles are unlikely to have influenced our findings as the Roche quantitative spike assay was performed last and demonstrated the highest prolonged levels of spike antibody of all tests used.
In summary, although serological tests may demonstrate high sensitivity 3-weeks after SARS-CoV-2 infection, this is far from the case with some tests 6-months after infection. The Abbott-N assay performed poorly at this time, whereas the Roche and MSD tests maintained a high sensitivity for the 200 days of the study. Tests that perform poorly over time will lead to spurious estimates in population level seroprevalence studies and findings from these studies should be adjusted to account for sensitivity of the test used and the time since infection. Test performance as time passes post infection should be considered before evaluating who is a candidate for booster vaccination or anti-SARS-CoV-2 antibody therapy.
[fig] Figure 2: Comparison of seropositivity and antibody dynamics between serological tests. The Roche-S assay target the spike antibody, the Abbott-N and the Roche-N assays target the N-antibody while the MSD assay targets the N-, the S-and the antibody to the Receptor Binding Domain (RBD) of the spike protein in parallel.(a) Kaplan-Meier curve and numbers at risk (the number of participants under follow up with serological tests available for analysis at that time point) for different serological tests. Y-axis represents the probability of remaining seropositive, while the X-axis shows days after symptom onset with numbers of participants under follow up shown in the table below. (b) Inferred posterior density distributions of the decay rate in a generalized linear mixed model. [/fig]
[fig] Figure 3: Comparison of antibody titers between the Abbott-N assay and the MSD-N assay. (a) The quantitative results for the MSD-N assay were compared to those of the Abbott-N test for each sample taken. Colours divide the samples depending on whether it was positive (green) or negative (red) for the MSD-N assay. Dotted red lines represent the seropositivity threshold for the Abbott-N assay (horizontal) and the MSD-N test (vertical). (b) ROC curve for the Abbott-N assay using the MSD-N test as gold standard. The x ~ y line represents the profile of a random classifier. Blue shaded area shows the 95% CI. [/fig]
[table] Table 2: Sensitivity of compared assays at 50, 100 and 150 days from symptom onset. [/table]
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Coronavirus disease 19 (COVID-19) during chemoradiation for locally advanced oropharyngeal squamous cell carcinoma (LA-OPSCC)
## Coronavirus disease 19 (covid-19) during chemoradiation for locally advanced oropharyngeal squamous cell carcinoma (la-opscc)
In the era of the pandemic the first goal is reducing COVID-19 risk for staff and patients and at the same time ensuring an optimal anti-cancer treatment avoiding delays.
Radiation therapy is a life-saving treatment and should be guaranteed to all patients with cancer for whom it is indicated.
Definitive chemo-radiotherapy (CRT) is the standard of care for locally advanced oropharyngeal squamous cell carcinoma (OPSCC). To continue curative CRT in patients affected by COVID-19 is subject of debate. Hereby we present a case report of OPSCC patient which was affected by COVID19 after the second Platinum cycle administration. In our multidisciplinary team after infectious disease assessment we decided to continue treatment because of curative intent and the young age of the patient. (1) Xy is a male 62 years old, heavy smokers, nothing in his medical history, who referred in From 25 th March 2020 the patient began to have fever (38.5°Celsius) and cough.
He performed a negative chest x-ray. For uncontrolled pain and inadequate oral intake it was agreed with the family doctor to increase pain therapy and start hydration at home On 27 March the fever was 38 °Celsius and a treatment with intravenous Ceftriaxone was started at home by the family doctor.
On 29th March, Xy accessed to the emergency room and was hospitalized for dysphagia (cancer and treatment -related) ,fever and cough.
Nasal and oropharyngeal swab revealed SARS-CoV-2 infection.
Blood examination were WBC 4.6K/mcl, Platelets144 K/mcl Haemoglobin 12.5 g/dL, D-Dimer 10ug/ml Reactive C protein 123mg/L pH =7.5; pCO2 35.1mmHg; pO2= 68.6mmHg. Given the stability of the patient, it was decided to resume radiation therapy on April 1st.
The treatment plan was modified by prescribing to the boost volume 6 fractions of 3 Gy instead of the planned 10 fractions of 2 Gy.
The aim was to achieve a radical dose for the patient but to reduce exposure to the staff of the Radiotherapy department. The patient was treated at the end of the working shift with appropriate protective devices for the staff. The bunker areas was sanitized at the end of the treatment session.
Xy received antibiotic treatment with piperacillin tazobactam for 10 days; low molecular weight heparin was administered prophylactically during the hospitalization.
Simultaneous supportive care was administered during the hospitalization; intravenous nutrition of about 1800 k-calories was administered from 29 th March to 20 th April with polyamine acids + glucose monohydrate + electrolytes + olive oil + medium chain triglycerides + fish oil with a high content of omega-3 acids + soybean oil for parenteral use, a reduced intravenous nutritional therapy of about 550 k-calories was administered to 23 rd April 2020.
Pain management consisted of continuous intravenous infusion of hydrochloride morphine 70 mg.
Pain therapy was turned to trans-dermal fentanyl to permit the patient to return home.
However pain control was insufficient therefore the patient was medicated with oral methadone with benefit.
Xy successfully completed radiotherapy on 7 th April, while the third Cis-platinum treatment was omitted. Total RT dose to gross tumor volume was 68 Gy, EQD2 69.5 Gy.
At the date of discharge (26 th April 2020) ENT examination revealed a clinical complete response.
The patient will perform a neck CT scan in July. Regarding these indications, we did not wait for the patient's recovery from the infection, while we respected the other recommendations.
The choice to continue RT treatment even during COVID 19 infection reflects the most recent indications of the ASTRO-ESTRO Consensus in which it emerged a strong agreement to continue RT in patients with SARS-CoV-2-related mild symptoms who had completed more than two weeks of treatment and an agreement to continue radiotherapy in patients with SARS-CoV-2-related mild symptoms, irrespective to the received treatment at that point. [bib_ref] Yom SSPractice recommendations for risk-adapted head and neck cancer radiotherapy during the..., Thomson [/bib_ref] We decided to omit the third cicle of concomitant chemotherapy because we considered it unsafe with the hypofractionated schedule choosed for RT.
Clinical assessment of patient's condition and rigid rules for radiotherapy staff and dedicated routes for this patient allowed to proceeding with treatment.
At this time, no clinical HNC-specific data on COVID-19 patients are available -but each choice requires an individualized risk/benefit assessment and a multidisciplinary agreement.
[table] Table 1: summarizes the results of blood exams. At the time of hospitalization, Xy had reached a radiotherapy (RT) dose of 50 Gy. The treatment was interrupted for two days to check for eventual evolution to a severe form of the Covid-19.In fact, it seems that cancer patients are at enhanced risk of serious morbidity, including the need for ventilator support or death (HR 3.56, [95% CI, 1.65 to 7.69])(2) [/table]
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Infantile hepatic hemangiomas associated with high-output cardiac failure and pulmonary hypertension
Background: Infantile hepatic hemangioma (IHH) is a rare endothelial cell neoplasm, which may be concurrent with severe complications and result in poor outcomes. Moreover, the coexistence of IHH and congenial heart disease is even rarer. Case presentation: We present a 10-day-old male born with IHH associated with patent ductus arteriosus (PDA), atrial septal defect (ASD) and pulmonary hypertension. Moreover, we reviewed a series of studies of IHH-associated high-output cardiac failure between 1974 and 2018, and summarized the treatment outcomes. Conclusions: Infantile hepatic hemangioma (IHH) has been known to induce high-output heart failure. There is no literature to summarize the severity of its impact on heart, which can lead to a high mortality rate. When IHH is detected by ultrasound, the heart should be evaluated to facilitate treatment. The outcomes of IHH associated with heart failure are good.
# Background
Infantile hepatic hemangioma (IHH) is a rare proliferative endothelial cell tumor. It appears to be a benign tumor, however, it may lead to poor outcomes because of severe complications such as congestive heart failure (CHF), which occurs in 15% of infants with this disease. More rarely, IHH is concurrent with congenital heart disease (CHD). Herein we present a patient with IHH, patent ductus arteriosus (PDA), and atrial septal defect (ASD). Meanwhile, we review and summarize the injury of IHH on the heart, and related outcomes.
## Case presentation
A 10-day-old boy was born at 38 weeks' gestation and had tachypnea at 65 breaths per minute. The liver margin was palpable 4 cm below the left costal margin. The heart rhythm was normal but a grade II-IV systolic murmur could be heard. Laboratory tests showed that his brain natriuretic peptide (BNP) level was greater than 5000 pg/ml. His CKMB was 110 U/L, C-reactive protein was 23.4 mg/L, alpha fetoprotein was greater than 1210 ng/ml, PTA 54%, INR 1.6, APTT 50 s, FIB 1.5 g/l, Ddimer 1064 μg/L, HGB, 123 g/L, MCH 36.4 pg, total bilirubin (BilT)196.7 umol/L, BilD 13.2 umol/L, ALT 44 U/L, and AST 23 U/L. Blood gas analysis revealed that the carbon dioxide pressure was raised to 53.4 mmHg and the oxygen partial pressure was 62.5 mmHg.
Echocardiography demonstrated a small PDA of 1.5-1.8 mm with right to left shunting, a large ASD with left to right shunting, and severe right atrial and right ventricular enlargement. A severe systolic pressure gradient of 70 mmHg suggested marked elevation of pulmonary artery pressure at the near systemic level.Color Doppler scanning of the liver displayed abundant blood flow in the lesion.The left hepatic vein was dilated to 8 mm with two great branches feeding the mass.The right hepatic artery and a branch arising from the abdominal aorta were also in close association with the lesion.
Contrast-enhanced computed tomography (CT) showed that the lesion enhanced irregularly in the left lobe of the liver.It was irregularly hypodense on plain scan with peripheral enhancement in the arterial phase and centripetal fill-in in the portal venous phase. In the delayed phases, the density of the leision was slightly higher than that of the liver parenchyma. The patient received diuretic therapy, fluid restriction, lowflow oxygen, and infection control for the management of the heart failure. After treatment his breathing difficulty improved. Then the patient was transferred to Beijing Children's Hospital for surgery and the adhesion
# Discussion and conclusions
IHH is an endothelial cell neoplasm, a benign tumor, which is usually clinically silent and slowly progressive during childhood. Although almost all are asymptomatic, a small subset can produce high-output cardiac failure and cause considerable mortality. Its pathologic changes are similar to that of hepatic artery to hepatic vein or hepatic artery to portal vein arteriovenous fistula. The prognosis of the disease is poor when complications present and the mortality rate can be as high as 90%. We reviewed a series of recent studies of IHH-associated high-output cardiac failure between 1974 and 2018, and summarized the treatment outcomes.
Heart failure was obviously a clinical relevant complication in the 25 cases which were presented in[2,. The age of diagnosis varied ranging from 1 day to 3.5 years. Eight patients (28%) presented with pulmonary hypertension including two mild, one moderate, three severe, and two unknown. Two of them were also associated with other congenital cardiac malformations which were illustrated in detail in. Cardiac function improved after treatment in 15 patients, achieving normal value in 2. Consequently the outcomes of IHH-associated high-output cardiac failure proved to be quite satisfying. Most of the patients discharged or achieved remarkable improvement through appropriate treatment, while only 4 of them failed to survive.
IHH can be classified as focal, multifocal, or diffuse. The diagnosis of IHH relies on ultrasonography (US), CT, and magnetic resonance imaging (MRI). CT and MRI can reveal discrete lesions in patients. Diffuse lesions require baseline determination of size, cardiac and thyroid function, and coagulation profile. When IHH is associated with heart disease, cardiac structure and function can be observed by echocardiography, which can identify intra-or extracardiac disease. There were 13 (52%) patients in our series that were diagnosed by echocardiography. In our case, the patient had a focal lesion, which is diagnosed primarily by ultrasonography and CT.
The pathological mechanism of CHF in IHH is associated with arteriovenous shunts in hemangiomas.
The arteriovenous shunts result in a decrease of systemic blood volume as well as increase of pulmonary blood volume, thus leading to the cardiac output increase. Furthermore, aggravated by the pulmonary hypertention it finally leads to high-output CHF. In fetal stage, high pulmonary vascular resistance and pulmonary pressure help maintain the fetal circulation, however, after birth the high pulmonary pressure will descend gradually within 3 months while systemic pressure of neonates will ascend with closure of oral foramen. Nevertheless, the existence of IHH can increase the load of right heart system and affect circulation transition from fetus to neonate, which further increase pulmonary vascular resistance and cause pulmonary hypertention. Various therapies have been reported to treat IHH, including drugs, embolization, ligation, and resection. (1) Drugs: steroid therapy functioned well in improving hemodynamics, reducing hepatic vascularity as well as deferring early emergency delivery in congestive heart failure fetus as recorded in literatures. There are no significant differences between single or combined drug use in the literature. (2) Embolization: embolization has been strongly suggested for provisional stabilization of fatal congestive heart failure combined with pharmacological therapy. (3) Ligation: ligation can reduce oxygen supply to hepatocytes and improve liver function. (4) Resection: surgery should be considered when medical management failed.Treatment methods and their outcomes as described in the literature are shown in. Among 25 patients, four ended in treatment failure (18%).
Early age of onset is typical of IHH with heart failure. When IHH is detected by US, echocardiography should also be performed timely for more detailed information about cardiac structure and function. For infants in lifethreatening and complicated conditions, US and echocardiography should be performed as early as possible to evaluate IHH associated with congestive heart failure and to facilitate treatment therapies. With regard to the treatment in our review, outcomes of IHH with heart failure are considered to be good. Availability of data and materials All data that was generated or analyzed during the current study are available from the corresponding author on reasonable request.
## Ethics approval and consent to participate
The study has been approved by the ethics committee of Shengjing Hospital of China Medical University, Shenyang, China. Consent for publication Written informed consent was obtained from the parents of the patient for publication of this case report and any identifying images. |
Automatic recognition of uterine contractions with electrohysterogram signals based on the zero-crossing rate
Uterine contraction (UC) is an essential clinical indicator in the progress of labour and delivery.Electrohysterogram (EHG) signals recorded on the abdomen of pregnant women reflect the uterine electrical activity. This study proposes a novel algorithm for automatic recognition of UCs with EHG signals to improve the accuracy of detecting UCs. EHG signals by electrodes, the tension of the abdominal wall by tocodynamometry (TOCO) and maternal perception were recorded simultaneously in 54 pregnant women. The zero-crossing rate (ZCR) of the EHG signal and its power were calculated to modulate the raw EHG signal and highlight the EHG bursts. Then the envelope was extracted from the modulated EHG for UC recognition. Besides, UC was also detected by the conventional TOCO signal. Taking maternal perception as a reference, the UCs recognized by EHG and TOCO were evaluated with the sensitivity, positive predictive value (PPV), and UC parameters. The results show that the sensitivity and PPV are 87.8% and 93.18% for EHG, and 84.04% and 90.89% for TOCO. EHG detected a larger number of UCs than TOCO, which is closer to maternal perception. The duration and frequency of UC obtained from EHG and TOCO were not significantly different (p > 0.05). In conclusion, the proposed UC recognition algorithm has high accuracy and simple calculation which could be used for real-time analysis of EHG signals and long-term monitoring of UCs.OPEN
Uterine contraction (UC) is the result of the electrical activity within the myometrium. As labour approaches, UCs become more intense and synchronized, eventually expel fetus [bib_ref] Accuracy of frequency-related parameters of the electrohysterogram for predicting preterm delivery, Lucovnik [/bib_ref] [bib_ref] Uterine electromyography characteristics for early diagnosis of mifepristone-induced preterm labor, Doret [/bib_ref] [bib_ref] Electrical and mechanical activity of the human uterus during labour, Wolfs [/bib_ref]. Therefore, UC monitoring provides essential clinical indicators in the progress of labour and delivery [bib_ref] Electrohysterography in the diagnosis of preterm birth: a review, Garcia-Casado [/bib_ref] [bib_ref] A validation of electrohysterography for uterine activity monitoring during labour, Jacod [/bib_ref] , especially for women at the risk of preterm delivery.
Manual palpation, which identifies UC by palpating the parturient's abdomen over the uterine, needs constant bedside presence which adds extra work for clinicians [bib_ref] Clinical assessment of uterine contractions, Cohen [/bib_ref]. The pregnant women can feel UCs but may not record them in time. External tocodynamometry (TOCO) is the most widely used technique to monitor uterine activity during pregnancy and delivery [bib_ref] The impact of maternal body mass index and gestational age on the..., Aina-Mumuney [/bib_ref]. However, TOCO is affected by the tightness of the belt and transducer position on the maternal abdomen [bib_ref] Electrical and mechanical activity of the human uterus during labour, Wolfs [/bib_ref]. Additionally, it is susceptible to motion artifacts and unreliable in obese women [bib_ref] Monitoring uterine activity during labor: a comparison of 3 methods, Euliano [/bib_ref]. Intrauterine pressure catheter (IUPC) directly measures the intrauterine pressure changes caused by UCs but is limited by its invasiveness which can cause ruptured membranes and infection [bib_ref] A comparison between electrical uterine monitor, tocodynamometer and intra uterine pressure catheter..., Hadar [/bib_ref].
Electrohysterogram (EHG) is a promising noninvasive technology considered to facilitate external uterine monitoring. With electrodes placed on the maternal abdomen, EHG can provide an objective evaluation of the myometrium activity by measurement of biopotentials underlying UCs [bib_ref] Clinical use of electrohysterography during term labor: a systematic review on diagnostic..., Vlemminx [/bib_ref]. EHG is more reliable and similar in accuracy to TOCO in detecting UCs compared to IUPC [bib_ref] Accuracy and reliability of uterine contraction identification using abdominal surface electrodes, Hayes-Gill [/bib_ref] and is applicable for long-term monitoring uterine activity throughout pregnancy and delivery. The EHG signal can be modelled as an action potential fast wave whose amplitude is modulated by the slow wave corresponding to the frequency of the contractions. During pregnancy, EHG signals are characterized by a slow cyclic pattern of bursts of action potentials followed by a period of quiescence [bib_ref] Propagation of electrical activity in uterine muscle during pregnancy: a review, Rabotti [/bib_ref]. It has been reported that the bursts of action potential spikes occurred in both intrauterine and abdominal electrical signals synchronized with the UCs identified by simultaneously recorded intrauterine www.nature.com/scientificreports/ pressure 13 . The slow wave expressed by the envelope of burst has an essential meaning for the analysis of uterine activity, and its amplitude is affected by various conditions [bib_ref] Quantitative analysis of contraction patterns in electrical activity signal of pregnant uterus..., Jezewski [/bib_ref] [bib_ref] Characterization of abdominally acquired uterine electrical signals in humans, using a non-linear..., Maner [/bib_ref]. Therefore, EHG burst associated with UC could be detected, and its envelope of slow wave is described to replace the routine clinical TOCO for long-term ambulatory uterine monitoring. Our team has explored the decision tree 15 and the convolutional neural network [bib_ref] Evaluation of convolutional neural network for recognizing uterine contractions with electrohysterogram, Hao [/bib_ref] to recognize EHG segments of UCs and non-UCs. However, to train these classifiers, EHG signals corresponding to UCs and non-UCs have to be segmented elaborately by manual referring to TOCO 17 . Moreover, a large number of EHG segments are required to improve the performance of these classifiers.
Nonlinear correlation analysis for burst extraction has been combined with fusion and elimination tests 18 or Gaussian mixture models to detect UCs. These approaches allowed to detect a great majority of the contractions but also many artifacts. The false detection rate is also high, which could be due to the incomplete reference from manual segmentation [bib_ref] An automatic method for the segmentation and classification of imminent labor contraction..., Tylcz [/bib_ref]. Recently, unsupervised clustering method was applied to classify different types of UCs using complete spectrum projection of EHG signals [bib_ref] Uterine contractions clustering based on electrohysterography, Esgalhado [/bib_ref]. Granger causal analysis of contraction and noncontraction was performed to extract EHG features. It was subsequently utilized by a quadratic discriminator classifier, which achieved high discriminatory power between term and preterm births [bib_ref] Granger causal analysis of electrohysterographic and tocographic recordings for classification of term..., Saleem [/bib_ref]. A deep neural network was developed for semi-automatic identification of term and preterm uterine recording [bib_ref] Deep neural network for semi-automatic classification of term and preterm uterine recordings, Chen [/bib_ref]. Although the good performance was achieved, manual segmentation contraction intervals and dummy intervals were required.
As a reliable and straightforward approach for on-line analysis, the root mean square (RMS) of the EHG signal within a sliding window was calculated to extract slow wave [bib_ref] Computer analysis of mechanical and electrical uterine activity, Ramondt [/bib_ref]. Besides, the method of higher-order zero crossings was proposed to discriminate between UC segments which relied upon the conclusion that the number of zero-crossings determined in a contraction segment significantly differs from a non-contraction segment [bib_ref] A fast algorithm for detecting contractions in uterine electromyography, Rudhakrishnan [/bib_ref]. The comparison study between EHG and TOCO showed that both methods demonstrate high agreement concerning the number of UCs recognized 13 . However, another paper reported that EHG was able to detect a higher number of UCs than TOCO identified by the experts with appropriate electrophysiological training [bib_ref] Computer analysis of mechanical and electrical uterine activity, Ramondt [/bib_ref]. Most studies compared their results with TOCO, which is not quite acceptable by obstetricians.
The duration and interval of UCs are not consistent at different gestational weeks (GWs) or even at the same recording. Automated detection of UCs is of great help for clinical evaluation and understanding the physiological activity of uterus during pregnancy. Most of the published papers focused on the use of pattern recognition techniques to extract features from EHG signals and evaluates various classifiers for detecting term and preterm delivery [bib_ref] Granger causal analysis of electrohysterographic and tocographic recordings for classification of term..., Saleem [/bib_ref] [bib_ref] Deep neural network for semi-automatic classification of term and preterm uterine recordings, Chen [/bib_ref] [bib_ref] Advanced artificial neural network classification for detecting preterm births using EHG records, Fergus [/bib_ref] [bib_ref] Dynamic neural network architecture inspired by the immune algorithm to predict preterm..., Hussain [/bib_ref] [bib_ref] Classification for uterine EMG signals: comparison between AR model and statistical classification..., Diab [/bib_ref]. However, manual UC segmentation is usually required for reference, which affects the classification accuracy. Besides, more data from the various dataset are in demand to test and improve the current methods. We should also notice that on-line analysis limits the complexity of the automatic recognition algorithm [bib_ref] Prognostic value of chosen parameters of mechanical and bioelectrical uterine activity in..., Zietek [/bib_ref]. Therefore, a fast and efficient algorithm is necessary to detect UCs with EHG signals in real-time.
In this paper, we propose a method for automatic recognition of UCs with EHG signals. ZCR related algorithm is used to highlight the EHG burst of UC, and its envelope is extracted to obtain TOCO-like signals for the convenience of clinicians. UCs from EHG signals are compared with maternal perception and TOCO. The number, duration, frequency and interval of UCs are provided for long-term monitoring of uterine activities and prediction of abnormalities in time. [fig_ref] Table 1: UC recognition results [/fig_ref] , 451 UCs were labelled with an average of 8 ~ 9 UCs for each pregnant woman during the 30-min recording. The number of UCs recognized by EHG is larger than TOCO, which is closer to maternal perception. Besides, the number of UCs that were correctly recognized (TP) by EHG is larger than TOCO. While the number of UCs and non-UCs that were falsely recognized (FP and FN) by EHG are smaller than TOCO. Both the sensitivity and PPV of EHG are higher than TOCO. shows the UC parameters obtained from EHG and TOCO. No significant difference was found in the UC parameters between EHG and TOCO (p > 0.05). www.nature.com/scientificreports/ [fig_ref] Table 3: UC parameters from preterm and term delivery [/fig_ref] shows the UC parameters of term and preterm delivery obtained from EHG and TOCO signals. No significant difference was found in the UC parameters between preterm and term delivery (p > 0.05), whether by EHG or by TOCO.
# Results
## Ucs recognition result. as shown in
## Results of preterm and term delivery.
The offset coefficient α. The offset coefficient α determined the degree to which the EHG signal was elevated, and therefore influenced the subsequent ZCR. As shown in [fig_ref] Figure 1: The influence of α on the recognition result [/fig_ref] , the sensitivity of UC recognition increases with α while PPV decreases with α. The score is the average of sensitivity and PPV. With the comprehensive consideration of the changes of sensitivity, PPV and score, we selected α between [bib_ref] Accuracy of frequency-related parameters of the electrohysterogram for predicting preterm delivery, Lucovnik [/bib_ref] [bib_ref] Uterine electromyography characteristics for early diagnosis of mifepristone-induced preterm labor, Doret [/bib_ref].
The exponent γ of ZCR power function. The exponent γ affected the ratio of UC to non-UC and the subsequent UC recognition. As shown in [fig_ref] Figure 2: The influence of γ on the recognition result [/fig_ref] , the sensitivity of UC recognition increases with γ while PPV decreases with γ. The score is the average of sensitivity and PPV. With the comprehensive consideration of the changes of sensitivity, PPV and score, we selected γ between [bib_ref] Electrical and mechanical activity of the human uterus during labour, Wolfs [/bib_ref] [bib_ref] Electrohysterography in the diagnosis of preterm birth: a review, Garcia-Casado [/bib_ref].
# Discussion
UCs are routinely and constantly monitored during pregnancy and delivery. The amplitude and frequency of UCs varied widely during the gestation period, and thus quantitative assessment of UCs can guide obstetricians to choose appropriate therapies. EHG signals have been investigated to monitor uterine activities in recent years. However, EHG signals are usually manually segmented for identifying UCs. This paper proposed a novel www.nature.com/scientificreports/ algorithm for automatic recognition of UCs from EHG signals without manual segmentation or referring to TOCO signal. With easy and fast on-line analysis, it provides TOCO-like signals acceptable to obstetricians and could be employed to monitor UCs clinically. The number of zero crossings has been shown to be related to the frequency content of the EHG signal, and the zero-crossings determined in UC segments significantly differ from the non-UCs 30 . However, zero-crossingbased techniques have not been further validated in subsequent studies. In this paper, ZCR was extracted to represent the variation of the EHG signal or the frequency content. Considering the amplitude information, we utilized the power of ZCR to modulate the EHG signal to reinforce the difference between the segments of UC and non-UC. Then, the envelope of EHG burst was achieved to recognize UC, and consequently, the parameters of UC were obtained.
We noticed most of the studies on UC detection with EHG signals compared their results with IUPC or TOCO [bib_ref] Automated electrohysterographic detection of uterine contractions for monitoring of pregnancy: feasibility and..., Muszynski [/bib_ref] [bib_ref] Electrohysterography for uterine monitoring during term labour compared to external tocodynamometry and..., Vlemminx [/bib_ref]. Although IUPC can provide accurate information of UCs, it is seldom applied to normal pregnant women in the clinical practice, and therefore the data from IUPC is quite limited. TOCO is more susceptible to the interference from maternal movement or shifting of the abdominal transducer, particularly unreliable in obese parturients. Maternal perception can also indicate UCs, which is first used as a reference in this study. Mostly, pregnant women can feel abdominal pain and tightness when UCs are coming. Even though not all UCs can be labelled by the pregnant women due to the increasing pain during the recording period, at least all the UCs labelled by the pregnant women are convincing and therefore could be taken as an evaluation criterion for UC recognition. To the best of our knowledge, only our team has investigated the time difference between the onset of UCs determined from TOCO and maternal perception [bib_ref] Comparison of the onset of uterine contractions determined from tocodynamometry and maternal..., Wang [/bib_ref]. The maternal perception can suggest a UC but not the onset, the peak or the end of a UC. Therefore, a UC identified by EHG burst or TOCO within a time interval to maternal perception could be regarded as a true UC. Once a UC was determined, its number, duration, interval and frequency could be obtained, which are indispensable parameters for clinical monitoring.
The proposed algorithm in this paper shows higher sensitivity and PPV in detecting UC than TOCO, the convention method of UC detection. The number of UCs detected with EHG is closer to maternal perception. Also, the published papers reported EHG presents higher consistency to IUPC than TOCO for monitoring uterine activities 9,32 . The UC parameters in this paper are similar to the published results [bib_ref] Clinical assessment of uterine contractions, Cohen [/bib_ref] [bib_ref] Quantitative analysis of contraction patterns in electrical activity signal of pregnant uterus..., Jezewski [/bib_ref] and are clinically reasonable. Besides, they are not significantly different between EHG and TOCO. For automatic UC detection, our results achieved by simple calculations are comparable to the algorithm of nonlinear correlation coefficient [bib_ref] Automated electrohysterographic detection of uterine contractions for monitoring of pregnancy: feasibility and..., Muszynski [/bib_ref].
UC parameters from both preterm and term delivery obtained by EHG and TOCO were reported in this study. However, UC parameters were not significantly different between preterm and term delivery, which could be due to fewer EHG recording from preterm delivery.
In a further study, more clinical data from various GWs and preterm delivery have to be collected to validate the proposed algorithm. Maternal perception has to be confirmed by obstetrician's palpation at the bedside to annotate UCs thoroughly.
In summary, with the advantage of high accuracy and simple calculation, the ZCR-based automatic UC recognition algorithm with the EHG signal is convincing. It has tremendous application potential for real-time and long-term UC monitoring.
# Methods
As shown in [fig_ref] Figure 3: Block diagram of recognition and evaluation of UCs with EHG and TOCO [/fig_ref] , the block diagram of automatic UC recognition includes EHG signal recording, EHG signal preprocessing, calculation of zero-cross rate (ZCR), calculation of the power of ZCR and envelope extraction of the modulated EHG. UCs were recognized by the modulated EHG and TOCO signals, respectively, and compared with maternal perception in terms of positive predictive value (PPV) and sensitivity. UC parameters, including number, frequency, duration and interval, were obtained from EHG and TOCO signals. [fig_ref] Table 4: Basic information of pregnant women [/fig_ref] , a total of 54 pregnant women with 33 to 41 GWs were recruited in Beijing Union Medical College Hospital in China. Four of them were preterm delivery (delivered before 37 GWs), and the others were term delivery (delivered after 37 GWs). The age, height, weight, average GWs, minimum GWs and maximum GWs of preterm and term pregnant women were given in [fig_ref] Table 4: Basic information of pregnant women [/fig_ref]. The study was approved by the Local Ethics Committee of Beijing Union Medical College Hospital and was conducted strictly according to the Declaration of Helsinki (1989) of the World Medical Association. The pregnant women were asked to sign consent after being informed of the aims, potential benefits and risks of the study.
## Ehg recording. as shown in
Eight-channel EHG signals and a TOCO signal were recorded simultaneously using a bespoke device in our lab. As shown in [fig_ref] Figure 4: The configuration of the 8-electrodes on the abdomen [/fig_ref] , electrodes 1 to 4 were placed on the fundus of the uterus, electrodes 5 and 6 symmetrically placed below the navel, electrodes 7 and 8 were placed on the uterine cervix, and the reference (R) and ground (G) electrodes were placed on the left and right ilium. The EHG and TOCO signals were sampled at 250 Hz for approximately 30 min. The pregnant woman pressed a button to label a UC she felt during the signal recording. Meanwhile, a researcher has been staying at the bedside to write UCs in a notebook during the recording. The UCs labelled by both of them were adopted for analysis. EHG signal preprocessing. The 8-electrode EHG signals were averaged and then preprocessed by a 4thorder Butterworth low-pass filter with the cut off frequency of 3 Hz, and then a median filter to remove the unwanted signals from maternal electrocardiograph, respiration, movement and power line interference.
Zero-cross rate of EHG signal. Each burst of EHG signal has been known to be a period of elevated electrical activity associated with a UC [bib_ref] Characterization of abdominally acquired uterine electrical signals in humans, using a non-linear..., Maner [/bib_ref]. The amplitude of the EHG signal is often influenced by electrode placement, GWs, as well as the individual difference. Therefore, the EHG amplitude and power spectral can- www.nature.com/scientificreports/ www.nature.com/scientificreports/ not be applied directly to evaluate UCs from different pregnant women. Only the variation of amplitude-based parameters is valuable in clinical application [bib_ref] Quantitative analysis of contraction patterns in electrical activity signal of pregnant uterus..., Jezewski [/bib_ref]. ZCR is the rate of sign-changes at which the signal changes from positive to zero to negative or from negative to zero to positive. ZCR is often used to extract the signal feature and has some relationship with frequency, which is useful in analyzing various physiological signals [bib_ref] Comparison of non-invasive electrohysterographic recording techniques for monitoring uterine dynamics, Alberola-Rubio [/bib_ref].
In this paper, ZCR was obtained by elevation of EHG amplitude and calculation in a sliding window.
Elevation of EHG signal. The EHG signal was elevated αE to highlight the EHG burst and reduce the impact of the non-UC segment. α is the offset coefficient and E is the mean of EHG amplitude. E was calculated by the formula (1).
where x(i) represents the EHG amplitude at the point i, n represents the length of the EHG signal in point.
The offset coefficient α ∈ [0, 5] was selected in this study, which was the tradeoff between retaining useful information and reducing interference.
Calculation of zero-crossing rate within a sliding window. A sliding window was applied to the elevated EHG signal with the moving step of 1 sampling point. ZCR within the window was calculated by the formula [bib_ref] Uterine electromyography characteristics for early diagnosis of mifepristone-induced preterm labor, Doret [/bib_ref].
where m is the number of zero-crossing points within the sliding window, and W is the length of the window.
Generally, a UC duration of 30-60 s was reported in the previous research [bib_ref] Comparison of non-invasive electrohysterographic recording techniques for monitoring uterine dynamics, Alberola-Rubio [/bib_ref]. Here, the length of the sliding window was set to 40 s to cover an EHG segment of UC. With the sampling rate of 250 Hz, the length of the sliding window was W = 10,000 points. shows the zero-crossing points in red and the sliding window in blue. b shows the ZCR curve, which the higher ZCR, the more uterine activity.
Power of the zero-crossing rate. The ZCR value was normalized to [0, 1] to overcome individual differences. Then, γth (γ > 1) power of ZCR or ZCR γ was calculated to strengthen the larger ZCR and attenuate the smaller ZCR, see . The power of ZCR was applied to modulate the preprocessed EHG signal to improve the ratio of UC to non-UC segments.
Recognition of UCs with the modulated EHG. As shown in ,b, the preprocessed EHG signal was modulated by ZCR γ to strength the bursts corresponding to UCs, and attenuate the EHG segments corresponding to non-UCs. Then the envelope of the modulated EHG signal was obtained by RMS, which is simple and applicable for on-line analysis. RMS was calculated by the formula (3).
where x(i) represents the amplitude of the modulated EHG signal at the point i, N stands for the length of 10 s EHG segment, here N = 2500. In this study, when the window length was 10 s (N = 2500), the envelope waveform obtained was the most similar to EHG signal.
A UC was identified if its amplitude was larger than the threshold Tha and its duration was larger than the threshold Thd, where Tha is the mean amplitude of EHG envelop, and Thd is 30 s in terms of UC duration. As shown in , a UC was correctly identified if its peak position in dotted line was within 20 s interval to the maternal perception in the dash line. , TOCO wave was smoothed by averaging multiple points in 40 s, then a UC was recognized with a similar method as EHG envelope.
## Recognition of ucs with toco. as shown in
Evaluation of UC recognition. In this paper, we assumed that the UCs labelled by maternal perception are reliable. All of the UCs obtained from EHG and TOCO were evaluated by their intervals to maternal perception. Only sensitivity and PPV were applied to evaluate the UC recognition results because the pregnant woman labelled not all UCs due to the acute pain during signal recording. The sensitivity and PPV were calculated by the formula (4) and (5): Calculation of UC parameters. The number of UCs during the recording period and within 10 min were counted respectively. The interval of UCs was defined as the time interval between the adjacent UC peaks. The duration of UCs was defined as the half-wave width of the envelope, as shown in [fig_ref] Figure 7: The definition of UC duration [/fig_ref].
[formula] (1) E = n−1 i=0 x(i) n (2) Z = m W × 100% (3) RMS = 1 N N−1 i=0 x(i) 2( [/formula]
Statistical analysis. The non-parametric t-test (Mann-Whitney U test) was performed using SPSS 22 (IBM Corporation, New York, United States) to assess the difference of UC parameters between EHG and TOCO, and between preterm and term delivery. P < 0.05 was considered statistically significant.
Ethical approval. The study was approved by the Local Ethics Committee of Beijing Union Medical College Hospital and was conducted strictly according to the Declaration of Helsinki (1989) of the World Medical www.nature.com/scientificreports/
[fig] Figure 1: The influence of α on the recognition result. [/fig]
[fig] Figure 2: The influence of γ on the recognition result. Scientific Reports | (2021) 11:1956 | https://doi.org/10.1038/s41598-021-81492-1 [/fig]
[fig] Figure 3: Block diagram of recognition and evaluation of UCs with EHG and TOCO. [/fig]
[fig] Figure 4: The configuration of the 8-electrodes on the abdomen. Scientific Reports | (2021) 11:1956 | https://doi.org/10.1038/s41598-021-81492-1 [/fig]
[fig] Figure 5, Figure 6: Strengthening of EHG burst. (a) A sliding window moving along the elevated EHG signal. The zero-crossing points are in red, and the sliding window is in blue. (b) The zero-crossing rate. (c) Power of zerocrossing rate. Recognition of UCs with EHG and TOCO. (a) Preprocessed EHG signal. (b) The modulated EHG signal. (c) Recognition of UCs with EHG envelope. (d) Recognition of UCs with TOCO signal. [/fig]
[fig] Figure 7: The definition of UC duration. Scientific Reports | (2021) 11:1956 | https://doi.org/10.1038/s41598-021-81492-1 [/fig]
[table] Table 1: UC recognition results. N/A: not available.Table 2. UC parameters obtained from EHG and TOCO. [/table]
[table] Table 3: UC parameters from preterm and term delivery. [/table]
[table] Table 4: Basic information of pregnant women. SD: standard deviation. [/table]
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An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition
Background: The evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents. Methods: One fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT) was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D) under varying conditions and the outcome of competition measured over 14 days by flow cytometry. Results: We demonstrate that ascorbic acid (vitamin C) can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus. Conclusions: This model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.
# Background
Cancer progression is an evolutionary process by which heterogeneous populations of neoplastic clones compete with each other and normal cells for space and resources [bib_ref] Cancer as an evolutionary and ecological process, Merlo [/bib_ref]. All interventions, whether preventive or therapeutic, are attempts to perturb this process of clonal evolution. Ultimately, if a treatment kills or disrupts neoplastic cells, some cell type must grow back in their place. Our interventions are implicit attempts to bias this competition in favor of normal cells. Successful prevention and therapeutic interventions can modulate the dynamics of competition in one of two ways, either 1) neoplastic cells may be negatively affected by a therapy or intervention, thus reducing the competitive advantage of these cells relative to normal cells. Most traditional interventions employ this strategy of reducing the fitness of neoplastic cells by killing or preventing proliferation. Alternatively, 2) the "normal" cells may gain a competitive advantage from a mitogen or survival factor added to the neoplastic environment that differentially affects cell fitness, allowing the normal cells to outcompete the neoplastic cells, a strategy we refer to as "benign cell boosters" [bib_ref] Cancer prevention strategies that address the evolutionary dynamics of neoplastic cells: simulating..., Maley [/bib_ref]. Computational models suggest this may be an effective strategy to harness clonal competition to prevent cancer [bib_ref] Cancer prevention strategies that address the evolutionary dynamics of neoplastic cells: simulating..., Maley [/bib_ref].
Clear documented examples of clonal expansion [bib_ref] Clonal expansion and loss of heterozygosity at chromosomes 9p and 17p in..., Galipeau [/bib_ref] [bib_ref] (INK4a) lesions are common, early abnormalities that undergo clonal expansion in Barrett's..., Wong [/bib_ref] [bib_ref] Many colorectal cancers are "flat" clonal expansions, Siegmund [/bib_ref] [bib_ref] Understanding the development of human bladder cancer by using a whole-organ genomic..., Majewski [/bib_ref] demonstrate that there is interaction and competition between heterogeneous clones within a neoplasm and those clones may displace normal cells in a tissue. Although competition between heterogeneous cell types is a fundamental property of progression and therapeutic intervention [bib_ref] Therapeutic perturbation of the tumor ecosystem in reconstructed heterogeneous mouse mammary tumors, Miller [/bib_ref] [bib_ref] Tumor subpopulation interactions affecting melphalan sensitivity in palpable mouse mammary tumors, Miller [/bib_ref] [bib_ref] Genetic and epigenetic heterogeneity in cancer: a genome-centric perspective, Heng [/bib_ref] , the mechanism of competition is incompletely understood and only a few studies [bib_ref] Interaction between two cellular subpopulations of a rat colonic carcinoma when inoculated..., Caignard [/bib_ref] [bib_ref] A primary tumor promotes dormancy of solitary tumor cells before inhibiting angiogenesis, Guba [/bib_ref] [bib_ref] Growth interaction in vivo between tumor subpopulations derived from a single mouse..., Miller [/bib_ref] have attempted to directly quantify the dynamics of competition between normal and neoplastic cells [bib_ref] Cell competition and its possible relation to cancer, Baker [/bib_ref].
Here, we define competition as interaction between two cell types such that the cell types exhibit behavior or dynamics when together that is not present when each cell type is grown alone. This is based on an ecological definition of competition, where the fitness of one population negatively affects the fitness of another, and can be the result of both changes in proliferative or death processes. Early work by Heppner and Miller demonstrated that subpopulations of mouse mammary tumor cells could affect each other's growth when reinjected into mice [bib_ref] Growth interactions between mammary tumor cells, Heppner [/bib_ref]. More recent studies of cell competition in cancer have found that cells containing a mutant tumor suppressor lgl or a mutant lgl-binding protein, mahj, can be competitively eliminated [bib_ref] Involvement of Lgl and Mahjong/VprBP in cell competition, Tamori [/bib_ref]. Indirect measures from human neoplasms suggest that oncogenic mutations may only increase clone relative fitness by 0.5% in clonal competition [bib_ref] Accumulation of driver and passenger mutations during tumor progression, Bozic [/bib_ref]. Transformed cells have also been found to exhibit different behavior when surrounded by normal cells compared to other transformed cells [bib_ref] Interactions between normal and transformed epithelial cells: Their contributions to tumourigenesis, Hogan [/bib_ref] [bib_ref] Characterization of the interface between normal and transformed epithelial cells, Hogan [/bib_ref] [bib_ref] Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of..., Kajita [/bib_ref]. In Drosophila, cells containing extra copies of the myc proto-oncogene can outcompete wild-type cells [bib_ref] dMyc transforms cells into super-competitors, Moreno [/bib_ref]. While there is certainly extensive interest in competition in cancer [bib_ref] Cell competition and its possible relation to cancer, Baker [/bib_ref] [bib_ref] Cell competition, Baker [/bib_ref] [bib_ref] Is cell competition relevant to cancer?, Moreno [/bib_ref] , cell competition plays an important role in other cellular systems, such as the developmental programme of Drosophila melanogaster [bib_ref] Drosophila myc regulates organ size by inducing cell competition, De La Cova [/bib_ref] [bib_ref] Steep differences in wingless signaling trigger myc-independent competitive cell interactions, Vincent [/bib_ref] [bib_ref] Soluble factors mediate competitive and cooperative interactions between cells expressing different levels..., Senoo-Matsuda [/bib_ref]. In cancer studies, most standard in vitro systems do not include normal cells or multiple neoplastic cell types and thus fail to model the process of competition that is the true target of our interventions. Here, we have developed a cell culture model system in which competition dynamics can be directly measured.
Barrett's esophagus (BE) provides an ideal model in which to test the evolutionary dynamics of competition. In an environment of chronic gastroesophageal reflux, in some patients, BE cells (specialized intestinal metaplasia) replace normal squamous tissue in the distal esophagus [bib_ref] Barrett's oesophagus and oesophageal adenocarcinoma: time for a new synthesis, Reid [/bib_ref]. Acid suppression alone is not sufficient to allow the squamous cells to outcompete Barrett's cells and cause regression, though the combination of wounding, via biopsies or ablation, along with acid suppression, can lead to regrowth of squamous tissue [bib_ref] Barrett's oesophagus and oesophageal adenocarcinoma: time for a new synthesis, Reid [/bib_ref] [bib_ref] Neosquamous epithelium does not typically arise from Barrett's epithelium, Paulson [/bib_ref] [bib_ref] History, molecular mechanisms, and endoscopic treatment of Barrett's esophagus, Spechler [/bib_ref]. BE is of clinical importance because it is the only known precursor of esophageal adenocarcinoma (EA) and is associated with a relative risk of esophageal adenocarcinoma of 30-125 compared to the general US population of similar age [bib_ref] Barrett's esophagus, dysplasia, and adenocarcinoma, Haggitt [/bib_ref]. However, the natural history of BE is typically nonprogressive, with the risk of progression to EA approximately 0.6-0.7%/year [bib_ref] Meta analysis: Cancer risk in Barrett's oesophagus, Thomas [/bib_ref] [bib_ref] The incidence of esophageal cancer and high-grade dysplasia in Barrett's esophagus: a..., Yousef [/bib_ref]. Thus, there may be an extensive time period in which to intervene in the process of progression to prevent EA and a unique opportunity for chemoprevention given that patients likely have BE for many years before EA develops. BE lacks a physiologically realistic animal model system for investigating potential chemopreventive agents, the best known being a surgical anastomosis model in the rat [bib_ref] Multilayered epithelium in a rat model and human Barrett's esophagus: similar expression..., Chen [/bib_ref] [bib_ref] Cytogenetic characterization and gene expression profiling in the rat reflux-induced esophageal tumor..., Bonde [/bib_ref] [bib_ref] Histopathologic evaluation of an animal model for Barrett's esophagus and adenocarcinoma of..., Buskens [/bib_ref] [bib_ref] Phenotype of columnar-lined esophagus in rats with esophagogastroduodenal anastomosis: similarity to human..., Su [/bib_ref] [bib_ref] Chemoprevention of esophageal adenocarcinoma by COX-2 inhibitors in an animal model of..., Buttar [/bib_ref]. Therefore, there is a need for a simple model system for screening compounds that may affect the establishment and progression of BE.
We have developed a sensitive in vitro model of competition between Barrett's esophagus and squamous esophageal cells that may be used to identify a new class of interventions, explicitly designed to modulate competition in favor of normal cells. Co-cultures of an esophageal squamous cell line with each of four BE cell lines were evaluated over 14 days for changes in the proportion of cells of each population under varying concentrations of the antioxidant vitamin C, as well as vitamin E and epidermal growth factor [bib_ref] Epidermal growth factor receptor kinase domain mutations in esophageal and pancreatic adenocarcinomas, Kwak [/bib_ref] [bib_ref] Gains and amplifications of c-myc, EGFR, and 20.q13 loci in the no..., Rygiel [/bib_ref]. In addition to exposure under normal cell culture growth conditions, we also examined the effect of daily acid pulses on vitamin C competitions to better replicate the reflux conditions of the lower esophagus in patients with BE. Results demonstrate that vitamin C reduces growth of BE cells relative to normal squamous cells, giving a competitive advantage to normal squamous cells at physiologically relevant concentrations of vitamin C. Under acidic conditions, the advantage conferred to normal cells by vitamin C is generally greater. This is consistent with recent epidemiological data suggesting that vitamin C may help prevent progression from Barrett's esophagus to esophageal adenocarcinoma [bib_ref] Meta-analysis of antioxidant intake and the risk of esophageal and gastric cardia..., Kubo [/bib_ref] [bib_ref] Dietary supplement use and risk of neoplastic progression in esophageal adenocarcinoma: a..., Dong [/bib_ref].
# Methods
## Cell lines
Four hTERT transformed Barrett's esophagus (BE) cell lines (gift of P. Rabinovitch, University of Washington, Seattle WA) were used in competition experiments. These cell lines, CP-A, CP-B, CP-C, and CP-D, were established from BE cells isolated from 4 different patients. The CP-A cell line (9p LOH, TP53 wt , 5q LOH) was established from an area of nondysplastic metaplasia and the CP-B (9p LOH, 17p LOH, TP53 mut ), CP-C (9p LOH, 17p LOH, TP53 mut ), and CP-D (9p LOH, 17p LOH, TP53 mut ) cell lines were established from areas of high-grade dysplasia. All 4 BE lines are aneuploid [bib_ref] Genetic analysis of long-term Barrett's esophagus epithelial cultures exhibiting cytogenetic and ploidy..., Palanca-Wessels [/bib_ref] [bib_ref] Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic..., Palanca-Wessels [/bib_ref]. Normal esophageal squamous cells that had been hTERT transformed (EPC2-hTERT) were also used (gift from A. Rustgi, University of Pennsylvania, Philadelphia PA). All BE cell lines were adapted to serum-free conditions in keratinocyte serum-free (KSF) medium supplemented with bovine pituitary extract and epidermal growth factor (EGF) (Invitrogen Corp. Carlsbad, CA) by serial passaging of cell lines into successively lower concentrations of serum until the cell lines survived without serum present. BE cell line identities were verified with the Identifiler PCR Amplification kit (Applied Biosystems).
## Cell labeling
Esophageal squamous cells (EPC2-hTERT) were transduced with an eGFP-expressing lentivirus (gift of M. Herlyn, Wistar Institute, Philadelphia, PA). Transduced cells were sorted twice by flow cytometry to purify eGFP expressing cells. The stability of the fluorescent tag was monitored throughout each experiment, and it was maintained at 100% of the original fluorescence for 14 days, the duration of competition experiments.
Labeled EPC2 cell lines were tested for evolutionary neutrality of the fluorescent label by competition between labeled and unlabeled populations of the EPC2 cells to confirm that the labeled cells do not generate a differential response to the mitogen of interest. We verified that there is no interaction between ascorbic acid and the eGFP fluorescent label (Additional File 1, [fig_ref] Figure 1: Cell culture competition [/fig_ref].
## Co-culture of squamous and be cells without acid
Competitions consisted of one unlabeled (non-fluorescent) BE cell line and one eGFP-labeled squamous cell line. These were seeded with a total of 2 × 10 5 cells (50% from each cell line) into a 60 mm plate (Greiner, USA Scientific). Initial seeding ratios were measured by flow cytometry. Cells were cultured in KSF medium (Invitrogen) with 50 U/mL penicillin and 50 ug/mL streptomycin supplemented with the agent of interest for 14 days, with media changes every 2-3 days. Vitamin C (ascorbic acid, Fisher Scientific) stocks were made every 5 days at 1000X the final concentration in PBS and filter sterilized (0.22 μm filter). Competitions were performed at 0, 50, 150, and 500 μM concentrations of ascorbic acid to approximate physiologically relevant conditions based on plasma and mucosal concentrations of this compound [bib_ref] A study to determine plasma antioxidant concentrations in patients with Barrett's oesophagus, Clements [/bib_ref] [bib_ref] Plasma and esophageal mucosal levels of vitamin C: role in the pathogenesis..., Fountoulakis [/bib_ref]. The effect of two other compounds, α-tocopherol (Sigma) and EGF (Gibco), was measured using an identical protocol (For methods, see Additional File 1, . Competitions were passaged by complete trypsinization every 3-4 days. 0.25% Trypsin (Gibco) was inhibited with addition of 250 mg/L soybean trypsin inhibitor (Gibco) in PBS. Trypsinized plates were examined by light microscopy to verify that no cells still adhered to the plate. For each experiment, there were 4 sampling points, starting 3-4 days after initial seeding. Competitions were reseeded with 20% of the cells after trypsinization. Flow cytometric analysis (Beckman Coulter EPICS XL flow cytometer) was performed on days 3, 7, 10 and 14 after seeding to discriminate between the fluorescent normal squamous cells and the non-fluorescent BE cells.
Acid Pulsing of co-cultured squamous and BE cells
Co-culture methods are as described above with the exception that for acid pulsed cells, competitions were pulsed for 6 minutes daily with pH 3.5 PBS at 37°C, rinsed twice with neutral pH PBS and fresh media was replaced in each plate. These competitions were reseeded with 15% of the cells after trypsinization and flow cytometric analysis (Beckman Coulter EPICS XL flow cytometer) performed on days 3, 7, 10 and 14 after seeding.
## Growth of cell lines in monoculture
For each of the 4 BE cell lines (CP-A, CP-B, CP-C, CP-D) and 1 esophageal squamous cell line (EPC2), 10,000 cells were seeded into a 6-well plate containing KSF media (Invitrogen) with or without 100 μM ascorbic acid. After 6 days of growth, cells were trypsinized and counted (Nucleocounter, New Brunswick Scientific).
## Immunofluorescence of proliferation and apoptosis
## Fixation of cells
Cells from competition experiments were fixed with 2% PFA in PBS for 20 minutes at room temperature, centrifuged at 485 × g for 5 minutes, and washed twice with PBS. PFA was made from a 16% stock solution (Electron Microscopy Sciences, Hatfield, PA) once per month and stored at 4°C in the dark. Fixed cells were stored in the dark at 4°C in PBS until analysis.
## Phospho-histoneh3
PHH3 was probed as a marker of proliferation. Fixed cells were blocked with 5% goat serum for 30 minutes at room temperature. After 2 rinses with a wash buffer of 0.1% saponin (Acros) in PBS, the primary antibody (Sigma H0412) was added to each sample at a dilution of 1:500 in the wash buffer. After 1 hour at room temperature, 3 washes were performed. The secondary antibody (goat anti-rabbit Alexa Fluor 488) was added at 1:100 dilution in wash buffer. This was followed by 3 more washes. Samples were analyzed by flow cytometry (Beckman Coulter EPICS XL flow cytometer). Postacquisition analysis was performed with Flowjo (Version 8.7.1).
## Active caspase-3
The cleaved, active form of Caspase-3 (Sigma C8487) was probed as a marker for apoptosis at a single time point at the end of the competition period. The protocol followed was the same as for PHH3, with a 1:500 dilution of the primary antibody and the same secondary antibody used at 1:100dilution. Samples were run on the flow cytometer (Beckman Coulter EPICS XL flow cytometer) and post-acquisition analysis was performed with Flowjo (Version 8.7.1).
## Ethidium homodimer-1
To measure total cell death, time lapse imaging of cells in monoculture and competition was performed using a nonspecific marker for cell death, ethidium homodimer-1 (Invitrogen). This assay has the advantage of measuring all forms of cell death, not just apoptosis. Competitions were established in 6-well plates using the methodology described above and 2.5 μM ethidium homodimer-1 added to the culture 24 hours following cell seeding. Immediately following the addition of the ethidium homodimer, plates were imaged using a Nikon TE300 inverted microscope equipped with a motorized XY stage and Environmental chamber (Temperature and CO 2 control surrounding entire microscope). Image Pro 6.2 was used for image acquisition and also controls the motorized stage, filter wheels, shutters, and lamp settings. Using the automated stage macro, twelve areas selected, 2 fields per well, locations in any X, Y, or Z direction saved. Two images from the same position in each well were taken every 15 minutes for a total of 48 hours with a Q-imaging retiga EX digital camera. Percent death statistics were calculated by manually tracing cell fates in each time laps experiment for 240-455 cells per condition.
# Statistical analysis
Statistical significance was evaluated using the R statistics software by a series of t-tests comparing the difference in proportion of BE cells between day 0 and day 14 at 0 μM and 500 μM Vitamin C. Using a Bonferroni correction for multiple testing, comparisons with a pvalue less than 0.00625 were considered significant.
# Results
## Competitions under varying levels of vitamin c
Four unlabeled BE cell lines (CP-A, CP-B, CP-C, and CP-D) were tested in co-culture competition [fig_ref] Figure 1: Cell culture competition [/fig_ref] against a stably-transduced eGFP-labeled normal esophageal squamous cell line (EPC2). The effect of vitamin C exposure varied between cell lines. There is a statistically significant reduction in BE cells at 500 μM ascorbic acid relative to the 0 μm treatment for CP-B and CP-C cell lines , demonstrating that the presence of Vitamin C gives the EPC2 squamous cells a relative competitive advantage. This trend is also apparent in the CP-A cell line , although this is not statistically significant under the conservative Bonferroni multiple testing correction. While we find that squamous cells more rapidly outcompete BE cells in the presence of vitamin C, this effect is often not dosedependent. The CP-B cell line shows a particularly noteworthy saturation effect in both the no-acid and acidpulsed competitions , 3B), with lower concentrations of ascorbic acid providing a greater competitive advantage to the squamous cells.
## Acid-pulsed competitions under varying levels of vitamin c
To better simulate the phenomenon of acid reflux associated with BE (a potential cause of the condition), competitions were exposed to pH 3.5 PBS for 6 minutes daily. With daily acid pulses, the difference between the 0 and 500 μM ascorbic acid treatments is generally magnified, with CP-A, CP-C, and CP-D cell lines all showing a decrease in BE cell lines with Vitamin C present . This effect occurred in a dose-dependent manner. No systematic effect of vitamin C on the CP-B cell line can be detected under acidic conditions. Again, it appears that high levels of vitamin C ameliorate the competitive advantage of the EPC2 cells compared with the BE cells as lower levels of vitamin C do show a substantial effect on competitions between EPC2 and CP-B cells . Generally, the addition of acid alone favors the BE cells, but the combination of acid and vitamin C results in conditions less favorable for BE cells and more favorable for squamous cells.
## Growth of cell lines in monoculture
Monoculture experiments [fig_ref] Figure 4: Effect of Vitamin C Exposure on Cell Number [/fig_ref] demonstrate that vitamin C reduces the net growth for both the squamous and Barrett's cell lines. It is important to note that the monoculture results alone do not explain the results of the competition. In monoculture, for example, the CP-C cell line grows as well or better than the EPC2 squamous line, but in competition the squamous cells quickly outcompete the CP-C cells. It is clear from examination of the monoculture data that the cell lines described here are not acting independently when grown in competition, therefore monoculture data does not accurately describe the co-culture environment. Cell culture competition models are able to capture interactions between different cell types in a way that a monoculture model cannot. This provides direct evidence that competition dynamics are present in the co-culture environment.
## Competitions under varying levels of vitamin e and egf
Competitions were also performed with varying levels of Vitamin E and EGF (Additional File 1, . EGF did not have a significant effect on the outcome of competition. In the case of vitamin E, very high levels of α-tocopherol strongly suppressed growth of both cell lines. Unexpectedly, the fitness of the squamous cells was more strongly suppressed by vitamin E than the BE cells with the end result that BE cells out-compete squamous cells at high concentrations of Vitamin E (Additional File 1, . Lower levels of Vitamin E did not affect the outcome of competition.
## Apoptosis and proliferation assays on vitamin c competitions
Cell culture competitions provide a window into the net growth of the cells under the conditions defined by the investigator, but they do not identify the Competition results. All four BE cell lines show a trend towards reduced proliferation with 500 uM Vitamin C compared to 0 uM controls, but the effect is only statistically significant for CP-B and CP-C cell lines when adjusted for multiple testing using a Bonferroni correction (p < 0.05/8 = 0.00625 required for significance). Mean (n = 3) ± standard error of the mean is shown.
## Mechanism of the fitness effect. while it is clear that vitamin c increases the overall proportion of squamous cells relative to barrett's cells, it is not clear if this is due to an increase in proliferation/survival of the squamous cells or a reduction in proliferation/survival in be cell lines.
To determine if the effects of vitamin C in the competition between EPC2 squamous cells and CP-C cells was due to changes in proliferation, differences in levels of phosphorylated serine 10 on histone H3 (PHH3) were measured by flow cytometry. Phosphorylated serine 10 on histone H3 is a marker of mitotic cells. Although the proportion of PHH3+ cells was extremely low, we were able to show reduced proliferation of the CP-C cells under 500 μM ascorbic acid conditions for two separate experiments [fig_ref] Table 1: CP-C Unlabeled BE cells vs [/fig_ref]. We did not find a systematic change in proliferation of EPC2 cells with ascorbic acid (data not shown). Several apoptosis assays (cleaved caspase-3, annexin-V+7AAD, TUNEL) were also performed; however, little apoptosis was detected in this system. Because the media which overlays the cells in the competitions is changed every 2-3 days, many apoptotic cells are likely lost as the competition progresses. The caspase-3 assays do hint at an increased apoptosis level for both EPC2 and CP-C cell lines but the total amount of apoptotic cells is very low (< 0.1%, Additional File 1, [fig_ref] Figure 4: Effect of Vitamin C Exposure on Cell Number [/fig_ref]. To overcome the limitation incurred by extrusion of apoptotic cells into the surrounding media, time lapse imaging of cells in competition in the presence of ethidium homodimer-1 was performed. No systematic differences in cell death in the presence of 500 μM vitamin C were detected, nor were there Acid-pulsed competition results. Competition experiments were subjected to daily acid pulsing to model gastric reflux in Barrett's esophagus. Under acid pulsing, CP-A, CP-C, and CP-D BE cell lines show a statistically significant reduction relative to normal squamous cells with 500 μM Vitamin C compared to 0 μM controls when adjusted for multiple testing using a Bonferroni correction (p < 0.05/8 = 0.00625 required for significance). CP-B cells show a reduction at lower concentrations of vitamin C but not at 500 μM. Mean (n = 3) ± standard error of the mean is shown. differences in total cell death between monoculture and competition (Additional File 1, .
We also confirmed that the effect was not due simply to serial passaging of the cells. We were initially concerned that if cell lines adhere to cell culture dishes at varying rates during reseeding, there might be differences in the lag time before exponential growth could resume that might account for differences seen between cell lines in competition. This was of particular concern with vitamin C, which can affect extracellular matrix components [bib_ref] The effect of cell-matrix interactions and aging on the malignant process, Labat-Robert [/bib_ref]. We found no significant differences in "lag time", the time between trypsinization and resumption of exponential growth, between the different cell lines following trypsinization and replating (data not shown).
# Discussion
We have developed a sensitive and robust in vitro model of the process of cellular competition, which is thought to drive the evolution of malignancy [bib_ref] Cancer as an evolutionary and ecological process, Merlo [/bib_ref]. The utility of our assay is demonstrated in proof of principle experiments with ascorbic acid, where we find that vitamin C increases the fitness of esophageal squamous cells relative to Barrett's esophagus cells.
The methodology for cell competition is based on bacterial batch culture competition models [bib_ref] Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000..., Lenski [/bib_ref] , in which cells are maintained in logarithmic growth and serially passaged. Human cell culture is more complicated than a simple bacterial batch culture system due to the substantially greater potential for interactions between cell lines via mechanisms such as diffusible growth factors, ECM interactions and contact inhibition, although these phenomena are certainly possible in bacterial systems as well [bib_ref] Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli, Rosenzweig [/bib_ref] [bib_ref] Chemical warfare between microbes promotes biodiversity, Czárán [/bib_ref].
The cell culture competition model allows us to capture the relative effect of a compound on 2 cell lines of interest simultaneously. It is this effect, rather than the absolute growth rate, that is most important physiologically as different cell types likely interact with one another. This phenomenon is of particular relevance in Barrett's esophagus, where there is a clear junction between Barrett's cells and normal squamous esophageal cells. It is important to note that in the experiments described here, the normal squamous esophageal cells outcompete the BE cells under almost all concentrations of vitamin C. Therefore, we are comparing the relative rate at which the squamous cells outcompete the BE cells under different concentrations of vitamin C. This baseline advantage of the squamous lines in this system is likely an artifact of the cell culture conditions used. Here, all cells are grown in the serum-free media most suitable for the growth of the normal esophageal squamous cells. The addition of serum in the media leads to increased relative fitness of BE cells compared to noserum controls in all cell lines tested. In 2 out of 3 cell lines, this increase is such that the BE cells outcompete the squamous cells as would be expected under in vivo conditions (Additional File 1, . Future models of competition might be developed with a more realistic microenvironment using three-dimensional organotypic cultures that include both extracellular matrix and stromal cells [bib_ref] Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in..., Andl [/bib_ref] [bib_ref] Stimulation of human colonic epithelial cells by leukemia inhibitory factor is dependent..., Kalabis [/bib_ref] [bib_ref] Life isn't flat: taking cancer biology to the next dimension, Smalley [/bib_ref].
The mechanism of the cancer preventive effect of vitamin C is thought to be via a reduction of reactive oxygen species (ROS). BE is associated with chronic inflammation, which can generate ROS, thought to contribute to the development of a variety of cancers. A meta-analysis and other recent studies have reported that intake of antioxidants such as vitamin C, E, and beta-carotene are inversely associated with risk of esophageal adenocarcinoma [bib_ref] Meta-analysis of antioxidant intake and the risk of esophageal and gastric cardia..., Kubo [/bib_ref] [bib_ref] Dietary supplement use and risk of neoplastic progression in esophageal adenocarcinoma: a..., Dong [/bib_ref]. While it is clear that there is some relationship between the concentration of antioxidants and BE [e.g. [bib_ref] A study to determine plasma antioxidant concentrations in patients with Barrett's oesophagus, Clements [/bib_ref] ], substantial further experimentation would be required to fully characterize vitamin C as a preventive agent. In general, patients with EA have been found to have lower intakes of antioxidants compared to control populations . Recent studies have shown that BE patients have significantly lower plasma levels of vitamin C (ascorbic acid), xanthophylls and other antioxidants [bib_ref] A study to determine plasma antioxidant concentrations in patients with Barrett's oesophagus, Clements [/bib_ref]. In addition to lower plasma levels, BE mucosa was found to contain significantly lower levels of vitamin C compared with matched normal squamous mucosa [bib_ref] Plasma and esophageal mucosal levels of vitamin C: role in the pathogenesis..., Fountoulakis [/bib_ref]. This reduced presence of exogenous antioxidants may enhance the effects of DNA-damaging oxygen radicals.
Reduction of vitamin C in patients with BE may come from both a diet low in antioxidants as well as a reduced absorption of vitamin C from the stomach [bib_ref] Meta-analysis of antioxidant intake and the risk of esophageal and gastric cardia..., Kubo [/bib_ref]. Vitamin C is unstable at non-acidic pH and patients on proton pump inhibitors, such as omeprazole, have more basic gastric fluid. In one study [53], individuals given 40 mg/day for 28 days of omeprazole had a 12.3% decrease in plasma vitamin C levels independent of dietary vitamin C intake, suggesting that there may be lowered bioavailability of vitamin C in patients currently prescribed proton pump inhibitors (PPI). It warrants further investigation whether the current standard of care for individuals with BE, PPI medication (with regular endoscopic surveillance), is promoting reduced levels of vitamin C and whether this may, in turn, promote expansion of the Barrett's segment.
We view these cell culture competition models as particularly useful for early screens of chemoprevention agents, particularly when there are few model systems available, as in the case of BE. Many drugs have subtle effects and this system allows for longitudinal measurement of the effects of various compounds on cell competition. Our model is able to integrate differential fitness effects on both normal and neoplastic cells, regardless of whether they act through decreasing the fitness of neoplastic cells or increasing the fitness of normal cells, and so may be useful for the discovery of new classes of drugs, such as benign cell boosters [bib_ref] Cancer prevention strategies that address the evolutionary dynamics of neoplastic cells: simulating..., Maley [/bib_ref]. In order to screen any large number of therapeutic agents, our model would have to be downscaled to run in 96or 384-well plates. This is a simple assay designed for an initial screen, performed on plastic in 2D culture, but it can provide preliminary data for more complex animal model testing and for exploration of epidemiologic results.
# Conclusions
We established a cell culture model that can capture the dynamics of competition between two interacting cell lines. Because it is this interaction between normal and neoplastic cells that is the basis for our cancer prevention and therapy interventions, this model can provide an initial screen of potential compounds. We show that ascorbic acid is a modulator of competition between esophageal squamous cells and Barrett's esophagus cells. The advantage of squamous cells relative to Barrett's cells is enhanced by the addition of acid pulses mimicking the reflux conditions of the distal esophagus.
# Additional material
[fig] Figure 1: Cell culture competition. Image of competition between Barrett's esophagus cell line CP-D (green) and normal squamous cell line EPC2 (red). Cells are grown until they begin to approach confluency and are then passaged to maintain logarithmic growth. Both cell lines are labeled here for imaging purposes, however, in standard competitions BE cell lines are unlabeled. Note that the green cells do not necessarily grow in contiguous patches, and so there must either be cell migration or a process of detachment and reattachment as those clones expand. The two cell types can interact both by physical contact and via secreted factors. [/fig]
[fig] Figure 4: Effect of Vitamin C Exposure on Cell Number. The squamous and Barrett's cell lines all show a reduction in average cell number with Vitamin C exposure compared to control conditions. Mean of n ≥ 6 ± standard error of the mean is shown. [/fig]
[table] Table 1: CP-C Unlabeled BE cells vs. EPC2 DsRed-labeled Squamous CellsExperiment Concentration Vitamin C Average CP-C % PHH3 + [/table]
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Role of Innate lymphoid Cells in Obesity and Insulin Resistance
Obesity, a growing chronic metabolic disease, greatly increases the risk of metabolic syndrome which includes type 2 diabetes, fatty liver and cardiovascular diseases. Obesity-associated metabolic diseases significantly contribute to mortality and reduce life expectancy. Recently, innate lymphoid cells (ILCs) have emerged as crucial regulators of metabolic homeostasis and tissue inflammation. This review focuses on the roles of ILCs in different metabolic tissues, including adipose tissue, liver, pancreas, and intestine. We briefly outline the relationship between obesity, inflammation, and insulin resistance. We then discuss how ILCs in distinct metabolic organs may function to maintain metabolic homeostasis and contribute to obesity and its associated metabolic diseases. The potential of ILCs as the therapeutic target for obesity and insulin resistance is also addressed.
# Introduction
Obesity is a chronic metabolic disease caused by the excessive accumulation of fat. The global prevalence of obesity is overgrowing. The World Health Organization (WHO) estimates that obese people worldwide have nearly tripled since 1975. From 1975 to 2014, the prevalence of obesity has increased from 3.2% to 10.8% in adult males and from 6.4% to 14.9% in adult females worldwide. And the prevalence of obesity among adolescents in the 5-19 years age group has dramatically increased from 1975 to 2016 worldwide. Specifically, the prevalence of obesity has increased from 0.7% to 5.6% in girls and 0.9% to 7.8% in boys [bib_ref] Worldwide Trends in Body-Mass Index, Underweight, Overweight, and Obesity From 1975 to..., Ncd-Risc [/bib_ref]. Obesity increases all-cause mortality in four continents [bib_ref] Comorbidities of Overweight and Obesity: Current Evidence and Research Issues, Pi-Sunyer [/bib_ref]. Metabolic syndrome is a condition characterized by obesity, insulin resistance, hypertension, and hyperlipidemia, which leads to the development of a range of diseases, including type 2 diabetes mellitus, cardiovascular disease, non-alcoholic steatohepatitis and cancer. These metabolic diseases are the leading causes of death nowadays. Obesity thus is a public health and clinical challenge worldwide. Exploring the pathophysiological mechanisms underlying obesity is thus critical for the development of efficient therapeutic strategies to combat this disease.
Insulin resistance, in short, is that insulin cannot function normally. The exact amount of insulin fails to increase the uptake and utilization of glucose in adipose tissue, liver and muscle. The etiology of insulin resistance is recognized as chronic tissue inflammation [bib_ref] Inflammatory Links Between Obesity and Metabolic Disease, Lumeng [/bib_ref]. Several potential mechanisms underlying the development of obesity-associated low-grade inflammation have been proposed. Firstly, obesity increases gut permeability, and gut microbiota-derived substances trigger the inflammation signals by activating receptors such as Toll-like receptor 4 [bib_ref] Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance, Saad [/bib_ref]. Secondly, obesity elevates various lipids in circulation such as free fatty acids, leading to subsequent activation of TLR2/NFkB pathways [bib_ref] A Subpopulation of Macrophages Infiltrates Hypertrophic Adipose Tissue and Is Activated by..., Nguyen [/bib_ref]. Besides, the perturbed phosphocreatine/creatine metabolism in the obese state results in increased transcription of multiple pro-inflammatory genes [bib_ref] Impaired Phosphocreatine Metabolism in White Adipocytes Promotes Inflammation, Maqdasy [/bib_ref]. Thirdly, obesity induces the rapid expansion of adipocytes, which induces adipocyte death, hypoxia and mechanical stress between the cell and the extracellular matrix (ECM), resulting in inflammation [bib_ref] A Subpopulation of Macrophages Infiltrates Hypertrophic Adipose Tissue and Is Activated by..., Nguyen [/bib_ref]. Inflammatory signaling such as NFkB and c-Jun N-terminal kinase (JNK) can directly or indirectly block insulin action. For example, JNK phosphorylates insulin receptor substrates at serine/threonine sites rather than the tyrosine site, thereby inhibiting downstream signals of insulin receptors [bib_ref] Ser/Thr Phosphorylation of IRS Proteins: A Molecular Basis for Insulin Resistance, Zick [/bib_ref]. The first evidence that obesity is connected with inflammation is the discovery that TNF-a is overexpressed and promotes insulin resistance in obese mice [bib_ref] Adipose Expression of Tumor Necrosis Factor-Alpha: Direct Role in Obesity-Linked Insulin Resistance, Hotamisligil [/bib_ref]. Later, macrophages and their proinflammatory polarization were shown as key risk factors in obesity [bib_ref] Obesity Is Associated With Macrophage Accumulation in Adipose Tissue, Weisberg [/bib_ref] [bib_ref] Signaling by IL-6 Promotes Alternative Activation of Macrophages to Limit Endotoxemia and..., Mauer [/bib_ref]. Subsequently, many other immune cells, such as eosinophils and mast cells, were found to participate in the lowgrade chronic inflammation in obesity [bib_ref] Inflammation, Metaflammation and Immunometabolic Disorders, Hotamisligil [/bib_ref] [bib_ref] Regulation of Metabolic Homeostasis in Health and Disease, Brestoff [/bib_ref] [bib_ref] Role of Innate and Adaptive Immunity in Obesity-Associated Metabolic Disease, Mclaughlin [/bib_ref] [bib_ref] Tissue Immunometabolism: Development, Physiology, and Pathobiology, Man [/bib_ref]. These immune cells orchestrate the local environment of metabolic organs and are connected with insulin resistance in obesity [bib_ref] Regulation of Metabolism by the Innate Immune System, Lackey [/bib_ref] [bib_ref] Inflammatory Mechanisms Linking Obesity and Metabolic Disease, Saltiel [/bib_ref] [bib_ref] An Integrated View of Immunometabolism, Lee [/bib_ref] [bib_ref] Chronic Adipose Tissue Inflammation: All Immune Cells on the Stage, Cildir [/bib_ref]. Targeting immune pathways in chronic inflammation may successfully prevent or treat obesity and insulin resistance.
Innate lymphoid cells (ILCs) are a recently identified group of innate lymphocytes which lack antigen-specific receptors expressed on T and B lymphocytes [bib_ref] Tissue Residency of Innate Lymphoid Cells in Lymphoid and Nonlymphoid Organs, Gasteiger [/bib_ref]. On the basis of developmental pathways, the ILC family have been divided into five subsets: natural killer (NK) cells, group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), lymphoid tissue inducer (LTi) cells, and group 3 ILCs (ILC3s) [bib_ref] Innate Lymphoid Cells: 10 Years on, Vivier [/bib_ref]. They are considered as the innate counterparts of T lymphocytes, which have been introduced in many reviews [bib_ref] Innate Lymphoid Cells-a Proposal for Uniform Nomenclature, Spits [/bib_ref] [bib_ref] Innate Lymphoid Cells. Innate Lymphoid Cells: A New Paradigm in Immunology, Eberl [/bib_ref] [bib_ref] The Biology of Innate Lymphoid Cells, Artis [/bib_ref]. ILCs reside in the intestine, lung, adipose tissue, liver, and pancreas, and react rapidly to environmental stimuli [bib_ref] Innate Lymphoid Cells as Regulators of Immunity, Inflammation and Tissue Homeostasis, Klose [/bib_ref]. Mature ILCs are activated by cytokines, alarmins, and inflammatory mediators from myeloid cells or epithelial cells. For example, NK cells express a range of NK cell receptors (NKRs) which recognize numerous ligands on target stressed cells [bib_ref] Functions of Natural Killer Cells, Vivier [/bib_ref] [bib_ref] Identification, Activation, and Selective In Vivo Ablation of Mouse NK Cells via..., Walzer [/bib_ref] [bib_ref] Tumor and Viral Recognition by Natural Killer Cells Receptors, Arnon [/bib_ref]. IL-12 activates ILC1s, while ILC2s are stimulated by IL-33 and IL-25 [bib_ref] ILC1 Confer Early Host Protection at Initial Sites of Viral Infection, Weizman [/bib_ref]. IL-33 induces strong activation of ILC2s through the receptor suppression of tumorigenicity 2 (ST2) [bib_ref] Innate Production of TH2 Cytokines by Adipose Tissue-Associated C-Kit +Sca-1+ Lymphoid Cells, Moro [/bib_ref] [bib_ref] Helper-Like Innate Lymphoid Cells in Humans and Mice, Guia [/bib_ref]. RORgt + ILC3s are activated by IL-23 and then produce IL-17 and IL-22 [bib_ref] Lymphoid Tissue Inducer-Like Cells Are an Innate Source of IL-17 and IL-22, Takatori [/bib_ref] [bib_ref] Interferon-l and Interleukin 22 Act Synergistically for the Induction of Interferon-Stimulated Genes..., Hernańdez [/bib_ref]. They quickly respond to stress signals and maintain tissue homeostasis. However, they may also participate in the progression of inflammation. Recently, studies have unveiled the role of ILCs in metabolism. The functions of ILCs in different metabolic tissues are being actively investigated in depth as a link between the immune system and the metabolic system.
Obesity-induced chronic low-grade inflammation occurs in multiple metabolic organs, including adipose tissue, liver, pancreas, and intestine. Inflammation can lead to tissue damage, necrosis, and fibrosis. ILCs in these metabolic organs function to maintain homeostasis or contribute to inflammation. Here, we focus on the roles of ILCs in obesity and insulin resistance, discuss how ILCs in different tissues regulate metabolic homeostasis to protect against obesity or how they contribute to inflammation and insulin resistance. Targeting ILCs and their associated immune pathways may represent a novel approach to treat obesity and insulin resistance.
## Ilcs in adipose tissue
Adipose tissue is a dynamic organ regulating the homeostasis of energy [bib_ref] Adipose-Tissue Plasticity in Health and Disease, Sakers [/bib_ref]. When energy intake exceeds energy expenditure, excess energy stores in white adipose tissue (WAT) in the form of triglycerides. In normal circumstances, insulin activates lipoprotein lipase and inhibits hormone-sensitive lipase and thus increases absorption and deposition of triglyceride in the adipose tissue after food intake. However, excessive fat accumulation leads to adipocytes hypertrophy and hyperplasia which results in inflammation and insulin resistance in the WAT [bib_ref] Why Does Obesity Cause Diabetes?, Klein [/bib_ref]. In contrast to the white adipocytes whose main function is storing triglyceride, beige adipocytes are thermogenic cells that can promote energy consumption [bib_ref] Beige Adipocytes Are a Distinct Type of Thermogenic Fat Cell in Mouse..., Wu [/bib_ref]. Beige adipocytes are inducible and plastic. When exposed to cold stimulation or b3adrenergic receptor agonists, the white adipose tissue can expend energy by increasing the number of beige adipocytes and improving their activity. Beige adipocytes exist in WAT and are differentiated from Myf5 negative adipose progenitor cells or transformed from mature white adipocytes. They can increase the body's energy expenditure and improve glucose and lipid metabolism, thus becoming promising targets for preventing and treating obesity and insulin resistance. Recently, Trim et al. have reviewed that leukocytes in the adipose tissue regulate the homeostasis of adipocytes and respond to the changes of nutrition and body temperature [bib_ref] Immune and Non-Immune Functions of Adipose Tissue Leukocytes, Trim [/bib_ref]. Here, we focus on the function of ILCs in the adipose tissue in both health and obese associated metabolic disease.
## Nk cells and ilc1s regulate the inflammation in adipose tissue
Recent studies showed that NK cells and ILC1s in the adipose tissue participate in developing inflammation and insulin resistance in obese mice [fig_ref] FIGURE 1 |: NK cells and ILC1s in the adipose tissue and liver [/fig_ref]. Diet-induced obesity (DIO) increases NK cell number and induces NK cells to produce IFN-g and TNFa in the visceral adipose tissue (VAT). IFN-g and TNFa induce type 1 macrophages (M1 macrophages) accumulation and promote insulin resistance. Ablation of NK cells prevents the differentiation of M1 macrophages, reduces inflammation, and restores insulin sensitivity [fig_ref] TABLE 1 |: Summary of ILCs depletion strategies [/fig_ref] [bib_ref] NK Cells Link Obesity-Induced Adipose Stress to Inflammation and Insulin Resistance, Wensveen [/bib_ref] [bib_ref] Adipose Natural Killer Cells Regulate Adipose Tissue Macrophages to Promote Insulin Resistance..., Lee [/bib_ref] [bib_ref] Systemic NK Cell Ablation Attenuates Intra-Abdominal Adipose Tissue Macrophage Infiltration in Murine..., O'rourke [/bib_ref] , while expansion of NK cells exacerbates DIO-induced inflammation and insulin resistance [bib_ref] Adipose Natural Killer Cells Regulate Adipose Tissue Macrophages to Promote Insulin Resistance..., Lee [/bib_ref]. Similarly, IL-12 acts on IL-12R, activating STAT4, and then induces production of IFN-g from ILC1s in DIO mice, resulting in the expansion of M1 macrophages and insulin resistance [bib_ref] Adipose-Resident Group 1 Innate Lymphoid Cells Promote Obesity-Associated Insulin Resistance, O'sullivan [/bib_ref]. Other than secreting cytokines, group 1 ILCs constrain macrophages through cytotoxicity as macrophages express stress ligand of activating receptor NKG2D. The killing ability of ILC1s is impaired in DIO mice, changing the proportion of M1 macrophages and anti-inflammatory M2 macrophages, leading to subsequent metabolic disorders [bib_ref] Adipose Type One Innate Lymphoid Cells Regulate Macrophage Homeostasis Through Targeted Cytotoxicity, Boulenouar [/bib_ref]. Furthermore, a recent study shows that obesity increases the number of a specific interleukin-6 receptor (IL6R) a + NK subpopulation in mice and humans. This specific NK cell population facilitates obesity and insulin resistance [bib_ref] IL-6/Stat3-Dependent Induction of a Distinct, Obesity-Associated Nk Cell Subpopulation Deteriorates Energy and..., Theurich [/bib_ref]. The exact site of origin, the precursors and the factors to stimulate IL6Ra + NK cells, remain unclear. Nevertheless, these results show that NK cells and ILC1s contribute to obesity and insulin resistance.
Human adipose tissue-resident ILC1s include two subsets, CD56 + CD127 lo ILC1like population and CD56 dim CD16 + peripheral NK-like subset cells [bib_ref] Adipose Type One Innate Lymphoid Cells Regulate Macrophage Homeostasis Through Targeted Cytotoxicity, Boulenouar [/bib_ref]. Increased number of adipose and circulating ILC1s has been detected in obese type 2 diabetes patients. Patients with higher levels of ILC1s are associated with a greater risk of type 2 diabetes [bib_ref] Type 1 Innate Lymphoid Cells Are Associated With Type 2 Diabetes, Liu [/bib_ref]. Bariatric surgery decreases circulating ILC1s numbers and improves metabolic disorders. Adipose tissue ILC1s of type 2 diabetes patients promote adipose fibrogenesis and CD11c + macrophage activation. Besides, IFNg + NK cells play a role in the progression of human obesity. IFNg + NK cells are positively correlated with inflammation in adipose tissue, plasma glucose levels, and insulin resistance [bib_ref] Ifng-Producing NK Cells in Adipose Tissue Are Associated With Hyperglycemia and Insulin..., Mogilenko [/bib_ref]. These data show that ILC1s in human adipose tissue promote adipose inflammation and fibrosis in obesity-related type 2 diabetes. NK cells in obese people are activated, stressed and fail to proliferate or lyse tumors [bib_ref] NK Cells in Childhood Obesity Are Activated, Metabolically Stressed, and Functionally Deficient, Tobin [/bib_ref] [bib_ref] Alteration of Natural Killer Cell Phenotype and Function in Obese Individuals, Viel [/bib_ref]. Obesity makes robust lipid accumulation in NK cells, inhibiting their mTOR signaling, blocking their cytotoxic effector functions [bib_ref] Metabolic Reprogramming of Natural Killer Cells in Obesity Limits Antitumor Responses, Michelet [/bib_ref]. Interestingly, physical exercise and caloric restriction can increase NK cell cytotoxicity in mice [bib_ref] NK Cell Maturation and Function in C57BL/6 Mice Are Altered by Caloric..., Clinthorne [/bib_ref] [bib_ref] Voluntary Running Suppresses Tumor Growth Through Epinephrineand IL-6-Dependent NK Cell Mobilization and..., Pedersen [/bib_ref]. These results collectively demonstrate that the obese environment impairs peripheral NK cells functions and suggest that metabolic reprogramming of NK cells may impair immune cell function, increasing the risk of obesity-related diseases.
Overall, NK cells and ILC1s contribute to insulin resistance in obesity by induction of M1 macrophages and adipose tissue fibrosis.
Targeting the pathways of NK cells and ILC1s in adipose tissue may provide new strategies for treating obesity and associated disease.
## Ilc2s regulate metabolic homeostasis in the adipose tissue
ILC2s limit obesity and insulin resistance and control the metabolic homeostasis in adipose tissue . ILC2s in adipose tissue increase the number of eosinophils and M2 macrophages by type 2 cytokines IL-5 or IL-13 [bib_ref] Innate Lymphoid Type 2 Cells Sustain Visceral Adipose Tissue Eosinophils and Alternatively..., Molofsky [/bib_ref]. Eosinophils in adipose tissue maintain M2 macrophages and thus promote insulin sensitivity and metabolic homeostasis [bib_ref] Eosinophils Sustain Adipose Alternatively Activated Macrophages Associated With Glucose Homeostasis, Wu [/bib_ref] [bib_ref] Eosinophils and Type 2 Cytokine Signaling in Macrophages Orchestrate Development of Functional..., Qiu [/bib_ref]. The deficiency of IL-5 significantly reduces visceral adipose tissue (VAT) eosinophils, increasing obesity and insulin resistance in high fat diet (HFD) fed mice [bib_ref] Innate Lymphoid Type 2 Cells Sustain Visceral Adipose Tissue Eosinophils and Alternatively..., Molofsky [/bib_ref]. Further, infiltration of ILC2s into VAT by IL-25 administration leads to weight loss and improves glucose tolerance in obese mice. Consistently, transferring ILC2s into obese mice also shows that ILC2s prevent diet-induced obesity [bib_ref] Cutting Edge: IL-25 Elicits Innate Lymphoid Type 2 and Type II NKT..., Hams [/bib_ref]. In addition, FIGURE 2 | ILC2s in the adipose tissue in lean and obesity state. ILC2s promote the accumulation of eosinophils and M2 macrophages through IL-5 or IL-13 and thus protect against insulin resistance. ILC2s can also directly promote the beigeing of subcutaneous WAT. ILC2s are regulated by the mesenchymal cells and adipocytes in the adipose tissue directly or indirectly. engagement of glucocorticoid-induced tumor necrosis factor receptor (GITR) on activated ILC2s with GITR agonist, DTA-1, induces type 2 cytokines by ILC2s. Experiments of Rag2 deficient mice injected with DTA-1 and adoptive transfer of adipose ILC2s to GITR -/mice injected with DTA-1 shows that engagement of GITR on ILC2s is protective against insulin resistance. Further, transfer experiment of IL5 -/or IL-13 -/-ILC2s shows that the protective effects of GITR engagement depends on IL-13 particularly [bib_ref] Costimulation of Type-2 Innate Lymphoid Cells by GITR Promotes Effector Function and..., Galle-Treger [/bib_ref]. Moreover, ILC2s are present in para-aortic adipose tissue. Diet-induced obesity reduced the number of ILC2s in para-aortic adipose tissue. Expansion of ILC2s improves the progression of atherosclerosis while ablation of ILC2s exacerbates atherosclerosis. Bone marrow transplantation experiments showed that the function of ILC2s on atherosclerosis is dependent on IL-5 and IL-13 [bib_ref] Type-2 Innate Lymphoid Cells Control the Development of Atherosclerosis in Mice, Newland [/bib_ref]. Thus, ILC2s regulate metabolic homeostasis partly through type 2 cytokines. ILC2s in white adipose tissue (WAT) contribute to thermogenesis. Strikingly, ILC2s activated by interleukin-33 (IL-33) are sufficient to promote WAT beigeing in thermoneutral mice. ILC2s secrete IL-13 which targets IL-4R in PDGFRa+ adipose precursor cells and promotes beige adipogenesis [bib_ref] Activated Type 2 Innate Lymphoid Cells Regulate Beige Fat Biogenesis, Lee [/bib_ref]. This research highlights the critical role of ILC2s and type 2 cytokines in regulating adipose precursor cell number and fate. Another study shows that ILC2s are present in human WAT and demonstrates that ILC2s in WAT are dysregulated in obesity. Notably, this study provides a novel mechanism by which IL-33-induced ILC2s drive white fat beigeing. It is not dependent on the eosinophil/IL-4Ra/ macrophage pathway or the adaptive immune system. Instead, ILC2s express proprotein convertase subtilisin/kexin type 1 (Pcsk1) which processes the production of methionineenkephalin (Met-Enk), which directly acts on adipocytes and promotes beige adipocyte formation [bib_ref] Group 2 Innate Lymphoid Cells Promote Beiging of White Adipose Tissue and..., Brestoff [/bib_ref]. In addition, cold exposure elevates the level of IL-33, ILC2s, and eosinophils in subcutaneous adipose tissue. Blocking the IL-33 signal reverses the expression of the thermogenic gene UCP1, highlighting that ILC2s are involved in cold-induced thermogenesis [bib_ref] IL-33-Driven ILC2/eosinophil Axis in Fat is Induced by Sympathetic Tone and Suppressed..., Ding [/bib_ref]. Interestingly, a recent study has reported that loss of ILC2s in adipose tissue drives thermogenic failure in aging. ILC2s are lost in aging, and an adoptive transfer experiment showed that adult ILC2s could help old mice resist cold [bib_ref] IL-33 Causes Thermogenic Failure In Aging by Expanding Dysfunctional Adipose ILC2, Goldberg [/bib_ref]. These studies shed light on the role of ILC2s in regulating metabolism and may represent a novel approach for treating obesity.
## Ilc2s interact with local cells in the adipose tissue
As tissue-resident cells, ILC2s interact with the stromal cells and adipocytes in adipose tissue to regulate metabolic homeostasis. White adipose tissue pluripotent mesenchymal cells produce IL-33, increasing the proliferation of ILC2s and the production of type 2 cytokine, thus promoting regulatory circuits that maintain WAT homeostasis [bib_ref] Stromal Cells Maintain Immune Cell Homeostasis in Adipose Tissue via Production of..., Mahlakõiv [/bib_ref] [bib_ref] Distinct Immunocyte-Promoting and Adipocyte-Generating Stromal Components Coordinate Adipose Tissue Immune and Metabolic..., Spallanzani [/bib_ref]. Studies by Shan et al. have further demonstrated (66) that IL-33 is only expressed in DPP4 + cells and its expression is directly regulated by the b1adrenergic receptor signaling pathway and CREB protein. Cold exposure rapidly stimulates DPP4 + cells to secrete IL-33, which in turn induces the proliferation and activation of ILC2s, thereby promoting white adipose beigeing. IL-33 increases death receptor 3 (DR3) expression on ILC2s and activates the NF-kB pathways, thus stimulating ILC2s and protecting against insulin resistance [bib_ref] DR3 Stimulation of Adipose Resident ILC2s Ameliorates Type 2 Diabetes Mellitus, Shafiei-Jahani [/bib_ref]. Besides, activation of ILC2 by IL-33 increases the expression of PPARg, which is indispensable for the proliferation and expression of cytokines of ILC2s. Inhibition of PPARg decreases expression of CD36 and uptake of fatty acids [bib_ref] Metabolic Regulation by Pparg is Required for IL-33-Mediated Activation of ILC2s In..., Fali [/bib_ref]. IL-33 increases the uptake of lipids and glucose of ILC2s to promote the proliferation of ILC2s in the context of allergendriven airway inflammation [bib_ref] Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation, Karagiannis [/bib_ref]. On the other hand, sST2, the soluble isoform of the IL-33 receptor ST2, secreted by adipocytes, attenuates the signaling of IL-33 and disrupts the ILC2 homeostasis in adipose tissue, thereby exacerbating obesityassociated insulin resistance. Zbtb7b, a negative regulator of adipocyte expression of sST2, maintains glucose homeostasis and prevents insulin resistance at a steady-state [bib_ref] The Obesity-Induced Adipokine Sst2 Exacerbates Adipose Treg and ILC2 Depletion and Promotes..., Zhao [/bib_ref]. In addition, the deficiency of ST2 decreases ILC2s in WAT, resulting in increased visceral fat, decreased browning, and impairment of glucose metabolism [bib_ref] ILC2s Improve Glucose Metabolism Through the Control of Saturated Fatty Acid Absorption..., Okamura [/bib_ref]. Meanwhile, adipokine Chemerin and its receptor chemokine-like receptor 1 (CMKLR1) inhibit adipocyte cAMP-PKA signaling, interfering with cold-induced IL-33 secretion and downstream ILC2 activation. This action thereby inhibits white adipose tissue beigeing, leading to obesity and metabolic disorders [bib_ref] The Chemerin-CMKLR1 Axis Limits Thermogenesis by Controlling a Beige Adipocyte/IL-33/Type 2, Lin [/bib_ref]. Other than IL-33, pluripotent mesenchymal cells express the intercellular adhesion molecule ICAM-1, while ILC2s express its ligand LFA antigen 1 (LFA-1). This direct interaction also promotes ILC2s and induces their production of cytokines, which induces mesenchymal cells to secrete eotaxin and support eosinophil recruitment [bib_ref] A Stromal Cell Niche Sustains ILC2-Mediated Type-2 Conditioning in Adipose Tissue, Rana [/bib_ref]. Besides, fibroblasts in adipose tissue express a classical cadherin, cadherin-11, that mediates cell-to-cell adhesion. In Cadherin-11-deficient mice, the stromal cells produced more IL-33 which increased the activity of adipose tissue ILC2s and M2 macrophages, thus reducing inflammation, fibrosis, and glucose intolerance [bib_ref] Stromal Cell Cadherin-11 Regulates Adipose Tissue Inflammation and Diabetes, Chang [/bib_ref]. Moreover, adipose mesenchymal cells express glial-derived neurotrophic factor (GNDF) upon stimulation by sympathetic nerve terminals through the b2adrenergic receptor. GNDF regulates adipose tissue-resident ILC2s, ameliorating high-fat diet-induced obesity [bib_ref] Neuro-Mesenchymal Units Control ILC2 and Obesity via a Brain-Adipose Circuit, Cardoso [/bib_ref]. Although murine intestinal ILC2s express the b2-adrenergic receptor (b2-AR), which negatively regulates ILC2s responses [bib_ref] b2-Adrenergic Receptor-Mediated Negative Regulation of Group 2 Innate Lymphoid Cell Responses, Moriyama [/bib_ref] , the adipose ILC2s are mainly regulated indirectly by the sympathetic signals through mesenchymal cells. These studies reveal that mesenchymal cells and adipocytes have a multifaceted dialogue with ILC2s to mainta in typ e 2 immune microenvironment in white adipose tissue.
ILC2s also interact with other immune cells through newly identified pathways in the adipose tissue. After IL-33 treatment, ILC2s interact with T cells via ICOSL-ICOS, promoting Treg cell accumulation. On the other hand, IFN-g treatment inhibits ILC2 activation and reduces the interaction of ILC2s and T cells, thus reducing Treg cell accumulation. Interestingly, this repression increases with HFD-induced obesity [bib_ref] Interleukin-33 and Interferon-g Counter-Regulate Group 2 Innate Lymphoid Cell Activation During Immune..., Molofsky [/bib_ref]. Besides, ILC2s express OX40 ligand (OX40L), which interacts with OX40 on T cells to sustain Treg cells and Th2 cells responses in adipose tissue after IL-33 induction [bib_ref] Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory..., Halim [/bib_ref]. Thus, ILC2s mediate type 2 immune responses, sustaining metabolic homeostasis in a lean state. In obesity, TNF triggers IL-33-dependent expression of PD-1 on ILC2s and further recruits and activates PD-L1 hi M1 macrophages. PD-1-PD-L1 pathway is responsible for ILC2 destabilization after HFD and results in impaired metabolism in obesity [bib_ref] PD-1 Is Involved in the Dysregulation of Type 2 Innate Lymphoid Cells..., Oldenhove [/bib_ref]. Besides, a hybrid cytokine IL233 with the activities of both IL-2 and IL-33 protects mice from obesitylinked diabetic nephropathy with a more significant accumulation of Tregs, ILC2s, M2 macrophages, and eosinophils in VAT [bib_ref] Novel Immunomodulatory Cytokine Regulates Inflammation, Diabetes, and Obesity to Protect From Diabetic..., Sabapathy [/bib_ref]. This evidence reveals the crosstalk between ILC2s and other immune cells in adipose tissue, providing novel targets to ameliorate obesity.
## Ilc3s are related to obesity in human adipose tissue
The function of ILC3s in adipose tissue is less studied. O'Sullivan has reported that ILC3s are absent in lean or obese mouse white adipose tissue [bib_ref] Adipose-Resident Group 1 Innate Lymphoid Cells Promote Obesity-Associated Insulin Resistance, O'sullivan [/bib_ref] [bib_ref] Single-Cell Sequencing of Human White Adipose Tissue Identifies New Cell States in..., Hildreth [/bib_ref]. Importantly, the frequency and density of ILC3s increases in obese white adipose tissue compared with healthy people. The frequency and density of ILC3s are positively correlated with patient BMI. What's more, they have identified a group of ILC precursor (ILCP) cells in human adipose tissue which give rise to ILC1s and ILC3s, but not ILC2s. Further studies are needed to verify whether ILC3s regulate the metabolic homeostasis in the lean state or contribute to the inflammation by expressing LIF, TNFSF13B and MIF in the obese state. The distinct function for ILC3s in adipose tissue between mice and human being suggests that ILCs may not be evolutionarily conserved.
## Ilcs in liver
Non-alcoholic fatty liver disease (NAFLD) is a growing chronic liver disease worldwide, which can lead to cirrhosis and even hepatocellular carcinoma [bib_ref] The Global Burden of Liver Disease: The Major Impact of China, Wang [/bib_ref] [bib_ref] A Cross-Sectional Study of the Public Health Response to Non-Alcoholic Fatty Liver..., Lazarus [/bib_ref]. The global prevalence of NAFLD is currently about 25%. Assessing the prevalence of NAFLD in different geographic regions revealed that NAFLD is prevalent on all continents, with South America (31%) and the Middle East (32%) having the highest prevalence, followed by Asia (27%), North America (24%), and Europe (23%), and Africa the lowest prevalence (13%) [bib_ref] Global Epidemiology of Nonalcoholic Fatty Liver Disease-Meta-Analytic Assessment of Prevalence, Incidence, and..., Younossi [/bib_ref]. The progression of NAFLD is closely related to insulin resistance and metabolic syndrome [bib_ref] Non-Alcoholic Fatty Liver Disease and Obesity: Biochemical, Metabolic and Clinical Presentations, Milićs [/bib_ref]. With the increasing prevalence of obesity, type 2 diabetes, and metabolic syndrome, NAFLD is expected to become the leading cause of cirrhosis requiring liver transplantation in the next decade [bib_ref] NAFLD in 2017: Novel Insights Into Mechanisms of Disease Progression, Khan [/bib_ref]. NAFLD includes a range of liver lesions, including simple steatosis, steatohepatitis (Non-alcoholic steatohepatitis, NASH), and fibrosis. During the progression of NAFLD, innate immune cells play a significant role (88).
## Nk cells and ilc1s regulate the progression of nafld
NK cells account for 30-50% of the total lymphocytes in the human liver [bib_ref] Natural Killer Cells in Liver Disease, Tian [/bib_ref]. They are important during the progression of NAFLD. Here we introduce the related studies of NK cells in different stages of NAFLD.
In the stage of HFD-induced liver steatosis, NK cells produce osteopontin, which induces hepatic ER stress and promotes insulin resistance. Ablation of NK cells with neutralizing antibody can improve HFD-induced ER stress, insulin resistance, and liver steatosis [bib_ref] NK Cells Induce Hepatic ER Stress to Promote Insulin Resistance in Obesity..., Wu [/bib_ref]. This study shows a pathogenic role of NK cells. On contrary, in the liver of obese mice fed 24 weeks on a high fat and sugar diet, NK cells are less cytotoxic, more like ILC1s, and seem to be protective against NAFLD, although the reduction of cytotoxicity increases the susceptibility to cancer. This shift of liver NK cells to ILC1s reflect the plasticity of NK cells. Reducing the cytotoxicity by perforin knockout alleviates the symptoms of NAFLD in mice [bib_ref] The Obese Liver Environment Mediates Conversion of NK Cells to a Less..., Cuff [/bib_ref].
Besides, NK cells prevent NASH progression to fibrosis by regulating liver macrophages polarization. In the NASH model of mice fed with a methionine and choline deficient (MCD) diet, DX5 + NKp46 + NK cells increased, which induced macrophages M1polarized through the production of IFN-g by NK cells. Accordingly, ablation of NKp46 + cells makes macrophage shift toward M2 phenotypes, which fail to clear damaged cells effectively, thereby promoting the development of fibrosis (91). Besides, genetic deletion of TNF-related apoptosis-inducing ligand (TRAIL) receptor reduces inflammatory macrophages in the liver and suppresses steatohepatitis in FFC (a diet high in saturated fat, cholesterol, and fructose)-fed mice [bib_ref] TRAIL Receptor Deletion in Mice Suppresses the Inflammation of Nutrient Excess, Idrissova [/bib_ref]. As the mice used in this study is whole body knockout of TRAILR, the reduction in hepatocyte lipoapoptosis may occur after the improved metabolic niche. And this research can't identify the tissue-specific roles of TRAIL signaling. Using tissue-and cell-specific TRAILR -/mice may address these problems. Despite the changes of NK cells in mouse model, in patients with NAFLD confirmed by biopsy, the number and function of NK cells is not altered, except for the increased expression of NKG2D on NK cells in NASH patients [bib_ref] Retained NK Cell Phenotype and Functionality in Non-Alcoholic Fatty Liver Disease, Stiglund [/bib_ref].
## Further studies exploring how nash affects nk cells in humans is needed.
Many studies reported the protective role of NK cells in liver fibrosis induced by carbon-tetrachloride (CCl 4 ). Melhem et al. reported that NK cells can improve liver fibrosis by killing activated hepatic stellate cells (HSC) [bib_ref] Anti-Fibrotic Activity of NK Cells in Experimental Liver Injury Through Killing of..., Melhem [/bib_ref]. HSC are dominant contributors to liver fibrosis and give rise to 82-96% of myofibroblasts [bib_ref] Fate Tracing Reveals Hepatic Stellate Cells as Dominant Contributors to Liver Fibrosis..., Mederacke [/bib_ref] [bib_ref] Senescence of Activated Stellate Cells Limits Liver Fibrosis, Krizhanovsky [/bib_ref]. Radaeva et al. employed the fibrosis model of mice fed with the 3,5-diethoxycarbonyl-1,4dihydrocollidine (DDC) diet or injected with CCl 4 . They further found that NK cells kill activated HSC dependent on retinoic acid early inducible 1/NKG2D and TRAIL. NK cells tend to lyse the activated HSC as the activated HSC rather than the quiescent HSC express the NKG2D ligand [bib_ref] Natural Killer Cells Ameliorate Liver Fibrosis by Killing Activated Stellate Cells in..., Radaeva [/bib_ref]. Other than ligand for NKG2D, murine and human HSC express the ligand for NKp46 of NK cells. NK cells kill HSC dependent on NKp46, and thus ameliorating liver fibrosis induced by CCl 4 [bib_ref] NKp46-Mediated Killing of Human and Mouse Hepatic Stellate Cells Attenuates Liver Fibrosis, Gur [/bib_ref]. Besides, IL-18 and TLR3 ligand activated NK cells kill HSC through the p38/PI3K/AKT-dependent pathway in vitro [bib_ref] Activated NK Cells Kill Hepatic Stellate Cells via P38/PI3K Signaling in a..., Li [/bib_ref]. These studies revealed that NK cells protect against the liver fibrosis by killing HSC.
Besides, NK cells are involved in the development of hepatocellular carcinoma [bib_ref] Natural Killer Cells in Liver Disease and Hepatocellular Carcinoma and the NK..., Liu [/bib_ref]. NK cells are important for the surveillance of hepatocellular carcinoma. In patients with hepatocellular carcinoma, the number of NK cells significantly decreased [bib_ref] Diminished Absolute Counts of CD56dim and CD56bright Natural Killer Cells in Peripheral..., Fathy [/bib_ref]. NK cells are regulated by monocytes and macrophages by CD48/2B4 axis in hepatocellular carcinoma [bib_ref] Monocyte/ macrophage-Elicited Natural Killer Cell Dysfunction in Hepatocellular Carcinoma Is Mediated by..., Wu [/bib_ref]. Besides, myeloid derived suppressor cells inhibit the cytotoxicity and production of cytokines from natural killer cells via the NKp30 receptor in hepatocellular carcinoma [bib_ref] Myeloid Derived Suppressor Cells Inhibit Natural Killer Cells in Patients With Hepatocellular..., Hoechst [/bib_ref]. Moreover, fibroblasts inducing NK cells dysfunction through production of prostaglandin E2 and indoleamine 2,3dioxygenase in hepatocellular carcinoma [bib_ref] Hepatocellular Carcinoma-Associated Fibroblasts Trigger NK Cell Dysfunction via PGE2 and IDO, Li [/bib_ref]. These studies revealed that multiple pathways lead to the dysfunction of NK cells and promote the occurrence and development of hepatocellular carcinoma.
Overall, there are several mechanisms of how NK cells protect against the progression of NAFLD [fig_ref] FIGURE 1 |: NK cells and ILC1s in the adipose tissue and liver [/fig_ref] although some studies reported NK cells play the opposite role. As for the role of ILC1s in the liver, a study reported that liver ILC1s is protective against acute liver injury. Intraperitoneally injected with 10% CCl 4 in corn oil at a dose of 10 uL per gram body weight activates liver ILC1s dependent on DNAM-1 and IL-7R. Activated ILC1s secrete IFN-g, which is regulated by Adenosine triphosphate (ATP)-purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7) signaling. And then, IFN-g induces hepatocytes expressing Bcl-Xl, thus protecting mice from CCl 4 -induced acute liver injury [bib_ref] Type 1 Innate Lymphoid Cells Protect Mice From Acute Liver Injury via..., Nabekura [/bib_ref].These results suggest that liver ILC1s are essential for protecting mice from acute liver injury. Notably, a recent study reported that unlike conventional NK cells which derive from the hematopoietic stem cells in adult bone marrow, liver ILC1s develop from Lin -Sca1 + Mac1 + pluripotent hematopoietic hepatocytes which are derived from fetal liver. IFN-g produced by liver ILC1s themselves promote their in situ development by acting on IFN-gR + liver precursor cells, which forms an IFN-g feedback loop [bib_ref] Liver Type 1 Innate Lymphoid Cells Develop Locally Via an Interferon-g-Dependent Loop, Bai [/bib_ref]. This study revealed that liver ILC1s are different from conventional NK cells in developmental pathway and emphasized the unique immune status of the liver. The role of liver ILC1s in the development of NAFLD still needs to be explored.
## Ilc2s and ilc3s are involved in the progression of nafld
Little is known on the role of ILC2s during hepatic steatosis and NASH. Main findings are related to fibrosis and tissue repair. In both humans and mice with hepatic fibrosis, IL-33 expression is increased. Further, IL-33 is able to cause rodent liver fibrosis. IL-33 activates liver ILC2s and induces ILC2s expansion. The proportion of ILC2s in ILCs is low in normal human liver. However, their number increases when liver fibrosis occurs and is directly related to the severity of the disease [bib_ref] Group 2 Innate Lymphoid Cells Exhibit Progressively Higher Levels of Activation During..., Gonzalez-Polo [/bib_ref]. Liver ILC2s secretes IL-13 when stimulated by IL-3, IL-25, and TSLP from hepatocytes, HSCs, and Kupffer cells in response to TLR3 stimulation [bib_ref] Composition and Functionality of the Intrahepatic Innate Lymphoid Cell-Compartment in Human Nonfibrotic..., Forkel [/bib_ref]. ILC2-derived IL-13 activates HSC through IL-4R and STAT6-dependent signaling and thus mediates hepatic fibrosis [bib_ref] Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis, Mchedlidze [/bib_ref] [bib_ref] Combinatorial Targeting of TSLP, IL-25, and IL-33 in Type 2 Cytokine-Driven Inflammation..., Vannella [/bib_ref]. These results suggest ILC2s are involved in liver fibrosis. Targeting ILC2s and modulation of IL-33 may be therapeutic strategies for treating liver fibrosis.
In CCl 4 -induced liver fibrosis, the proportion of IL-22 + ILC3 and IL-17A + ILC3 subsets markedly increased. A Co-culture experiment with LX-2 cells showed that ILC3s directly promote LX-2 fibrogenesis by IL-17A and IL-22 [bib_ref] Type 3 Innate Lymphoid Cell: A New Player in Liver Fibrosis Progression, Wang [/bib_ref]. However, a recent study using Rorc gfp/gfp mice and in vitro primary hepatocytes showed that ILC3s protect from HFD induced steatohepatitis and IL-22 from ILC3s increases lipid metabolism and suppresses apoptosis [bib_ref] Group 3 Innate Lymphoid Cells Protect Steatohepatitis From High-Fat Diet Induced Toxicity, Hamaguchi [/bib_ref]. Besides, IL-22 can regulate lipogenesis related genes and prevent liver steatosis [bib_ref] Amelioration of High Fat Diet Induced Liver Lipogenesis and Hepatic Steatosis by..., Yang [/bib_ref]. IL-22-Fc treatment restores liver insulin sensitivity, decreased hepatic triglyceride and cholesterol levels, and ameliorated liver steatosis in dietinduce obesity and db/db mice. IL-22-Fc directly functioned on hepatocytes to induce Stat3 activation in vitro [bib_ref] Interleukin-22 Alleviates Metabolic Disorders and Restores Mucosal Immunity in Diabetes, Wang [/bib_ref]. This study indicates that ILC3s can protect mice from liver steatosis through producing IL-22. However, whether ILC3s in the liver play a protective or promoting role in the progression of NAFLD is worth further investigation.
## Ilcs in pancreas
Pancreas contains exocrine glands and endocrine glands. Exocrine glands secret pancreatic juice, which has a strong digestion capacity. Endocrine function is performed by specialized cells located in the pancreas. These cells aggregate into clusters and are dispersed in the pancreas, called pancreatic islets. There are four types of hormone-secreting cells in the pancreatic islet, a, b, d, and F cells. Among them, a cells secrete glucagon and b cells secrete insulin. Insulin plays a wide and complex physiological role in regulation of glucose and lipid metabolism. Type 1 diabetes mellitus has an early onset autoimmune disorder, which leads to failure of insulin secretion. In contrast, Type 2 diabetes mellitus is associated with obesity and insulin resistance. In the early stages of insulin resistance, b-cells compensate by secreting more insulin and increasing b-cell proliferation. As insulin resistance and inflammation prolong, b-cell stress impairs glucose tolerance. Finally, b-cell failure leads to type 2 diabetes. As there are few studies about NK cells and ILC1s in the pancreas in obesity and insulin resistance, we introduce the role of ILC2s and ILC3s in the pancreas.
## Ilc2s regulate the pancreas function
During obesity, the inflammation in pancreatic islets makes b cells fail to secrete insulin. In lean mice, IL-33 from islet mesenchymal cells activates ILC2s in the pancreas. Activated ILC2s secrete colony-stimulating factor 2 and IL-13, therefore inducing retinoic acid (RA) from macrophages and dendritic cells. Local RA signals promote b cell function and increase insulin secretion. Obesity impairs the IL-33-ILC2 signal and islet function, which can be rescued by IL-33 injection [bib_ref] Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion Through Myeloid Cell Retinoic..., Dalmas [/bib_ref]. Besides, ILC2s activate tumor immunity to restrict pancreas-specific tumor growth [bib_ref] ILC2s Amplify PD-1 Blockade by Activating Tissue-Specific Cancer Immunity, Moral [/bib_ref]. These results suggest that ILC2s can promote insulin secretion. Selective activation of type 2 immunity may be a therapeutic strategy for treating diabetes.
## Ilc3s alter the pancreatic function
AHR ligands from gut microbiota induce pancreatic ILC3s secreting IL-22 which induces pancreatic endocrine cells expressing b-defensin 14 (mBD14). mBD14 stimulates B cells secreting IL-4, promoting regulatory macrophages and T cells to inhibit autoimmune diabetes. This study identified crosstalk between ILCs and endocrine cells in pancreas associated with autoimmune diabetes. Besides, IL-22 administration inhibits islets' oxidative stress and ER stress, restoring insulin secretion and glucose homeostasis in obese mice [bib_ref] Glycemic Control in Diabetes Is Restored by Therapeutic Manipulation of Cytokines That..., Hasnain [/bib_ref]. This study indicates that ILC3s and IL-22 in the pancreas may play a role in preventing obesity-associated type 2 diabetes. However, this hypothesis still needs to be verified.
## Ilcs in intestine
The gut is an extensive immune system due to exposure to many microorganisms and ingested antigens. The gut microbiota is altered in obesity and its associated metabolic disease, known as dysbiosis [bib_ref] Host-Gut Microbiota Metabolic Interactions, Nicholson [/bib_ref] [bib_ref] Gut Microbiota Composition and Activity in Relation to Host Metabolic Phenotype and..., Holmes [/bib_ref] [bib_ref] The Gut Microbiome and Metabolic Syndrome, Dabke [/bib_ref]. One major consequence of dysbiosis is defects in the gut barrier, increasing the leakage of bacterial products and contributing to chronic low-grade inflammation and insulin resistance [bib_ref] Metabolic Endotoxemia Initiates Obesity and Insulin Resistance, Cani [/bib_ref] [bib_ref] Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity..., Cani [/bib_ref] [bib_ref] Intestinal Mucosal Adherence and Translocation of Commensal Bacteria at the Early Onset..., Amar [/bib_ref]. As sensor of the microbiota, the intestinal immune system was an essential regulator of obesity-related insulin resistance [bib_ref] Regulation of Obesity-Related Insulin Resistance With Gut Anti-Inflammatory Agents, Luck [/bib_ref] [bib_ref] Jejunal T Cell Inflammation in Human Obesity Correlates With Decreased Enterocyte Insulin..., Monteiro-Sepulveda [/bib_ref] [bib_ref] The Gut Microbiota Regulates Intestinal CD4 T Cells Expressing Rorgt and Controls..., Garidou [/bib_ref]. As there are few studies about NK cells and ILC1s in the intestine in obesity, we introduce ILC2s and ILC3s in the intestine and focus on the function and regulation of intestinal ILC3 in metabolism.
## Ilc2s in the intestine induce obesity
Despite that ILC2s in adipose tissue have the potential to limit obesity, a recent study suggested that ILC2s in the gut induce obesity [bib_ref] Innate Lymphoid Cells in the Induction of Obesity, Sasaki [/bib_ref]. Il2rg −/− Rag2 −/− mice lacking ILCs, T and B cells resist HFD-induced obesity compared with Rag2 −/− mice lacking T and B cells. Adoptive transfer experiment has showed that supplementation of ILC2s from the small intestine could render Il2rg -/-Rag2 -/mice prone to HFD-induced obesity. IL-2 from ILC2s in the small intestine may thus be critical to the induction of obesity and insulin resistance. These results also suggest that the role of ILCs in the regulation of obesity and associated metabolic disease is tissue-specific. The detailed effect of intestinal ILC2s on obesity still needs to be further investigated.
## Intestinal ilc3s produce cytokines to regulate metabolism
ILC3s are abundant in the intestine. Gut ILC3 cells produce the cytokine interleukin-22 (IL-22), which exerts essential roles in eliciting an innate immune response [bib_ref] Interleukin-22 Induces Interleukin-18 Expression From Epithelial Cells During Intestinal Infection, Muñoz [/bib_ref] , maintaining mucosal barrier integrity [bib_ref] Border Patrol: Regulation of Immunity, Inflammation and Tissue Homeostasis at Barrier Surfaces..., Sonnenberg [/bib_ref] , and assuring gut homeostasis [bib_ref] Interleukin-22 Protects Intestinal Stem Cells Against Genotoxic Stress, Gronke [/bib_ref] [bib_ref] Interleukin-22 Promotes Intestinal-Stem-Cell-Mediated Epithelial Regeneration, Lindemans [/bib_ref] [bib_ref] Innate Lymphoid Cells Promote Anatomical Containment of Lymphoid-Resident Commensal Bacteria, Sonnenberg [/bib_ref]. Notably, IL-22 from ILC3s has been demonstrated to improve metabolic disorders. IL-22 from ILCs and CD4 + T cells is reduced in obesity under immune challenges. Mice deficient in the IL-22 receptor are more prone to metabolic disorders. Injection of IL-22 can reverse many metabolic symptoms in obese mice. The beneficial effects of IL-22 include preserving gut permeability, reducing endotoxemia and inflammation, regulating lipid metabolism, and improving insulin sensitivity [bib_ref] Interleukin-22 Alleviates Metabolic Disorders and Restores Mucosal Immunity in Diabetes, Wang [/bib_ref]. Moreover, IL-22 from ILC3s improves the Polycystic ovary syndrome (PCOS) phenotype. Mice transplanted with stool from PCOS patients display a reduced percentage of IL-22 + ILC3s and develop insulin resistance. Administration of glycodeoxycholic acid induces IL-22 secretion from ILC3s through GATA3, which improves the disorder. The mechanisms of IL-22-mediated improvements likely involve promoting adipose tissue browning and inhibiting inflammation [bib_ref] Gut Microbiota-Bile Acid-Interleukin-22 Axis Orchestrates Polycystic Ovary Syndrome, Qi [/bib_ref]. Interestingly, exhaustive exercise decreases the proportion of ILC3s and mRNA levels of IL-22 in lamina propria, which destroys intestinal barrier integrity and aggravates intestinal inflammation [bib_ref] Exhaustive Exercise Induces Gastrointestinal Syndrome Through Reduced Ilc3 and Il-22 in Mouse..., Hou [/bib_ref]. However, IL-22 can decrease the expression of lipid transporter in the small intestine, which impairs lipid metabolism [bib_ref] Innate and Adaptive Lymphocytes Sequentially Shape the Gut Microbiota and Lipid Metabolism, Mao [/bib_ref]. Consistently, a study using single-cell RNA sequencing has identified a population of DC cells, named CIA-DCs, as the major source of IL-22 binding protein (IL-22BP). Mice lacking IL-22BP demonstrate an increase in functional IL-22. This alteration is associated with the concurrent reduction in the expression of lipid transporters, leading to a decrement in lipid resorption and subsequent change in body fat homeostasis [bib_ref] Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22bp-Producing Dendritic..., Guendel [/bib_ref]. Interestingly, mice feeding carbohydrate diet express higher levels of enzymes and transporters required for carbohydrate digestion and absorption, compared with mice feeding protein diet. gdT cells regulate this process by inhibiting IL-22 production by ILC3s. Treating Organoids with IL-22 reduced the carbohydrate transcriptional program [bib_ref] gd T Cells Regulate the Intestinal Response to Nutrient Sensing, Sullivan [/bib_ref]. These studies thus indicate that IL-22 from ILC3s regulates nutrition absorption. Overall, the fact that IL-22 from ILC3s regulates metabolism homeostasis highlights the link between metabolism and immunity and provides a new avenue for therapeutic intervention of metabolic diseases.
Although ILC populations and their potentiality of secreting IL-22 are nearly intact in the colon of obese mice, the upstream cytokine IL-23, which activates ILC3 to produce IL-22, is reduced in obese mice after pathogenic bacteria infection [bib_ref] Interleukin-22 Alleviates Metabolic Disorders and Restores Mucosal Immunity in Diabetes, Wang [/bib_ref]. Lack of IL-23-IL-22 signaling damaged the intestinal barrier, increasing the concentration of lipopolysaccharide (LPS) in plasma. In mice fed HFD, the relative proportion of IL22-producing NKp46 + CD4 − ILC3s is reduced despite the increase in the total cell numbers of ILC3s in the colon [bib_ref] Regulation of Obesity-Related Insulin Resistance With Gut Anti-Inflammatory Agents, Luck [/bib_ref]. The impairment of IL-23-ILC3-IL22 signaling may partly lead to obesity and insulin resistance.
Another essential cytokine produced by ILC3s is IL-17. IL-17 regulates the migration of intestinal neutrophils, protects the gut barrier, reduces systemic LPS, and improve metabolic syndrome [bib_ref] Interleukin-17/Interleukin-17 Receptor Axis Elicits Intestinal Neutrophil Migration, Restrains Gut Dysbiosis and Lipopolysaccharide..., Peŕez [/bib_ref]. Intestinal IL-17-secreting ILCs can also promote hostmicrobiota mutualism, preventing liver inflammation and dysfunction of lipid metabolism [bib_ref] Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22..., Martıńez-Loṕez [/bib_ref]. However, Teijeiro et al. have reported (141) that IL-17A promotes diet-induced obesity and metabolic syndrome. Disruption of IL-17 production or knockdown of IL-17 receptor inhibits diet-induced obesity and metabolic disorders, promoting adipose tissue beigeing, thermogenesis and energy expenditure. Mechanistically, IL-17A induces phosphorylation of the serine 273 site of PPARg in adipocytes in a Cyclin-dependent Kinase 5 (CDK5)dependent manner, which subsequently modifies the expression of obesity-associated genes. Interestingly, mothers exposed to HFD render the offspring having more IL-17 + ILC3s through microbiota, and increasing the offspring's susceptibility to intestinal injury. Further, the IL-17 blockade reversed the susceptibility to inflammation [bib_ref] Maternal High-Fat Diet Results in Microbiota-Dependent Expansion of ILC3s in Mice Offspring, Babu [/bib_ref]. Thus, whether IL-17 + ILC3s in the intestine are beneficial or adverse to metabolism remains paradoxical, which may depend on specific conditions.
## Regulation of intestinal ilc3s
Multiple signaling pathways regulate ILC3 responses in the intestine . Among these modulations, mTOR complex 1 (mTORC1) is critical for the proliferation of ILC3s and production of IL-22 and IL-17A after activation and Citrobacter rodentium infection [bib_ref] ILC3s Integrate Glycolysis and Mitochondrial Production of Reactive Oxygen Species to Fulfill..., Luccia [/bib_ref]. Moreover, the capacity of ILC3s presenting antigen to T cells is reduced by IL-23, which is also dependent on mTORC1 phosphorylation [bib_ref] Microbiota-Induced Tissue Signals Regulate ILC3-Mediated Antigen Presentation, Lehmann [/bib_ref]. Recent studies further reveal that both mTORC1 and mTORC2 control ILC3 cell numbers and ILC3-driven inflammation during colitis. mTOR signaling influences ILC3s in the intestine, leading to subsequent alteration in metabolic homeostasis. Notably, another study has reported that activation of ILC3s upon low oxygen challenge occurs via a HIF-1a-dependent mechanism instead of mTOR-signaling [bib_ref] Hypoxia Enhances ILC3 Responses Through HIF-1a-Dependent Mechanism, Fachi [/bib_ref]. P38 MAPK pathway also regulates the production of GM-CSF by ILC3s after activation of death receptor 3 (DR3) signaling [bib_ref] Activation of DR3 Signaling Causes Loss of ILC3s and Exacerbates Intestinal Inflammation, Li [/bib_ref]. PI3K-AKT or ERK signaling regulates the activation of ILC3s by Lysophosphatidylserine (LysoPS) from apoptotic neutrophils [bib_ref] GPR34-Mediated Sensing of Lysophosphatidylserine Released by Apoptotic Neutrophils Activates Type 3 Innate..., Wang [/bib_ref]. IL-17D acts via the CD93 on ILC3s to regulate the production of IL-22 [bib_ref] Interleukin-17D Regulates Group 3 Innate Lymphoid Cell Function Through Its Receptor CD93, Huang [/bib_ref] , whereas IL-7 activates ILC3s to secret IL-22 through aryl hydrocarbon receptor (AHR) and STAT3 [bib_ref] Fungal-Induced Glycolysis in Macrophages Promotes Colon Cancer by Enhancing Innate Lymphoid Cell..., Zhu [/bib_ref]. However, a recent study has reported that ILC3driven tissue repair is IL-22 and STAT3 independent. Instead, this occurs through activation of Src family kinases [bib_ref] Yap1-Driven Intestinal Repair Is Controlled by Group 3 Innate Lymphoid Cells, Romera-Hernańdez [/bib_ref].
Functions of ILC3s in the intestine are influenced by rhythmicity. Environmental light signals regulate intestinal ILC3s functions and further regulate the homeostasis of the intestine and the lipid metabolism in mice [bib_ref] Light-Entrained and Brain-Tuned Circadian Circuits Regulate ILC3s and Gut Homeostasis, Godinho-Silva [/bib_ref]. This concept is further supported by two distinct reports [bib_ref] A Circadian Clock Is Essential for Homeostasis of Group 3 Innate Lymphoid..., Teng [/bib_ref] [bib_ref] Circadian Rhythm-Dependent and Circadian Rhythm-Independent Impacts of the Molecular Clock on Type..., Wang [/bib_ref]. What's more, food intake affects the functions of ILC3s. ILC3s express vasoactive intestinal peptide receptor type 2 (VIPR2). Food induced-VIPR2 activation inhibits the secreting of IL-22 of ILC3 and epithelial anti-microbial response, thus enhancing the growth of segmented filamentous bacteria and increasing lipid absorption [bib_ref] Feeding-Dependent VIP Neuron-ILC3 Circuit Regulates the Intestinal Barrier, Talbot [/bib_ref]. In contrast, another study finds that VIP markedly increased the production of IL-22 of ILC3s. Lack of VIPR2 impaired the production of IL-22 of ILC3s and made mice more susceptible to DSS-induced gut injury [bib_ref] The Neuropeptide VIP Confers Anticipatory Mucosal Immunity by Regulating ILC3 Activity, Seillet [/bib_ref]. Meanwhile, VIP regulates the recruitment of intestinal ILC3s by increasing the gut-homing receptor CCR9 indirectly [bib_ref] Vasoactive Intestinal Peptide Promotes Host Defense Against Enteric Pathogens by Modulating the..., Yu [/bib_ref]. Whether the VIP-VIPR2 pathway in ILC3s inhibits or stimulates the production of IL-22 is still controversial.
Nutritional signals also regulate ILC3s. GPR183 and its ligand 7a,25-dihydroxycholesterol (7a,25-OHC) regulate the migration of ILC3s. GPR183-deficient mice have lower IL22 + ILC3s in the intestine and increased susceptibility to enteric bacterial infection [bib_ref] Anti-Microbial Functions of Group 3 Innate Lymphoid Cells in Gut-Associated Lymphoid Tissues..., Chu [/bib_ref]. Vitamin D/vitamin D receptor (VDR) signaling regulates the proliferation and function of ILC3 [bib_ref] Vitamin D/Vitamin D Receptor Signaling Is Required for Normal Development and Function..., He [/bib_ref]. Since VDR is also a receptor of bile acids [bib_ref] Microbial Bile Acid Metabolites Modulate Gut Rorg+ Regulatory T Cell Homeostasis, Song [/bib_ref] , bile acids may regulate gut ILC3s through VDR. Besides, colonic ILC3s express the receptor, Ffar2, which can sense microbial metabolites. Ffar2 activation by short-chain fatty acid (SCFA) increases IL-22 + ILC3s via an AKT and STAT3 axis and modulates gut homeostasis [bib_ref] Metabolite-Sensing Receptor Ffar2 Regulates Colonic Group 3 Innate Lymphoid Cells and Gut..., Chun [/bib_ref] [bib_ref] Dietary Fiber Metabolites Regulate Innate Lymphoid Cell Responses, Sepahi [/bib_ref] [bib_ref] Acetate Coordinates Neutrophil and ILC3 Responses Against C. Difficile Through FFAR2, Fachi [/bib_ref]. What's more, in Vitamin A-deficient mice, ILC3s are markedly reduced, which makes mice more susceptible to acute bacterial infection [bib_ref] Adaptation of Innate Lymphoid Cells to a Micronutrient Deficiency Promotes Type 2..., Spencer [/bib_ref]. Besides, erythritol can increase the number of ILC3s in the small intestine and markedly decrease metabolic disorders such as insulin resistance [bib_ref] Erythritol Ameliorates Small Intestinal Inflammation Induced by High-Fat Diets and Improves Glucose..., Kawano [/bib_ref]. Whether erythritol influences ILC3s directly or indirectly by short-chain fatty acids still needs further study.
Commensal microbes or their products also regulate the intestinal ILC3s. Symbiotic microbiota represses the production of IL-22 from ILC3s (167), while it indirectly induces the production of GM-CSF and IL-2 from ILC3s by increasing the interleukin-1b (IL-1b) from macrophages. GM-CSF and IL-2 in turn help maintain Treg cell numbers and intestinal homeostasis [bib_ref] Microbiota-Dependent Crosstalk Between Macrophages and Ilc3 Promotes Intestinal Homeostasis, Mortha [/bib_ref] [bib_ref] Innate Lymphoid Cells Support Regulatory T Cells in the Intestine Through Interleukin-2, Zhou [/bib_ref]. Besides, gut microbiota regulates ILC3s through bile acid metabolism [bib_ref] Microbial Bile Acid Metabolites Modulate Gut Rorg+ Regulatory T Cell Homeostasis, Song [/bib_ref] [bib_ref] Bile Acid Metabolites Control TH17 and Treg Cell Differentiation, Hang [/bib_ref]. Glycodeoxycholic acid induces intestinal ILC3s to secrete IL-22, improving insulin resistance [bib_ref] Gut Microbiota-Bile Acid-Interleukin-22 Axis Orchestrates Polycystic Ovary Syndrome, Qi [/bib_ref]. Interestingly, ketogenic diets alleviate colitis and reduce the activation of ILC3s [bib_ref] Ketogenic Diet Alleviates Colitis by Reduction of Colonic Group 3 Innate Lymphoid..., Kong [/bib_ref]. Furthermore, single-cell RNA-seq reveals that ILC3s integrate signals from the microbiota to alter phenotypic and functional plasticity [bib_ref] The Spectrum and Regulatory Landscape of Intestinal Innate Lymphoid Cells Are Shaped..., Gury-Benari [/bib_ref]. The gut microbiome and metabolic syndrome are closely linked [bib_ref] The Gut Microbiome and Metabolic Syndrome, Dabke [/bib_ref]. Whether gut microbiota influences the metabolism by regulating ILC3s still needs to be investigated.
Overall, ILC3s are involved in the development of obesity and insulin resistance through the production of IL-22 and IL-17. The signals regulating ILC3s may provide novel therapeutic approaches against obesity and metabolic disorders.
# Conclusion and perspectives
Multiple immune cells in the metabolic organs play diverse roles [fig_ref] TABLE 2 |: Summary of reported roles for adipose tissue immune cells [/fig_ref]. Here, we focus on the functions of ILCs in different metabolic organs in obesity and insulin resistance. In adipose tissue, NK cells and ILC1s trigger macrophage M1 polarization and thus contribute to inflammation, insulin resistance, and even adipose tissue fibrosis.ILC2s produce type 2 cytokines, orchestrate type 2 immunity and maintain metabolic homeostasis. Besides, ILC2s promote white adipose tissue beigeing, increasing energy expenditure and protecting against obesity and insulin resistance. However, the homeostasis of ILC2s is disrupted in obesity. ILC3s are present in human adipose tissue but not mice adipose tissue. The frequency and density of ILC3s increase with the BMI of obese patients. However, further analyses are required to clarify the function and mechanism of ILC3s in human adipose tissue. In the liver, NK cells and ILC1s prevent fibrosis, while ILC2s are profibrotic. These observations suggest a tissue-specific action for ILCs. Whether ILC3s promote or inhibit the progression of NAFLD is worth further investigation. In the pancreas, ILC2s and ILC3s regulate the development of type 2 and type 1 diabetes, respectively. Whether ILC3s in the pancreas are relevant to type 2 diabetes requires further investigation. In the intestine, ILC3s may either improve metabolic disorders through the production of IL-22 or promote metabolic disease by producing IL-17. The intestinal ILC3s are regulated by internal and external signals, which may further influence the [bib_ref] Brown Adipose Tissue Harbors a Distinct Sub-Population of Regulatory T Cells, Medrikova [/bib_ref] [bib_ref] Adipose Tissue-Resident Regulatory T Cells: Phenotypic Specialization, Functions and Therapeutic Potential, Cipolletta [/bib_ref] promote [bib_ref] Brown Adipose Tissue Harbors a Distinct Sub-Population of Regulatory T Cells, Medrikova [/bib_ref] gd T cells IL-17A and IL-17F inhibit [bib_ref] gd T Cells and Adipocyte IL-17RC Control Fat Innervation and Thermogenesis, Hu [/bib_ref] [bib_ref] gd T Cells Producing Interleukin-17A Regulate Adipose Regulatory T Cell Homeostasis and..., Kohlgruber [/bib_ref] promote [bib_ref] gd T Cells and Adipocyte IL-17RC Control Fat Innervation and Thermogenesis, Hu [/bib_ref] iNKT cells IL-2, IL-4, IL-10 inhibit [bib_ref] Regulatory iNKT Cells Lack PLZF Expression and Control Treg Cell and Macrophage..., Lynch [/bib_ref] [bib_ref] iNKT Cells Induce FGF21 for Thermogenesis and Are Required for Maximal Weight..., Lynch [/bib_ref] promote ( homeostasis of the intestine and the metabolism. ILC2s in the gut induce obesity through IL-2. Despite these findings, numerous questions remain unsolved relating to the roles of ILCs on metabolic disease. For example, can ILC3s affect metabolic homeostasis in response to the altered gut microbiota? Do ILC1s suppress adipose tissue beigeing by inducing macrophages' M1 polarization? What's the specific role of intestinal ILC2s and ILC3s in developing obesity and insulin resistance? Is there a specific population of ILC3s in the adipose tissue, and if yes, what function do they serve? Addressing these relevant questions will shed new light on the immune regulation of metabolism. Further research investigating the mechanisms of how ILCs influence metabolism may provide novel approaches for intervention of obesity and insulin resistance.
# Author contributions
HC: Original draft, review, and editing. LS and LF: Review and editing. YY and WZ: Supervision, review, and editing. All authors contributed to the article and approved the submitted version.
# Funding
[fig] FIGURE 1 |: NK cells and ILC1s in the adipose tissue and liver. (A) NK cells and ILC1s produce IFNg and TNFa to induce insulin resistance in obesity by inducing M1 macrophages and adipose tissue fibrosis. (B) NK cells in the liver prevent liver fibrosis by killing hepatic stellate cells (HSC) or inducing macrophages M1 polarization. In the obese liver, NK cells are more like ILC1s (39). The reduction of NK cell cytotoxicity may benefit the liver in NAFLD. [/fig]
[table] TABLE 1 |: Summary of ILCs depletion strategies. [/table]
[table] TABLE 2 |: Summary of reported roles for adipose tissue immune cells. [/table]
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Re-Examination of 30-Day Survival and Relapse Rates in Patients with Thrombotic Thrombocytopenic Purpura-Hemolytic Uremic Syndrome
Background and ObjectivesThrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are characterized by microangiopathic hemolytic anemia and thrombocytopenia. Interestingly, markedly different survival rates have been reported despite increases in survivability. We studied TTP-HUS 30-day mortality and relapse rates of patients who received TPE at our institution and compared them to published data.Patients and MethodsRetrospective study analyzed 30-day mortality and relapse rates attributed to TTP-HUS from 01/01/2008 to 12/31/2012 and compared them to comparable literature reporting mortality and survival. Studies describing other etiologies for TPE and different mortality time interval were excluded.ResultsFifty-nine patients were analyzed and all were initially treated with TPE and corticosteroids. Eleven patients were classified as not having TTP-HUS due to testing or clinical reassessment which ruled in other etiologies, and 18/59 patients had ADAMTS13 activity <10%. Of remaining patients, 36/48 (75%) were diagnosed as idiopathic and 12/48 (25%) as secondary TTP-HUS. Patients received a mean of 12 TPEs (range 1-42); 42/48 (87.5%) patients had ADAMTS13 activity measured; complete response obtained in 39/48 (81.2%) patients (platelet count >100 x 10 9 /L); partial response in 4/48 (8%); and 5/48 (10.4%) did not have increases in platelet counts (2/5 of these patients died within the study period). Forty percent of patients obtained platelet counts >150 x 10 9 /L. Overall 30-day mortality for our patient cohort was 6.7% (4/59). Comparison of our mortality rate to combined data of five published studies of 16% (92/571) showed a significant difference, p = 0.04. Our relapse rate was 18.6% (11/59) similar to previous reports.
# Introduction
Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are rare diseases characterized by microangiopathic hemolytic anemia (MAHA) and thrombocytopenia. HUS is characterized by three clinical signs: kidney failure, hemolytic anemia, and thrombocytopenia while TTP has been defined by a diagnostic pentad of thrombocytopenia, hemolytic anemia, fever, neurologic changes and renal compromise [bib_ref] Thrombotic thrombocytopenic purpura: report of 16 cases and review of the literature, Amorosi [/bib_ref]. However, the pentad associated with TTP is today mostly academic since most patients do not present with all symptoms at once [bib_ref] Therapeutic plasma exchange in patients with thrombotic thrombocytopenic purpura: a retrospective multicenter..., Korkmaz [/bib_ref].
At the center of the pathology initial reports listed deficiency in the von Willebrand Factor (vWF) metalloprotease ADAMTS13 as a marker of disease and as a potential factor that may aid in the differentiation of TTP from HUS [bib_ref] von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome, Furlan [/bib_ref] [bib_ref] Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura, Tsai [/bib_ref]. However, even though ADAMTS13 differences were originally described, not all patients presenting with the clinical diagnosis of TTP have a deficiency in this metalloprotease leading to the suggestion that it lacks importance in the diagnostic workup of these disease entities [bib_ref] Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic..., Remuzzi [/bib_ref] , and as predictor of clinical response to therapy which should be based mostly on clinical criteria [bib_ref] ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features..., Vesely [/bib_ref].
Ever since the results of the clinical trial by the Canadian Apheresis Study Group showed the benefits of therapeutic plasma exchange (TPE) vs. simple high volume plasma infusion, the survival of TTP patients has improved significantly from the high mortality seen a quarter of a century ago [bib_ref] Antiplatelet treatment of thrombotic thrombocytopenic purpura, Amorosi [/bib_ref] , and has led to a seven fold improvement in outcomes including those TTP patients with severe renal compromise [bib_ref] Comparison of plasma exchange with plasma infusion in the treatment of thrombotic..., Rock [/bib_ref] [bib_ref] Thrombotic thrombocytopenic purpura: outcome in 24 patients with renal impairment treated with..., Rock [/bib_ref]. In another study evaluating the efficacy of TPE vs. plasma infusion, the former was shown to be more beneficial in patients receiving disease-relief by plasma infusion but who did not respond well to the increased volume leading to the use of TPE in this subgroup [bib_ref] Severe ADAMTS13 deficiency in adult idiopathic thrombotic microangiopathies defines a subset of..., Coppo [/bib_ref].
Complications can occur in response to TPE, and the disease can still be fatal and patients can relapse despite aggressive therapy. However, reports for the last two decades have shown markedly differing survival and relapse rates during the critical treatment period with TPE. In the present study we determined the 30-day mortality rate and relapse of TTP-HUS patients at a large tertiary academic medical center and compared these to published literature reporting similar data.
# Materials and methods
## Study design and data collection
All patient information was anonymized and de-identified prior to analysis. This retrospective study evaluated the 30-day survival and relapse rates in patients with suspected TTP-HUS at University Hospitals Case Medical Center (UHCMC), a tertiary academic medical center, from January 1 st 2008 to December 31 st 2012. All individuals given a presumptive diagnosis of TTP-HUS by the hematology clinical service, referred to the Apheresis Center (AC) at UHCMC, and who were transferred to be treated with TPE by the AC were included in the study. None of the patients received TPE at transferring institutions. Patients who had undergone apheresis for other clinical indications were excluded. The inpatient and outpatient records of all patients were identified through AC records and the hospital's electronic medical record.
Two reviewers using standardized forms that we developed, independently collected and verified the accuracy of the data gathered which included: demographic information, duration of TPE treatment, number of TPE procedures, volume replaced, complete blood count (platelet count, hemoglobin/ hematocrit), creatinine at presentation and end of therapy, ADAMTS13 activity, neurological symptoms and/ or fever, 30 day-mortality and relapse, and presence of other co-morbidities. In order to ascertain and compare disease severity, a Clinical Severity Score (CSS) that has been previously described was used [bib_ref] Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS): a 24-year clinical experience with 178..., Levandovsky [/bib_ref]. Briefly, the four parameters used in clinically scoring patients were: neurologic symptoms, renal insufficiency, platelet counts and hemoglobin concentration, if available at the time of presentation. Score ranged from 0 to 8 points. Clinical diagnostic criteria followed recommendations previously outlined [bib_ref] How I treat patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, George [/bib_ref].
Patients were grouped into two categories: idiopathic if they had no other illness that is recognized to be associated with the symptoms at presentation; or secondary TTP-HUS if the patients had an identified trigger leading to the symptoms (e.g. stem cell transplantation, pregnancy/ postpartum, drugs, infections, autoimmune diseases, or malignancy-associated). The study was approved by the Institutional Review Board of UHCMC, Cleveland, OH.
## Patient outcome definitions
Remission, response and relapse were defined as previously described [bib_ref] Improved survival with plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic..., Lara [/bib_ref] [bib_ref] Survival and relapse in patients with thrombotic thrombocytopenic purpura, Hovinga [/bib_ref]. Briefly, remission was defined as an increase in platelet counts within a 30-day period after completion of TPE. Complete response was defined as a platelet count greater than 100 x 10 9 /L for two consecutive days (used for data comparison since three of four studies found in literature review use this platelet count to define recovery [bib_ref] Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS): a 24-year clinical experience with 178..., Levandovsky [/bib_ref] [bib_ref] Improved survival with plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic..., Lara [/bib_ref] [bib_ref] Therapeutic plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the..., Kim [/bib_ref] ; partial response as a platelet count in the 50-100 x10 9 /L range; and no response as platelet counts below 50 x10 9 /L. Survival was defined as achievement of remission. Mortality from TTP was defined as occurring within 30-days of stopping TPE. Relapse was defined as a decrease in platelet count below 100x10 9 /L, consistent with TTP after achievement of remission. All patients received corticosteroids at different times during their clinical presentations.
## Therapeutic plasma exchanges
All procedures were performed as previously described [bib_ref] Immature platelet fraction can help adjust therapy in refractory thrombotic microangiopathic hemolytic..., Kier [/bib_ref]. Briefly, all patients were initiated on daily TPE as soon as the presumed diagnosis of TTP-HUS was clinically made. TPE was performed daily with 1-1.5 plasma volume exchange using the COBE Spectra Apheresis System (Terumo BCT, Lakewood, CO). When indicated, patients were transfused to a hematocrit of 23% to increase the efficiency of TPE. Pre-and post-TPE fibrinogen, pro-thrombin time, partial thrombin time, ionized calcium and daily CBC were obtained. Patients were pre-medicated with 650 mg acetaminophen and 25 mg of diphenhydramine prior to TPE. All procedures used ABO-type-specific fresh-frozen plasma as replacement fluid and citrate dextrose-A solution as anticoagulation.
## Adamts13 activity
Forty one out of 48 (85.4%) patients had a sample collected for baseline ADAMTS13 activity prior to initiation of TPE but activity results were not available at the time of TPE initiation. ADAMTS13 activity assay was performed at the Blood Center of Wisconsin (BCW) (Milwaukee, WI) by the fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity as previously described [bib_ref] FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay, Kokame [/bib_ref]. When appropriate, metalloprotease inhibitor level was also measured by BCW.
## Literature search and study selection
A comprehensive literature search strategy was conducted to identify potential articles published in English on PUBMED from 1970 to 2013. Following search keyword combinations were used: TTP-HUS, mortality/outcome, and relapse. Our search algorithm is shown in [fig_ref] Fig 1: Study selection dendrogram used for data comparison [/fig_ref] Selected articles were examined by two reviewers independently. Inclusion criteria were: 1) Patients with initial diagnosis of TTP-HUS, 2) 30-days mortality rate, and 3) relapse rate reported.
# Statistical analysis
All statistics were performed using Prism 6 (GraphPad Software Inc., La Jolla, CA). Since the number of studies we found in the literature review was small and we did not have all of their raw numerical data, we were unable to perform a meta-analysis so we limited our analysis to comparisons of their results to ours. Mortality and relapse rates comparisons with similar combined data from five relevant articles were performed using a two-tailed Fisher test with a p< 0.05 set for significance.
# Results
## Uhcmc patients
A total of 59 patients were analyzed; 11 patients had TPE discontinued as requested by the clinical team due to other diagnoses that explained these patients' presentations. These patients were eventually diagnosed with disseminated intravascular coagulation, acute myeloid leukemia with marrow involvement, scleroderma, idiopathic thrombocytopenic purpura, malignant hypertension and sepsis/ bacteremia. Patients responded to treatment modalities which addressed each of these diagnoses after discontinuation of TPE.
The remaining 48 patients were diagnosed with TTP-HUS and received daily TPEs. The demographic characteristics of these patients at diagnosis are summarized in [fig_ref] Table 1: UHCMC study patients' characteristics at initial presentation with TTP-HUS [/fig_ref]. Idiopathic TTP-HUS was diagnosed in 36 patients (75%) and secondary causes in 12 patients (25%). Thirty-three (69%) of patients were female, mean age of 49 (range 16-85); while 15 (31%) patients were males with mean age of 47.5 (range 28-76). There were a number of co-morbidities in our patient cohort which were as follows: two patients were pregnant/ postpartum (4%); 3 patients had a drug-associated presentation (6%); 2 patients had autoimmune disease (4%); 1 patient had a systemic infection (2%); 4 patients had malignancy/ metastasis (8%); and 1 patient had received a renal allograft (2%). Thirty-day mortality among TTP-HUS patients was 1/48 patients (2%). However, overall mortality for our patient cohort was 4/59 (6.7%) since 3 additional patients died of causes unrelated to TTP-HUS: one of these patients died due to complications of engraftment failure and graft vs. host disease (outside 30 day study period); a second died of multi-organ failure due to sepsis and bacteremia in the setting of disseminated terminal Burkitt's lymphoma complicated by renal allograft rejection; and a third died of heart failure due to worsening ejection fraction and heart disease. Only one of the four patients who died in our cohort had an autopsy with findings that were consistent with a TTP presentation [bib_ref] Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome are distinct pathologic entities. A..., Hosler [/bib_ref] and had ADAMTS13 deficiency (<5%). One male patient was treated with two cycles of TPE 3 months apart due to relapse and a second male was treated three times with TPE within a year from initial diagnosis (data not shown).
## Patients' laboratory findings
As shown in [fig_ref] Table 2: Patient outcomes at UHCMC and comparison with relevant studies describing 30-day mortality... [/fig_ref] , mean platelet count at presentation was 46 x 10 9 / L (range 5 x 10 9 / L-307 x 10 9 / L). Mean hemoglobin was 9.0 g/dl (range 2.5-13.4 g/dl); mean creatinine concentration was 2.6 mg/dL (range 0.5-11.3 mg/dL). Mean CSS for our cohort was 4. Of note, baseline values and clinical severity score at our institution may not reflect the values/score at initial presentation because a few patients were transferred to our institution after steroid therapy was initiated at outside hospitals.
All patients started receiving and underwent TPE at our institution performed according to the manufacturer's recommendations as well as our standard operating procedure. The daily procedures were done until achieving the target platelet level. Study patients received a mean of 12 TPEs (range 1-42); complete response was obtained in 81.2% of patients (N = 39/48) (platelet count > 100 x 10 9 /L); partial response in 8.3% of patients (N = 4/48) and 10.4% had no response (N = 5/48). Forty percent (N = 19/48) of the patients obtained platelet counts greater than 150 x 10 9 /L.
## Comparison with published literature
We pooled the data of five studies that met the selection criteria [fig_ref] Fig 1: Study selection dendrogram used for data comparison [/fig_ref] [fig_ref] Table 2: Patient outcomes at UHCMC and comparison with relevant studies describing 30-day mortality... [/fig_ref]. These five studies [bib_ref] Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS): a 24-year clinical experience with 178..., Levandovsky [/bib_ref] [bib_ref] Improved survival with plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic..., Lara [/bib_ref] [bib_ref] Therapeutic plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the..., Kim [/bib_ref] [bib_ref] The Oklahoma Thrombotic Thrombocytopenic Purpura-Hemolytic Uremic Syndrome (TTP-HUS) Registry: a community perspective..., George [/bib_ref] [bib_ref] Hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: outcome with plasma exchange, Roberts [/bib_ref] ] described a total of 660 patients, 571 of which were used to calculate the mortality rate. Clinical Severity Score was derived only for two of the studies. Mean number of TPE are reported by 3/5 studies and practically all patients used in the statistical analysis received TPE. As shown in [fig_ref] Fig 2: Thirty-day mortality and relapse in TTP-HUS [/fig_ref] our overall mortality rate was significantly lower than the combined reported rate of all studies (4/59 (6.7%) vs. 92/571 (16%) respectively; p = 0.04). Our relapse rate, 11/59 (18.6%), was not significantly different from that which has been reported for the other studies used in our analysis (range 12-28%). However, when only those patients with TTP-HUS are taken into account the mortality rate (1/48) was more significant (p = 0.0053).
# Discussion
We have defined the clinical outcome of relapse and 30-day mortality in a population of 48 patients with a diagnosis of TTP-HUS. TPE was used as therapy for all patients whether the condition was idiopathic or secondary. Of interest, our 30-day mortality rate was significantly lower than what has been previously published but our relapse rate was similar to published reports. The marked difference in mortality rates may be difficult to dissect but in order to (1/48); however, overall mortality for patient cohort was 6.7% (4/59) (3 patients died of causes unrelated to TTP-HUS diagnosis (engraftment failure and graft vs. host disease, multi-organ failure due to sepsis and bacteremia, and heart failure due to worsening ejection fraction). Pooled 30-day mortality rate is 16.1% (92-571), p = 0.04. Relapse rate between our entire patient cohort (18.6%) and that of pooled data (14.3%). minimize confounding biases we compared our cohort findings to studies also reporting 30-day mortality rates and similar patient populations to ours since we were unable to perform a meta-analysis due to the small number of studies and limited raw data provided in these publications An exhaustive search for treatment modalities/ interventions in published literature which included patients with either TTP or HUS found that patients with TTP tend to respond to TPE vs. patients with typical/ diarrheal HUS who respond best to supportive therapy that includes dialysis [bib_ref] Interventions for haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura, Michael [/bib_ref]. At our institution, all patients were given immunosuppression in the form of steroids which was given and discontinued at different intervals during TPE regimen as previously described [bib_ref] Immature platelet fraction can help adjust therapy in refractory thrombotic microangiopathic hemolytic..., Kier [/bib_ref]. This is similar to what has been described for a similar size patient cohort [bib_ref] Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS): treatment outcome, relapses, prognostic factors. A..., Dervenoulas [/bib_ref]. Therefore, the combination of immunosuppression and TPE may account for our improved survival. However, it did not have a role in decreasing relapse rates. In any case this is the therapeutic approach taken by most institutions (standard of care) and it is unlikely to explain our mortality differences with published reports. As a result, inherent differences in the patient cohorts which lead to marked differences in mortality may begin to explain our observations.
Studies which have grouped patients under TTP-HUS as a single clinical entity find that responses differ depending on the elements identified in the clinical presentation and patients' gender [bib_ref] Therapeutic plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the..., Kim [/bib_ref]. Likewise, ABO blood group may be an independent risk factor for patients with TTP who have severe ADAMTS13 deficiency [bib_ref] Blood group O and black race are independent risk factors for thrombotic..., Terrell [/bib_ref]. As a result, potential reasons for these reported differences include heterogeneity of patients diagnosed with TTP-HUS and serious underlying co-morbid conditions.
Since the majority of our patients had idiopathic TTP-HUS of which half had severe ADAMTS13 deficiency (TTP diagnosis), may make our better survival even more significant. Of interest, a recent report from our group showed that the response to TPE in patients with ADAMTS13 deficiency follows similar kinetics in platelet count recovery. There is overlap between the two syndromes and the distinction between HUS and TTP based on routine laboratory and clinical presentation is at times challenging which has led to the unifying diagnosis of TTP-HUS to describe this patient population. For these reasons the term unifying both diagnoses into a single entity has been introduced [bib_ref] Thrombotic thrombocytopenic purpura and related disorders, Ruggenenti [/bib_ref]. However, if two potentially pathologically different disease entities were grouped, it could lead to differences in mortality rates and give creed to alternative suggestions for the markedly lower mortality rate that we observed in our patient cohort. In addition, unlike other studies included in our analysis, most of our patients had ADAMTS13 activity measured which was helpful in sub-stratifying our patient cohort. However, even subdividing patients according to ADAMTS13 activity still yielded a lower mortality rate than those prior reports.
The grouping of TTP and HUS into a unifying single pathologic entity may not represent a uniform way of studying, reporting and comparing these patients that is reproducible. A great deal of debate has placed by some investigators these two potential diseases under the same clinical umbrella as manifestations of a single disease continuum [bib_ref] How I treat patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, George [/bib_ref] [bib_ref] Thrombotic thrombocytopenic purpura and related disorders, Ruggenenti [/bib_ref] [bib_ref] TTP: variable expression of a single entity, Remuzzi [/bib_ref]. However, grouping these two clinical entities may oversimplify these two complex syndromic presentations that fails to recognize previously reported markedly different histopathologic findings in a large cohort of autopsied patients with either TTP or HUS which showed non-overlapping pathological signs, presentations that were distinct, and do not support the view that they represent a continuum of the same disease process [bib_ref] Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome are distinct pathologic entities. A..., Hosler [/bib_ref]. Similarly, a meta-analysis looking at both clinical entities found that TTP patients respond to TPE leading to restoration of platelet count while HUS patients do not fully benefit from plasma therapy [bib_ref] Long-term renal prognosis of diarrhea-associated hemolytic uremic syndrome: a systematic review, meta-analysis,..., Garg [/bib_ref].
Molecularly, ADAMTS13 deficiency has to happen in the context of a "second hit" which is necessary for the clinical presentation to occur [bib_ref] Animal models for thrombotic thrombocytopenic purpura, Vanhoorelbeke [/bib_ref]. These could come in the form of both inhibitory and non-inhibitory antibodies to ADAMTS13 which could begin to explain why not all patients present with severe metalloprotease deficiency and that may also depend on the subjects' genetic susceptibility as seen in different strains of ADAMTS13 knockout mice [bib_ref] Animal models for thrombotic thrombocytopenic purpura, Vanhoorelbeke [/bib_ref]. However, all ADAMTS13 deficient mice have shown a pro-thrombotic phenotype. The second hit could be in the form of an increase in vWF as shown in a TTP mouse model in which disease severity improved with the use of recombinant human ADAMTS13 [bib_ref] A new mouse model mimicking thrombotic thrombocytopenic purpura: correction of symptoms by..., Schiviz [/bib_ref]. Likely, disease dynamics of TTP and HUS may prove to be independent of each other with few if any intermediates mediating both pathologic presentations. In the case of TTP the correlation with ADAMTS13 deficiency may be stronger than the metalloprotease's link to HUS [bib_ref] Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS): a 24-year clinical experience with 178..., Levandovsky [/bib_ref]. Of note, though there is a wide variety of animal models that are used to determine the pathologic mediators behind TTP and HUS, these models have not yielded any shared molecule that is involved in the pathology of both [bib_ref] Animal models for thrombotic thrombocytopenic purpura, Vanhoorelbeke [/bib_ref]. More basic research is needed to dissect these possibilities further.
Additionally, promptness of TPE initiation is essential for patient survival [bib_ref] Timing of plasma exchange therapy for thrombotic thrombocytopenia purpura: a brief clinical..., Colflesh [/bib_ref] since in those whose diagnosis is delayed are more likely to die than those promptly treated [bib_ref] Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS): treatment outcome, relapses, prognostic factors. A..., Dervenoulas [/bib_ref]. Another possibility for our better outcomes is that at our institution TPE could be initiated earlier than others, based on clinical practice that patients with MAHA and thrombocytopenia of unknown etiology is suspected of TTP-HUS until proven otherwise. This is however unlikely. Along these lines, it has been proposed that adults with idiopathic disease characterized by unexplained thrombocytopenia, MAHA, normal international normalized ratio, partial thromboplastin time and D-dimer may have TTP-HUS [bib_ref] Thrombotic microangiopathy: current knowledge and outcomes with plasma exchange, Clark [/bib_ref]. However, this algorithm may be more based in caution than in actual evidence that these two disease entities are isoforms of a single syndromic presentation due to valid concerns that failure to timely treat a TTP diagnosis will lead to a potentially poor and complicated clinical outcome [bib_ref] Thrombotic microangiopathy: current knowledge and outcomes with plasma exchange, Clark [/bib_ref].
Differences in survival could also be due to lack of uniformity in plasma exchange practice, apheresis equipment used (COBE, membrane filtration method [bib_ref] Therapeutic plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the..., Kim [/bib_ref] ; Staphylococcal protein A absorption column; [bib_ref] Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS): a 24-year clinical experience with 178..., Levandovsky [/bib_ref] Fenwal CS-3000 Blood Separator and Haemonetics model V50 [bib_ref] Improved survival with plasma exchange in patients with thrombotic thrombocytopenic purpura-hemolytic uremic..., Lara [/bib_ref] ; use of plasma infusion only [bib_ref] The Oklahoma Thrombotic Thrombocytopenic Purpura-Hemolytic Uremic Syndrome (TTP-HUS) Registry: a community perspective..., George [/bib_ref] ; and as adjunctive therapy [bib_ref] Treating TTP/HUS with plasma exchange: a single centre's 25-year experience, Forzley [/bib_ref]. Our institution has a TPE protocol, which is universally applied for all patients presenting with suspected TTP-HUS; additionally, as a tertiary referral center for others in our region, patients may begin adequate treatment prior to transfer or may not have survived their disease prior to transfer. These findings may begin to explain the observed discrepancies among studies.
In summary, our findings reinforce that early diagnosis; promptness of TPE initiation and use of TPE as standard treatment in the setting of TTP-HUS is essential to avoid the high 30-day mortality rate. Differences in survival may be due to lack of uniformity among the patient cohorts reported by us and others. Survival of TTP-HUS patients continues to improve; however, the marked differences in survivability need to be addressed with prospective studies that take into account the actual differences between these two disease presentations and negate the grouping of both under a single pathological entity.
[fig] Fig 1: Study selection dendrogram used for data comparison. All five studies reported 30-day mortality and relapse in their study cohorts. doi:10.1371/journal.pone.0127744.g001 Re-Examination of 30-Day Survival and Relapse in TTP-HUS PLOS ONE | DOI:10.1371/journal.pone.0127744 May 22, 2015 [/fig]
[fig] Fig 2: Thirty-day mortality and relapse in TTP-HUS. Thirty-day mortality attributed to TTP-HUS was 2% [/fig]
[table] Table 1: UHCMC study patients' characteristics at initial presentation with TTP-HUS. [/table]
[table] Table 2: Patient outcomes at UHCMC and comparison with relevant studies describing 30-day mortality and relapse.In the overall cohort of 59 patients, those with severe ADAMTS13 deficiency (10%) required more TPE than those patients with enzyme deficiency >10% (mean 19.1 vs. 7.2). If the patients with ADAMTS13 deficiency are further subdivided into moderate deficiency (11%-40%), mild deficiency (41%-66%) and no deficiency (67%) there was no difference in the number of TPE in each subgroup (data not shown). For those patients who had a diagnosis of TTP-HUS and enzyme activity >10% the mean number of TPE received was 8.2. [/table]
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Glial functions in the blood-brain communication at the circumventricular organs
CITATION Miyata S (2022) Glial functions in the blood-brain communication at the circumventricular organs. Front. Neurosci. 16:991779.
The circumventricular organs (CVOs) are located around the brain ventricles, lack a blood-brain barrier (BBB) and sense blood-derived molecules. This review discusses recent advances in the importance of CVO functions, especially glial cells transferring periphery inflammation signals to the brain. The CVOs show size-limited vascular permeability, allowing the passage of molecules with molecular weight <10,000. This indicates that the lack of an endothelial cell barrier does not mean the free movement of blood-derived molecules into the CVO parenchyma. Astrocytes and tanycytes constitute a dense barrier at the distal CVO subdivision, preventing the free diffusion of blood-derived molecules into neighboring brain regions. Tanycytes in the CVOs mediate communication between cerebrospinal fluid and brain parenchyma via transcytosis. Microglia and macrophages of the CVOs are essential for transmitting peripheral information to other brain regions via tolllike receptor 2 (TLR2). Inhibition of TLR2 signaling or depletion of microglia and macrophages in the brain eliminates TLR2-dependent inflammatory responses. In contrast to TLR2, astrocytes and tanycytes in the CVOs of the brain are crucial for initiating lipopolysaccharide (LPS)-induced inflammatory responses via TLR4. Depletion of microglia and macrophages augments LPSinduced fever and chronic sickness responses. Microglia and macrophages in the CVOs are continuously activated, even under normal physiological conditions, as they exhibit activated morphology and express the M1/M2 marker proteins. Moreover, the microglial proliferation occurs in various regions, such as the hypothalamus, medulla oblongata, and telencephalon, with a marked increase in the CVOs, due to low-dose LPS administration, and after high-dose LPS administration, proliferation is seen in most brain regions, except for the cerebral cortex and hippocampus. A transient increase in the microglial population is beneficial during LPS-induced inflammation for attenuating sickness response. Transient receptor potential receptor vanilloid 1 expressed in astrocytes and tanycytes of the CVOs is responsible for thermoregulation upon exposure to a warm environment less than 37 - C. Schematic representation revealing CVOs locations in the adult rodent brain. Insets indicate that the CVOs are characterized by dense and thick laminin + (green) capillaries and dense GFAP + (red) astrocytic and tanycytic networks. Scale bar = 50 µm. AP, area postrema; CP, choroid plexus; GFAP, glial fibrillar acidic protein; ME, median eminence; NH, neurohypophysis; OVLT, organum vasculosum of the lamina terminalis; PG, pineal gland; SFO, subfornical organ. The illustration is rearranged with permission from Elsevier B.V. [bib_ref] Microglia are continuously activated in the circumventricular organs of mouse brain, Takagi [/bib_ref].
signal transduction from the periphery to the brain, and (3) Maintenance of body fluid and thermal homeostasis by glial Na x channel and transient receptor potential vanilloid 1 (TRPV1), respectively.
## Size-limited vascular permeability at fenestrated capillaries
The BBB is important for normal brain physiology and protects neuronal tissues by limiting the ingress of bloodderived bioactive and toxic hydrophilic molecules. Disturbance of the BBB leads to the accumulation of these molecules within the parenchyma causing severe brain damage [bib_ref] Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders, Zlokovic [/bib_ref] [bib_ref] Development, maintenance and disruption of the blood-brain barrier, Obermeier [/bib_ref]. Mice deficient in tight junction proteins such as claudin-5 or ZO-1 die due to blood-derived toxic molecules accessing the brain [bib_ref] Endothelial claudin: Claudin-5/TMVCF constitutes tight junction strands in endothelial cells, Morita [/bib_ref] [bib_ref] Conversion of zonulae occludentes from tight to leaky strand type by introducing..., Furuse [/bib_ref]. However, CVO vasculature lacks the typical BBB, and possesses fenestrated features unlike those in other brain regions [bib_ref] A new method for visualization of endothelial cells and extravascular leakage in..., Miyata [/bib_ref] [bib_ref] Different vascular permeability between the sensory and secretory circumventricular organs of adult..., Morita [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] [bib_ref] Advances in understanding of structural reorganization in the hypothalamic neurosecretory system, Miyata [/bib_ref]. For example, levels of glutamate, an excitatory transmitter, in plasma and brain is in the range of 50-100 and 0.5-2 µmol/l, respectively [bib_ref] The blood-brain barrier and glutamate, Hawkins [/bib_ref]. Even after oral ingestion of large quantities of glutamate, only small changes in glutamate plasma concentration occur [bib_ref] Plasma and erythrocyte amino acid levels in normal adult subjects fed a..., Stegink [/bib_ref]. The subcutaneous administration of high-dose monosodium glutamate increases fivefold increase of glutamate levels in the SFO and AP [bib_ref] Uptake of exogenous glutamate and aspartate by circumventricular organs but not other..., Price [/bib_ref]. The administration of monosodium glutamate also induces neuronal loss in the ME and arcuate nucleus (Arc) of adult [bib_ref] Acute lesioning and rapid repair of hypothalamic neurons outside the blood-brain barrier, Yulyaningsih [/bib_ref] [bib_ref] MyD88 deficiency, but not gut microbiota depletion, is sufficient to modulate the..., Heiss [/bib_ref] and the CVOs of neonatal rodents [bib_ref] Nature and extent of brain lesions in mice related to ingestion of..., Lemkey-Johnston [/bib_ref]. Many studies demonstrate the free diffusion of LMW molecules into the CVOs parenchyma: fluorescein (MM = 332), fluorescein isothiocyanate (FITC; MM = 390), Evans blue (MM = 961), and Dextran 3,000 (MW = 3,000) [bib_ref] A size selective vascular barrier in the rat area postrema formed by..., Willis [/bib_ref] [bib_ref] Different vascular permeability between the sensory and secretory circumventricular organs of adult..., Morita [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] [bib_ref] Structural reconstruction of the perivascular space in the adult mouse neurohypophysis during..., Nishikawa [/bib_ref]. The molecular weight of these tracer molecules is within the range of bioactive neuropeptides, amino acids, or amines; e.g., adenohypophyseal hormone-releasing hormones such as thyrotropin-releasing hormone (minimum size at MW = 362.4) to growth-hormone-releasing hormone (maximum size at MW = 5040.4). In contrast to LMW molecules, blood-derived high-molecular-weight (HMW) molecules, more than MW 10,000, do not freely enter the CVOs parenchyma [bib_ref] A size selective vascular barrier in the rat area postrema formed by..., Willis [/bib_ref] [bib_ref] Different vascular permeability between the sensory and secretory circumventricular organs of adult..., Morita [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. LMW soluble molecules (<3 nm molecular radius) are presumed to move passively through endothelial intercellular space in BBB-lacking capillaries [bib_ref] Signaling mechanisms regulating endothelial permeability, Mehta [/bib_ref] [bib_ref] Regulation of endothelial permeability via paracellular and transcellular transport pathways, Komarova [/bib_ref]. Thus, the CVOs endothelial intercellular space exhibits a size-limited vascular permeability lower than approximately MW 10,000 [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] [bib_ref] Advances in understanding of structural reorganization in the hypothalamic neurosecretory system, Miyata [/bib_ref]. However, it is often misunderstood that the fenestrated capillaries of the CVOs allow molecules of any size to enter the parenchyma.
High-molecular-weight molecules cannot pass freely through fenestrate capillaries in the CVOs. HMW molecules such as hormones and cytokines reach the brain parenchyma beyond the BBB via transcytosis [bib_ref] The crosstalk between brain and periphery: Implications for brain health and disease, Gonçalves [/bib_ref]. Recently, more than a thousand proteins, including various cytokines and growth factors, readily permeated brain parenchyma with the transcytosis system by BBB-specific transcriptional programs [bib_ref] Physiological blood-brain transport is impaired with age by a shift in transcytosis, Yang [/bib_ref]. Autoradiographic and fluorescent microscopic techniques show heterogenous transcytosis of blood proteins within the brain, especially prominent at the basolateral hypothalamus, including the ME, Arc, and ventromedial hypothalamic nucleus [bib_ref] Physiological blood-brain transport is impaired with age by a shift in transcytosis, Yang [/bib_ref]. Blood-derived leptin activates leptin receptors in tanycytes of the ME and is transported along the tanycytic cellular process and secreted into the CSF . Blood-derived horseradish peroxidase extensively accumulates between the inner and outer BM in the NH and ME, but it also reaches the brain parenchyma, possibly via mannose receptor-mediated transcellular routes [bib_ref] Receptor-mediated and fluid-phase transcytosis of horseradish peroxidase across rat hepatocytes, Ellinger [/bib_ref] [bib_ref] The design of barriers in the hypothalamus allows the median eminence and..., Rodríguez [/bib_ref].
## Astrocytic and tanycytic barrier instead of endothelial blood-brain barrier
The basement membrane of fenestrated capillaries is permeable to blood-derived LMW molecules, which easily reach the CVOs parenchyma; however, they do not diffuse out of the CVO, as shown in Figures 2A-C [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. The capillaries of central subdivisions in the sensory CVOs possess a higher vascular permeability than those of distal subdivisions due to the lack of tight-junction protein expression in the endothelial cells [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. Astrocytes in the distal regions of the sensory CVOs become hypertrophic and their density is remarkably high [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. Tanycytes in the OVLT and SFO, but not in the AP, extend cellular processes into the surrounding neuronal somata and fenestrated capillaries, connecting tightly [bib_ref] Sodium sensing in the brain, Noda [/bib_ref] [bib_ref] Anatomical organization of the rat organum vasculosum laminae terminalis, Prager-Khoutorsky [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. Astrocytes and tanycytes often express several kinds of tight junction proteins (Figures 2D,E; [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] , and electron microscopy reveals junctions between intimately apposed astrocytic or tanycytic cellular membranes; [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. Astrocytes are reported to express ZO-1 and occludin in the periphery of infarct areas during the stroke recovery process to prevent ingress of neurotoxic molecules [bib_ref] Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed..., Yang [/bib_ref] [bib_ref] Early inhibition of MMP activity in ischemic rat brain promotes expression of..., Yang [/bib_ref]. Thus, the astrocytes and tanycytes, densely located at distal CVO subdivisions, are an alternative to the endothelial BBB and prevent blood-derived LMW molecules from diffusing into neighboring brain regions.
Inability of blood-derived high-molecular-weight molecules to cross fenestrated capillaries into the parenchyma In contrast to the high vascular permeability of bloodderived LMW molecules in the CVOs fenestrated capillaries, HMW molecules, MW > 10,000, can cross the outer basement membrane but do not diffuse into parenchyma past the inner basement membrane and accumulate between these membranes [bib_ref] Different vascular permeability between the sensory and secretory circumventricular organs of adult..., Morita [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] [bib_ref] Structural reconstruction of the perivascular space in the adult mouse neurohypophysis during..., Nishikawa [/bib_ref]. Continuous capillaries in the typical BBB-containing brain vasculature possess a narrow perivascular space surrounded by inner and outer basement membrane generally fused, whereas fenestrate capillaries in the CVO have a large and complex perivascular space [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. Increased collagen type I expression is detected on the inner basement membrane compared with the outer basement membrane, whereas laminin expression is increased in the outer basement membrane compared with the inner [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] [bib_ref] Structural reconstruction of the perivascular space in the adult mouse neurohypophysis during..., Nishikawa [/bib_ref]. These features are also seen in the kidney glomerulus, the glomerular basement membrane contributing to the size-limited kidney filtration barrier that separates proteins and LMW molecules such as glucose and urea [bib_ref] Glomerular basement membrane and related glomerular disease, Chen [/bib_ref] [bib_ref] The glomerular basement membrane, Miner [/bib_ref]. Mutations in either laminin or collagen type IV result in proteinuria, often leading to nephrotic syndrome and renal failure [bib_ref] Glomerular basement membrane and related glomerular disease, Chen [/bib_ref]. Thus, like typical BBB-containing capillaries, fenestrated capillaries of the CVOs prevent parenchyma access to HMW molecules beyond the inner basement membrane. Blood-derived albumin in the brain parenchyma causes neurodegeneration [bib_ref] Beta-caryophyllene attenuates short-term recurrent seizure activity and blood-brain-barrier breakdown after pilocarpineinduced status..., Mallmann [/bib_ref] , demonstrating the neurotoxicity of blood-derived HMW molecules. However, this evidence does not proscribe all HMW molecule access to the parenchyma, as recent studies found that active transport of blood-derived HMW molecules into the brain parenchyma occurs by ligand-specific receptor-mediated and non-specific caveolar transcytosis [bib_ref] Receptor-mediated and fluid-phase transcytosis of horseradish peroxidase across rat hepatocytes, Ellinger [/bib_ref] [bib_ref] Physiological blood-brain transport is impaired with age by a shift in transcytosis, Yang [/bib_ref] [bib_ref] The crosstalk between brain and periphery: Implications for brain health and disease, Gonçalves [/bib_ref].
## Tanycytic transcytosis mediated cerebrospinal fluid-brain communication in the circumventricular organs
Tanycytes are specialized ependymal cells interfacing with the CSF and have a long basal process that terminates at the capillary [bib_ref] The fiber glia of selacean brain, Horstmann [/bib_ref] [bib_ref] Hypothalamic tanycytes: A key component of brain-endocrine interaction, Rodríguez [/bib_ref]. Ependymal cells in the Arc and ME are the best-known tanycytes and have a single, long cellular process projecting to discrete hypothalamic parenchyma and fenestrated capillaries [bib_ref] Cellular organization of the lateral and postinfundibular regions of the median eminence..., Rodríguez [/bib_ref] [bib_ref] Hypothalamic tanycytes: A key component of brain-endocrine interaction, Rodríguez [/bib_ref]. Tanycytes also exist at the ventricular borders of the OVLT and SFO [bib_ref] Differential distribution of tight junction proteins suggests a role for tanycytes in..., Mullier [/bib_ref]. A honeycomb distribution pattern of tight junction proteins is observed at tanycytes around the CVO proximal cell bodies, possibly to prevent the diffusion of blood-derived molecules into the CSF [bib_ref] Differential distribution of tight junction proteins suggests a role for tanycytes in..., Mullier [/bib_ref]. Hypothalamic tanycytes also reduce the release into circulation of thyrotropinreleasing hormone by activating the tanycytic receptor for Vascular permeability and tight junctions in the CVOs of the adult mouse brain. Strong fluorescence of blood-derived fluorescein isothiocyanate is seen at the central subdivision of the sensory CVOs, but fluorescence is weak at distal subdivisions and no fluorescence is seen in the neighboring brain region beyond dense astrocytic and tanycytic barriers (A-C). Tight junction protein occludin is seen at parenchyma close to fenestrated capillaries and tanycytic layers (D,E). An electron microscopic image reveals the tight junction-like structure between the plasma membrane of neighboring cells making complete contact (F). Scale bar = 50 µm (A,C,D) and 100 nm (F). AP, area postrema; CC, central canal; D3V, dorsal 3rd ventricle; FITC, fluorescein isothiocyanate; GFAP, glial fibrillar acidic protein; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ. Photographs are rearranged with permission from Springer Nature [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref].
thyrotropin-releasing hormone . However, circulating leptin is recognized by leptin receptors on tanycytes in the ME, then extracellular signal-regulated kinase signaling of tanycytes promotes leptin transport, activating neurons in the mediobasal hypothalamus . Tanycytes participate in the release of gonadotropin-releasing hormone by the spatial relationship between the tanycytes and gonadotropin-releasing hormone-secreting terminals [bib_ref] Hypothalamic tanycytes: Potential roles in the control of feeding and energy balance, Bolborea [/bib_ref]. Hypothalamic tanycytes also reduce the release of thyrotropin-releasing hormone into the circulation by activating the tanycytic receptor for thyrotropin-releasing hormone . Active transcytosis occurs at tanycytes in the CVOs of the adult mouse brain, as in the Arc [bib_ref] Transcytosis of tanycytes in the circumventricular organs of adult mouse, Okamoto [/bib_ref]. The CVOs can, by tanycytic transcytosis, transport bioactive molecules between the CSF and CVO, suggesting that the long cellular processes of tanycytes act as communication routes between the CSF and brain parenchyma.
## Circumventricular organ tanycytes are neural stem cells
Tanycytes in the ME and Arc are shown to be a neurogenic niche in adult mammals [bib_ref] α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive..., Robins [/bib_ref]. Tanycytes in the spinal cord are NSCs that, remarkably, proliferate and differentiate into astrocytes and oligodendrocytes upon spinal injury [bib_ref] Spinal cord injury reveals multilineage differentiation of ependymal cells, Meletis [/bib_ref]. Recently we have found two types of NSCs in the CVOs; astrocyte-and tanycyte-like NSCs [bib_ref] Neurogenesis in the circumventricular organs of adult mouse brains, Hourai [/bib_ref] [bib_ref] Characterization of neural stem cells and their progeny in the sensory circumventricular..., Furube [/bib_ref]. Radial glial cells in the ventricle zone are NSCs that, during cortical development, produce new neurons and glial cells [bib_ref] Molecular pathways underlying projection neuron production and migration during cerebral cortical development, Ohtaka-Maruyama [/bib_ref]. Tanycytes in the adult mammalian brain are NSC precursors, ultimately derived from radial glia during development [bib_ref] Hypothalamic tanycytesmasters and servants of metabolic, neuroendocrine, and neurogenic functions, Goodman [/bib_ref]. The CSF provides important functions to support brain homeostasis, morphogenesis, and proliferation during development [bib_ref] The choroid plexuses and their impact on developmental neurogenesis, Johansson [/bib_ref]. In adults, the CSF actively synthesizes and secretes factors that promote the proliferation of quiescent and transit-amplifying NSCs in the subventricular zone . Aging speed is controlled primarily by hypothalamic NSCs via levels of exosomal miRNAs in the CSF . Moreover, continuous angiogenesis occurs in the CVOs of adult mice, possibly reconstructing neurons, glia, and fenestrated capillaries [bib_ref] VEGF-dependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice, Furube [/bib_ref] [bib_ref] Proliferation of endothelial cells in the choroid plexus of normal and hydrocephalic..., Asami [/bib_ref]. Thus, tanycytes, throughout the adult mammalian brain, are accepted as NSCs, with their proliferation and differentiation potentially regulated by factors Frontiers in Neuroscience 05 frontiersin.org from the CSF and fenestrated capillaries, depending on body conditions. The circumventricular organs are a possible communication route between the periphery and brain
Peripheral immune response transmits information to the brain to induce physiological and behavioral responses, but communication between the peripheral immune signal molecules or PAMPs and brain parenchyma is not well understood. Hypotheses proposed as to how peripheral inflammatory signals and PAMPs in circulation are transferred to the brain parenchyma to stimulate associated neural circuits include; 1) cytokine-dependent prostaglandin E 2 (PGE 2 ) synthesis and release at endothelial cells of brain vasculature, 2) activation of parenchymal cells in the CVOs by bloodderived molecules across fenestrated capillaries, 3) activation of peripheral nerves by cytokines and PGE 2 [bib_ref] Neural mechanisms of inflammationinduced fever, Blomqvist [/bib_ref]. However, it is difficult to conclude that a single route transmits peripheral inflammatory information to the brain, as neuroinflammation is associated with various physiological changes such as body temperature, locomotor activity, food and water intake, and metabolism [bib_ref] Sleep and brain infections, Tesoriero [/bib_ref]. Therefore, several routes may work in conjunction to regulate complex physiological inflammation responses.
This section focuses on glial cells in CVOs with a specialized niche for the blood-brain interface to transport information from the periphery to the brain. The CVOs express a range of inflammation-associated receptors for cytokines, PAMPs, neuropeptides, and PGE 2 [bib_ref] The sensory circumventricular organs of the mammalian brain, Mckinley [/bib_ref] [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref]. In comparison to other brain regions, peripheral inflammatory stimulation in the CVOs causes faster and stronger transcriptional activation of a vast number of proinflammatory molecules; such as nuclear factor-κ B (NF-κB; [bib_ref] Induction of inhibitory factor kappa B alpha mRNA in the central nervous..., Quan [/bib_ref] [bib_ref] An essential role of interleukin-1b in mediating NF-κB activity and COX-2 transcription..., Laflamme [/bib_ref] [bib_ref] Toll-like receptor 4 on nonhematopoietic cells sustains CNS inflammation during endotoxemia, independent..., Chakravarty [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref] [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref] , the signal transducer and activator of transcription factor 3 (STAT3; [bib_ref] Nuclear translocation of the transcription factor STAT3 in the guinea pig brain..., Rummel [/bib_ref] [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref] [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref] , and Fos [bib_ref] Temporal and spatial relationships between lipopolysaccharide-induced expression of fos, interleukin-1 β and..., Konsman [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref] [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref] [bib_ref] Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression..., Takagi [/bib_ref]. Microsomal prostaglandin E synthase-1, an inducible catalytic enzyme for converting PGH 2 to PGE 2 , is highly expressed in autonomic relay structures such as the CVOs, preoptic area (POA), Arc, and paraventricular nucleus [bib_ref] Distribution of microsomal prostaglandin E synthase-1 in the mouse brain, Eskilsson [/bib_ref]. Importantly, microsomal prostaglandin E synthase-1 expression is observed in endothelial cells, pericytes, and neurons of the CVOs. Sickness responses induced by interleukin-1β (IL-1β) are not derived from cerebral parenchymal vasculature but depend on a relatively small number of fenestrated capillaries located in the CVOs [bib_ref] Interleukin-1B signaling in fenestrated capillaries is sufficient to trigger sickness responses in..., Knoll [/bib_ref]. Strong expression of receptors against cytokines such as IL-6, IL-1β, and tumor necrosis factor-α (TNF-α) is also demonstrated in the CVOs [bib_ref] Type 1 interleukin-1 receptor in the rat brain: Distribution, regulation, and relationship..., Ericsson [/bib_ref] [bib_ref] Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear..., Nadeau [/bib_ref] [bib_ref] Molecular aspects of fever and hyperthermia, Roth [/bib_ref]. IL-6-deficient mice do not exhibit fever generation in response to peripheral administration of lipopolysaccharide (LPS) in contrast to IL-1β and TNF-αdeficient animals. Also, remarkably high expression of TLR2 and TLR4 mRNA occurs in the CVOs .
The AP forms the dorsal vagal complex with the solitary nucleus (Sol) and plays a role in the emetic reflex, immune responses, cardiovascular regulation, and energy homeostasis [bib_ref] Vagal control of digestion: Modulation by central neural and peripheral endocrine factors, Rogers [/bib_ref] [bib_ref] The area postrema: A brain monitor and integrator of systemic autonomic state, Price [/bib_ref]. The Arc and AP are the most critical brain regions for circulating factors involved in regulating food intake and energy homeostasis [bib_ref] The sensory circumventricular organs of the mammalian brain, Mckinley [/bib_ref] [bib_ref] The area postrema: A brain monitor and integrator of systemic autonomic state, Price [/bib_ref] [bib_ref] The receptive function of hypothalamic and brainstem centres to hormonal and nutrient..., Riediger [/bib_ref]. The AP is known to be implicated in nausea and vomiting to at least visceral stimulation [bib_ref] The area postrema and vomiting, Miller [/bib_ref]. Blood levels of growth/differentiation factor 15 are increased in sickness-associated events such as bacterial and viral infection, LPS, lithium chloride, and pregnancy . Growth/differentiation factor 15 receptor-expressing excitatory neurons in the AP mediate aversion responses to visceral LPS and lithium chloride [bib_ref] Area postrema cell types that mediates nausea-associated behaviors, Zhang [/bib_ref]. TNF-α stimulates neurons in the AP to elicit sympathetic excitation and blood pressure increases [bib_ref] In renovascular hypertension, TNF-α type-1 receptors in the area postrema mediate increases..., Korim [/bib_ref]. Thus, the AP is chiefly concerned with inflammation-induced sickness responses, such as decreased food intake, nausea, and hypertension.
The OVLT is located close to the POA that regulates body temperature, and sizeable electrolytic lesion to the anteroventral 3rd ventricle (AV3V) region, including the OVLT and SFO, attenuates fever after peripheral administration of LPS [bib_ref] Suppression of fever after lesions of the anteroventral third ventricle in guinea..., Blatteis [/bib_ref] [bib_ref] Evidence for the involvement of the organum vasculosum laminae terminalis in the..., Stitt [/bib_ref]. The quantitative autoradiography of [ 3 H] PGE 2 shows the highest binding density in the AV3V wall surrounding the OVLT. Subsequent studies indicate that fever-generation abnormalities come from side effects due to body fluid deficiency, electrolyte, and cardiovascular homeostasis [bib_ref] The organum vasculosum laminae terminalis in immune-to-brain febrigenic signaling: A reappraisal of..., Romanovsky [/bib_ref]. In contrast to the OVLT, an electrical lesion to the SFO prevents LPS-induced fever. Direct injection of IL-1β into the SFO increases body temperature and salt intake [bib_ref] Effects of interleukin-1 beta injections into the subfornical organ and median preoptic..., Cerqueira [/bib_ref].
When the SARS-CoV-2 virus reaches the brain, the subsequent neurological damage results in dysfunctions of long-term body condition [bib_ref] Persistent neurologic symptoms and cognitive dysfunction in nonhospitalized Covid-19 "long haulers, Graham [/bib_ref]. SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) on brain endothelial cells and induces inflammatory responses and activation of microglia and astrocytes to disrupt the integrity of the BBB [bib_ref] The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D..., Buzhdygan [/bib_ref] [bib_ref] ACE2, circumventricular oragns and the hypothalamus, and COVID-19, Ong [/bib_ref]. Infection of SARS-CoV-2 to brain neurons also induces the release of inflammatory molecules and ROS and free radical formation [bib_ref] NOX2 activation in COVID-19: Possible implications for neurodegenerative diseases, Sindona [/bib_ref]. ACE2 is highly expressed in the CVOs and brain regions connecting to the CVOs, such as paraventricular nucleus and Sol [bib_ref] Differential expression of neuronal ACE2 in transgenic mice with overexpression of the..., Doobay [/bib_ref]. The CVOs are essential Frontiers in Neuroscience 06 frontiersin.org brain regions to control angiotensin-dependent regulation of osmotic thirst, the release of arginine-vasopressin (AVP) and oxytocin (OXT) release, and blood pressure [bib_ref] Circumventricular organs; integrators of circulating signals controlling hydration, energy balance, and immune..., Ferguson [/bib_ref]. In addition to ACE2, SARS-CoV-2 virus interacts directly with TLR4 to cause an immune response and increase cell surface expression of ACE2 [bib_ref] Immunoinformatics approach to understand molecular interaction between multi-epitopic regions of SARS-CoV-2 spike-protein..., Bhattacharya [/bib_ref] [bib_ref] COVID-19 and Toll-like receptor 4 (TLR4): SARS-CoV-2 may bind and activate TLR4..., Aboudounya [/bib_ref] [bib_ref] SARS-CoV-2 spike protein interacts with and activates TLR4, Zhao [/bib_ref]. TLR4 protein is highly expressed in astrocytes and tanycytes in the CVOs of adult mice [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref]. These facts suggest that the CVOs are possibly the primary route of entry of SARS-CoV-2 virus into the brain.
In conjunction with viral and bacterial infection, the CVOs are also involved with parasitic infection. African trypanosomes first infect the ME fenestrated capillaries, then move, by rotating flagella and protease secretion, into adjacent hypothalamic regions such as the Arc and suprachiasmatic nucleus [bib_ref] Protease activated receptor signaling is required for African trypanosome traversal of human..., Grab [/bib_ref] [bib_ref] Characterization of different proteolytic activities in Trypanosoma brucei brucei, Huet [/bib_ref] [bib_ref] Mechanisms of CNS invasion and damage by parasites, Kristensson [/bib_ref] [bib_ref] Species-specific adaptations of trypanosome morphology and motility to the mammalian host, Bargul [/bib_ref].
## Toll-like receptor 2 in microglia and macrophages of the circumventricular organs
Toll-like receptors recognize PAMPs, which play crucial roles in early innate recognition and host inflammatory responses against invading disease-causing microbes [bib_ref] Toll-like receptors: Critical proteins linking innate and acquired immunity, Akira [/bib_ref] [bib_ref] Assembly and localization of Toll-like receptor signalling complexes, Gay [/bib_ref]. Studies show that expression of TLR2 and TLR4 is remarkably higher in the CVOs of the adult brains than in other brain regions [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref] [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref]. Circulating LPS has limited access to the brain parenchyma in non-CVOs region due to the BBB, but it can reach the parenchymal in the CVOs possibly via the lipoprotein transport system [bib_ref] Lipopolysaccharide enters the rat brain by a lipoproteinmediated transport mechanism in physiological..., Vargas-Caraveo [/bib_ref]. Moreover, a TLR2 ligand zymosan also reaches the parenchymal in the CVOs, although the transport system is unclear [bib_ref] Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression..., Takagi [/bib_ref]. These shreds of evidence suggest peripheral endotoxins can activate parenchymal TLR2 and TLR4 in the CVOs. Moreover, TLR2 and TLR4 have crucial functions in modulating inflammatory responses of the brain via endogenous ligands such as heat-shock proteins, high mobility group box 1, hyaluronic acids, and fibronectin [bib_ref] TLR2 and TLR4 in the brain injury caused by cerebral ischemia and..., Wang [/bib_ref] [bib_ref] Role of astroglial toll-like receptors (TLRs) in central nervous system infections, injury..., Li [/bib_ref]. However, the specialized spatial niches suggest that TLR2 and TLR4 ligands, derived from pathogens in the blood, activate TLR2 and TLR4 in the CVOs. This indicates that the CVOs can directly detect PAMPs, and integrate and relay information to deep brain regions, and initiate brain neuroinflammation.
Among TLR family members, TLR2 recognizes a variety of TLR2 molecules from viruses, fungi, bacteria, and parasites by forming a heterodimer with TLR1 or TLR6 [bib_ref] The role of TLR2 in infection and immunity, Oliveira-Nascimento [/bib_ref]. TLR2 heterodimers generally initiate a MyD88-dependent intracellular signaling pathway so that NF-κB is activated and translocated to the nucleus to modulate gene transcription of cytokines [bib_ref] The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors, Kawai [/bib_ref]. The activation of TLR2 in immune cells by lipoproteins is a significant factor in Gram-positive sepsis [bib_ref] Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus..., Takeuchi [/bib_ref] [bib_ref] Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active..., Hashimoto [/bib_ref]. In the CVOs, TLR2 mRNA expression was higher than in other brain regions , and TLR2expressing cells are identified as microglia and macrophages in the CVOs ; [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref]. The systemic administration of macrophage-activating lipopeptide-2 induces the activation of STAT3 in a subpopulation of parenchyma cells in the CVOs, together with fever generation [bib_ref] Macrophage-activating lipopeptide-2 (MALP-2) induces a localized inflammatory response in rats resulting in..., Knorr [/bib_ref]. Peripheral administration of the LMW TLR2 agonist Pam3CSK4 caused NF-κB activation in microglia and macrophages and Fos expression in astrocytes, tanycytes, and neurons in the CVOs . Peripheral and intracerebroventricular (i.c.v.) injection of a TLR2 ligand, Pam3CSK4, leads to sickness responses, including anorexia, hypoactivity, and fever, depending on NF-κB and PGE 2 [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref].
A robust and temporal elevation of the TLR2 mRNA expression occurs in the CVOs after the systemic injection of a TLR4 agonist LPS [bib_ref] Molecular insights on the cerebral innate immune system, Rivest [/bib_ref] [bib_ref] Regulation of innate immune responses in the brain, Rivest [/bib_ref] , indicating crosstalk between TLR4 and TLR2. Pam3CSK4-induced fever is augmented by pretreatment of LPS ; [bib_ref] Activation of microglia and macrophages in the circumventricular organs of the mouse..., Murayama [/bib_ref]. The expression of TLR2 is reported in both perivascular macrophages and parenchyma microglia of the CVOs [bib_ref] Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression..., Takagi [/bib_ref]. The CVO perivascular structure is largely different from that of the general brain; Firstly, a broad perivascular space is present between the inner and outer basement membranes in the CVOs, whereas brain capillaries generally fuse, thus lacking perivascular space [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] [bib_ref] Advances in understanding of structural reorganization in the hypothalamic neurosecretory system, Miyata [/bib_ref]. Secondly, blood-derived HMW molecules accumulate in the perivascular space [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref] [bib_ref] Advances in understanding of structural reorganization in the hypothalamic neurosecretory system, Miyata [/bib_ref] [bib_ref] Structural reconstruction of the perivascular space in the adult mouse neurohypophysis during..., Nishikawa [/bib_ref]. Perivascular macrophages possess crucial functions such as maintaining vascular permeability, pathogen phagocytosis, antigen representation, and limiting endothelial cell production of PGE 2 to avoid unnecessary inflammation [bib_ref] Perivascular macrophages in health and disease, Lapenna [/bib_ref]. IL-1β signaling in fenestrated capillaries of the CVOs is sufficient to trigger sickness responses in mice [bib_ref] Interleukin-1B signaling in fenestrated capillaries is sufficient to trigger sickness responses in..., Knoll [/bib_ref].
The depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) has been shown to attenuate fever caused by the i.c.v. administration of Pam3CSK. I.c.v injection of Clod-Lips reduced zymosan-induced Fos expression in astrocytes, tanycytes, and neurons in the CVOs, while a Clod-Lips injection attenuated zymosaninduced hypothermia ; [bib_ref] Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression..., Takagi [/bib_ref]. Thus, microglia and macrophages are primary cells in initiating faster and stronger transcriptional activation of the CVOs, at least partially via direct activation of TLR2 and transmitting peripheral information to neurons in The expression of TLR2 at microglia and macrophages in the CVOs and TLR2 agonist-induced fever and hypothermia in the adult mouse. Triple labeled immunohistochemistry shows prominent TLR2 expression at Iba1 + microglia (arrows) in parenchyma and Iba1 + macrophages (arrowheads) in laminin + capillary of the CVOs (A-D). LPS pretreatment promotes fever generation induced by a LMW TLR2 agonist Pam3CSK4 the CVOs to cause sickness responses in TLT2-dependent neuroinflammation.
## Tlr4 in astrocytes and tanycytes in the circumventricular organs
The expression of TLR4 mRNA is higher in the CVOs than in other brain regions [bib_ref] Toll-like receptor 4 on nonhematopoietic cells sustains CNS inflammation during endotoxemia, independent..., Chakravarty [/bib_ref]. TLR4 protein is expressed in astrocytes and tanycytes in the CVOs of adult mice; [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref]. The peripheral administration of LPS upregulates mRNA levels of CD14, which helps bind TLR4 to LPS-binding protein in the CVOs [bib_ref] The bacteria endotoxin lipopolysaccharide has the ability to target the brain in..., Lacroix [/bib_ref] [bib_ref] Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear..., Nadeau [/bib_ref]. Peripheral administration of LPS activates STAT3 [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref] and NF-κB signaling [bib_ref] An essential role of interleukin-1b in mediating NF-κB activity and COX-2 transcription..., Laflamme [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref] in astrocytes and tanycytes of the CVOs. The i.c.v. injection of LPS also causes STAT3 signaling activation in astrocytes and tanycytes of the CVOs [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref] [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref]. There is no apparent difference in the activation of NF-κB and STAT3 between astrocytes and tanycytes after peripheral administration of LPS [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref] [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref]. In TRM8 KO mice, [bib_ref] Role of TRPM8 in switching between fever and hypothermia in adult mice..., Shiraki [/bib_ref] [bib_ref] Role of TRPM8 in cold avoidance behaviors and brain activation during innocuous..., Kasuga [/bib_ref] i.c.v. injection of an LPS antagonist LPS-RS attenuates NF-κB signaling in the astrocytes and tanycytes of the CVOs after peripheral administration of LPS [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref]. Thus, the cellular phenotype for faster and more robust activation of transcriptional activation is largely different between TLR2 and TLR4 in the CVOs; TLR2 in microglia and macrophages, and TLR4 in astrocytes and tanycytes. Thus, astrocytes are important in initiating neuroinflammation in the CVOs at least partially by TLR4 and transmitting peripheral information to neurons in the CVOs.
The microglial and macrophage depletion by Clod-Lips enhanced the fever induced by peripheral administration of LPS [bib_ref] Dual roles for perivascular macrophages in immune-tobrain signaling, Serrats [/bib_ref]. The depletion of microglia and macrophages with the antagonist to the colony-stimulating The expression of TLR4 at astrocytes/tanycytes of the adult mouse brain. Immunohistochemistry shows robust and specific TLR4 expression in the CVOs (open arrows), Arc (open arrowhead), and CC (solid arrowhead) (A-D). Double-labeling immunohistochemistry reveals that TLR4 is expressed at vimentin + tanycytes (arrowheads) and GFAP + astrocytes (arrows) (E-I). Scale bars = 50 µm. 3V, 3rd ventricle; AP, area postrema; Arc, arcuate nucleus; DAPI, 4 ,6-diamidino-2-phenylindole; D3V, dorsal 3rd ventricle; GFAP, glial fibrillar acidic protein; ME, median eminence; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ; TLR4, toll-like receptor 4. Photographs are rearranged with permission from Elsevier B.V. [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref].
factor-1 receptor and transgenic mouse fractalkine receptor does not attenuate LPS-induced sickness responses such as bodyweight decrease, locomotor activity, and exacerbates voluntary wheel running [bib_ref] Microglia depleation fails to abrogate inflammation-induced sickness in mice and rats, Vichaya [/bib_ref]. Moreover, it is shown that the depletion of microglia and macrophages does not prevent proinflammatory cytokine expression and exacerbates some cytokines in LPS-induced inflammation [bib_ref] Microglia depleation fails to abrogate inflammation-induced sickness in mice and rats, Vichaya [/bib_ref]. Astrocytes have emerged as key contributors to the innate immune response of the brain to infections, neurodegenerative disorders, and injuries [bib_ref] Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators, Bsibsi [/bib_ref] [bib_ref] Astrocytes are active players in cerebral innate immunity, Farina [/bib_ref] [bib_ref] Role of astroglial toll-like receptors (TLRs) in central nervous system infections, injury..., Li [/bib_ref]. These results suggest that TLR4-dependent sickness responses in acute inflammation are caused independently of microglia activation.
In contrast, we cannot deny the possibility that microglia participate in acute and chronic neuroinflammation during LPS-induced inflammation. For example, microglia inhibitor minocycline promotes recovery from sickness behaviors and reduces mRNA expression of proinflammatory cytokines upon LPS stimulation without changing plasma IL-1β levels [bib_ref] Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia, Henry [/bib_ref]. Minocycline treatment decreases STAT3 activation in astrocytes and tanycytes of the OVLT and AP, but not SFO, upon peripheral administration of LPS [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref]. Central injection of TLR4 agonists induces morphological activation of microglia [bib_ref] Toll-like receptor 4 agonist and antagonist lipopolysaccharides modify innate immune response in..., Vargas-Caraveo [/bib_ref]. The treatment of LPS antagonist RS-LPS results in a markedly elongated morphology of microglia in the three CVOs, a shape generally seen in severe brain injury and infection [bib_ref] Toll-like receptor 4 agonist and antagonist lipopolysaccharides modify innate immune response in..., Vargas-Caraveo [/bib_ref]. Microglial proliferation in the brain is ubiquitous in chronic inflammation and exerts beneficial effects by attenuating sickness responses under LPS-induced chronic inflammation [bib_ref] Microglial proliferation attenuates sickness responses in adult mice during endotoxin-induced inflammation, Torii [/bib_ref]. Thus, the functional significance of microglia and macrophages in TLR4-dependent inflammation is largely different from TLR2, although they may work in conjunction with astrocytes during neuroinflammation.
## Unique properties of microglia and macrophages in the circumventricular organs
Microglia are resident macrophage-like cells of mesoderm origin and are distinct from other types of neuroglia in the brain. Although microglia are considered quiescent under normal physiological conditions, activated only upon an immune challenge or mechanical damage, recent evidence indicates that they control neuronal function and homeostasis without requiring these stimuli [bib_ref] Microglial dysregulation in psychiatric disease, Frick [/bib_ref]. For example, microglia are involved in synaptic pruning and the generation of neurons and oligodendrocytes . Microglia exhibit a ramified morphology, a so-called "resting state" under physiologically normal conditions, but rapidly transform to amoeboid after infection of pathogens, toxins, mechanical injury, or radiation, together with dramatically increased proinflammatory cytokine gene expression and promotion of phagocytosis for dying cells or pathogens [bib_ref] Microglia: Active sensor and versatile effector cells in the normal and pathologic..., Hanisch [/bib_ref] [bib_ref] Heterogeneity of microglial activation in the innate immune response in the brain, Colton [/bib_ref]. In the CVOs, surprisingly, microglia adopt the amoeboid form with fewer branched cellular processes even under normal conditions, whereas those in other brain regions exhibit the ramified form with well-branched and dendritic cellular processes ; [bib_ref] Microglia are continuously activated in the circumventricular organs of mouse brain, Takagi [/bib_ref]. Moreover, the density of microglia and macrophages is more significant in the CVOs than in other brain regions ; [bib_ref] Microglia are continuously activated in the circumventricular organs of mouse brain, Takagi [/bib_ref].
In addition to the amoeboid shape, microglia and macrophages in the CVOs express higher levels of M1 marker proteins CD16/32 and CD86 and M2 marker proteins CD206 and Ym1 than other brain regions (Figures 5G-I; [bib_ref] Microglia are continuously activated in the circumventricular organs of mouse brain, Takagi [/bib_ref]. Peripheral macrophages are classified into two activated states; the M1 phenotype, which shows a classical proinflammatory response by the production of inflammatory cytokines and reactive oxygen species, antigen presentation, and the removal of pathogens [bib_ref] Macrophage Polarization, Murray [/bib_ref] , and the M2 phenotype that expresses mediators or receptors to down-regulate, repair, or protect the body from inflammation . M1/M2 classification of macrophages has also been applied to microglia in the brain, although the M1/M2 categories have limitations [bib_ref] A polarizing question: Do M1 and M2 microglia exist?, Randsohoff [/bib_ref]. This demonstrates that microglia and macrophages in the CVOs are continuously activated, considering the amoeboid morphology and M1 and M2 protein expression.
The reason for continuous activation of microglia and macrophage is not well understood. Endothelial cells in the CVOs lack tight junction proteins, allowing blood-derived molecules into the parenchyma [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. CD163-expressing macrophages in AP perivascular space sequester dextran 10 kDa (molecular weight 10,000) within 5 min of administration [bib_ref] A size selective vascular barrier in the rat area postrema formed by..., Willis [/bib_ref]. Perivascular macrophages in the pineal gland express M1 marker, MHC II and display phagocytosis activity [bib_ref] The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell, Møller [/bib_ref]. In considering the function of these M1 and M2 marker proteins, activated microglia and macrophages phagocytize neurotoxic blood-derived molecules or cells to maintain CVO parenchyma microenvironment [bib_ref] Molecular mechanisms that influence the macrophage m1-m2 polarization balance, Wang [/bib_ref]. Another possible activated microglia role is in structural dynamics such as neurogenesis and angiogenesis. Continuous angiogenesis with proliferation and apoptosis of endothelial cells occurs in the CVOs of adult mice [bib_ref] VEGF-dependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice, Furube [/bib_ref] [bib_ref] Proliferation of endothelial cells in the choroid plexus of normal and hydrocephalic..., Asami [/bib_ref]. Conditioned media of resting-state microglia inhibits brain endothelial cell proliferation, whereas that from the activated-state microglia facilitates proliferation [bib_ref] Microglial activation state exerts a biphasic influence on brain endothelial cell proliferation..., Welser [/bib_ref]. Activated microglia increase vascular branching density in the retina. Thus, activated microglia may regulate endothelial cell proliferation or removal of apoptotic endothelial cells. Activated microglia in the subventricular zone of developing brains promote neurogenesis and oligodendrogenesis [bib_ref] Microglia enhance neurogenesis and oligodendrogenesis in the early postnatal subventricular zone, Shigemoto-Mogami [/bib_ref]. Differentiation of NPCs with M1 microglia supernatant leads to neurogenesis, while exposure to M2 supernatants leads to oligodendrogenesis [bib_ref] Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from..., Butovsky [/bib_ref] [bib_ref] M2 microglia promotes neurogenesis and oligodendrogenesis from neural stem/progenitor cells via the..., Yuan [/bib_ref]. Activated microglia are likely involved in controlling of oligodendrogenesis and neurogenesis by acting on NSCs and/or their progenitor cells.
## Inflammation-induced microglial proliferation in the brain
Microglia are long-lived cells in adult mammalian brains, and their density remains stable under normal physiological conditions, although their turnover occurs several times in a lifetime through the spatial and temporal coordination of proliferation and apoptosis [bib_ref] Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the..., Askew [/bib_ref]. Robust microglial proliferation often occurs under severe pathological brain conditions such as stroke-induced brain injury, spinal cord injury (Bellver-Landete et al.,, traumatic brain injury [bib_ref] Microglial depletion with CSF1R Inhibitor during chronic phase of experimental traumatic brain..., Henry [/bib_ref] , and Alzheimer's disease [bib_ref] Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of..., Olmos-Alonso [/bib_ref]. Recently, we revealed that LPSinduced microglial proliferation occurs in brain regions, such as the hypothalamus, medulla oblongata, and telencephalon, with marked increases in the CVOs, by LPS administration at a low dose of 100 µg/kg; [bib_ref] Brain regiondependent heterogeneity and dose-dependent difference in transient microglia population increase during..., Furube [/bib_ref]. Moreover, the single peripheral administration of 1 mg/kg LPS induced robust microglial proliferation in most brain regions, except for the cerebral cortex and hippocampus, in the adult mouse [bib_ref] Characterization of neural stem cells and their progeny in the sensory circumventricular..., Furube [/bib_ref] [bib_ref] Brain regiondependent heterogeneity and dose-dependent difference in transient microglia population increase during..., Furube [/bib_ref]. The CVOs are the blood-brain interface to initiate early stages of brain inflammation; prominent activation of STAT3 [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref] and NF-κB signaling [bib_ref] TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic..., Muneoka [/bib_ref] , and increased expression of Fosand cytokine mRNA [bib_ref] Induction of inhibitory factor kappa B alpha mRNA in the central nervous..., Quan [/bib_ref]. On the other hand, a proliferation of microglia does not occur in the cerebral cortex and hippocampus after peripheral LPS administration of 100 and 1 mg/kg. This indicates increased proliferation of microglia during LPS-induced inflammation in a region-and dose-dependent manner, demonstrating proliferation sensitivity variations of microglia. Microglial proliferation peaks 24-72 h after intraperitoneal LPS injection accompanied by increased microglial density, but the density returns to normal levels within 3 weeks; [bib_ref] Brain regiondependent heterogeneity and dose-dependent difference in transient microglia population increase during..., Furube [/bib_ref]. Microglial proliferation is also observed during inflammation induced by the toll-like receptor 2 (TLR2) agonist zymosan and PGE 2 [bib_ref] Microglial proliferation attenuates sickness responses in adult mice during endotoxin-induced inflammation, Torii [/bib_ref]. These results indicate that microglial proliferation Activated state of microglia in the CVOs even under physiologically normal conditions. Microglia morphology in the CVOs, such as the OVLT and AP, is amoeboid, whereas in the medial preoptic area and cerebral cortex, it is dendritic (A-D). Quantitative analysis reveals shorter microglial cellular processes in the CVOs than other brain regions (E). The density of the CVOs microglia is significantly higher than that of other brain regions (F). Double-labeling immunohistochemistry shows that CX3CR1 + microglia express an M2 marker protein CD206 (arrowheads) in the SFO and AP (G,H). Quantitative analysis shows the percentage of CD206 expression in CX3CR1 + microglia is higher in the CVOs than that of other brain regions (I). 10N, dorsal motor nucleus of vagus nerve; 12N, hypoglossal nucleus; AP, area postrema; Cx, cerebral cortex; D3V, dorsal 3rd ventricle; DG, dentate gyrus; fi, fimbria; ME, median eminence; MnPO, median preoptic area; MPA, medial preoptic area; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ; Sol, solitary nucleus; vhc, ventral hippocampal commissure; VMH, ventromedial hypothalamic nucleus. Scale bars = 10 (A) and 50 µm (G). Scale bars = 50 µm. Yellow and blue bars indicate the CVOs and non-CVO regions, respectively. Photographs are rearranged with permission from Elsevier B.V. [bib_ref] Microglia are continuously activated in the circumventricular organs of mouse brain, Takagi [/bib_ref].
is a general event of neuroinflammation rather than a specialized one in severe neurodegenerative diseases accompanied by neuronal death.
The inhibition of LPS-induced microglial proliferation with continuous i.c.v. infusion of AraC causes a persistent reduction in body weight and intake of food and water and prolongs LPS-induced sickness responses, such as lower locomotor activity and core body temperature; [bib_ref] Microglial proliferation attenuates sickness responses in adult mice during endotoxin-induced inflammation, Torii [/bib_ref]. Thus, a transient increase in the microglial population is beneficial during endotoxin-induced inflammation as it attenuates sickness response. The selective elimination of microglia with a colony-stimulating factor 1 receptor (CSF 1 R) antagonist increased neuronal death in an acute stroke modeland spinal cord injury [bib_ref] Microglia are an essential component of the neuroprotective scar that forms after..., Bellver-Landete [/bib_ref]. In contrast to the neuroprotective function in acute injury, the elimination of microglia with a CSF 1 R antagonist attenuates neurodegeneration during the chronic phase of traumatic brain injury [bib_ref] Microglial depletion with CSF1R Inhibitor during chronic phase of experimental traumatic brain..., Henry [/bib_ref] and Alzheimer's [bib_ref] Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of..., Olmos-Alonso [/bib_ref]. Therefore, microglia appear to exert beneficial or deleterious effects depending on brain disease, beneficial in acute inflammation and deleterious in chronic inflammation.
Fluid homeostasis by osmolality and Na + sensitive sensor in the circumventricular organs Animals with the electrolytic lesion of the OVLT and SFO, namely AV3V region, show a decreased response to dehydration or hypertonic saline ingestion, exhibiting reduced fluid intake, Fos expression, and AVP and OT secretion [bib_ref] Callahan dissociation between vasopressin and oxytocin mRNA and peptide secretion after AV3V..., Morris [/bib_ref] [bib_ref] Effect of AV3V lesions on Fos expression and cell size increases in..., Miyata [/bib_ref] [bib_ref] Anteroventral third ventricle (AV3V) lesions alter c-fos expression induced by salt loading, Rocha [/bib_ref] [bib_ref] Alterations in the central vasopressin and oxytocin axis after lesion of a..., Oliveira [/bib_ref] , indicating that AV3V region is crucial for the reception and integration of osmotic signals. TRPV1 is the first in the thermosensitive TRP family of proteins or non-selective cation channels [bib_ref] The capsaicin receptor: A heat-activated ion channel in the pain pathway, Caterina [/bib_ref] [bib_ref] TRP channels and pain, Julius [/bib_ref]. TRPV1 is activated by noxious high temperatures (>42 - C), pH, positive voltages, and chemicals such as capsaicin [bib_ref] TRP channels and pain, Julius [/bib_ref]. Moreover, TRPV1 is involved in mechanical Remarkable increase in proliferation of microglia and macrophages in the CVOs and neighboring brain regions in adult mouse brain after LPS administration. Intraperitoneal administration of 100 µg/kg LPS induces a robust increase of Iba1 + BrdU + proliferating microglia and macrophages in the OVLT (arrowheads) and medial preoptic area (MPA; arrows) (A), the SFO (arrowheads) and ventral hippocampal commissure (vhc; arrows) (B), and the AP (arrowheads) and solitary nucleus (Sol; arrows) (C). High magnification reveals many Iba1 + BrdU + proliferating microglia and macrophages in the CVOs (D-F). Scale bars = 100 (A) and 50 µm (D). Photographs are rearranged with permission from Springer Nature [bib_ref] Brain regiondependent heterogeneity and dose-dependent difference in transient microglia population increase during..., Furube [/bib_ref]. The density of PU.1 + microglia and macrophages is significantly elevated in the CVOs and neighboring brain regions on the 5th and 10th day after administration of 100 µg/kg and 1 mg/kg LPS, but returns to almost normal levels on the 20th day (G). The i.c.v. infusion of the mitotic inhibitor AraC significantly delays body weight recovery after 5 mg/kg administration (H). 10N, dorsal motor nucleus of vagus nerve; 12N, hypoglossal nucleus; AP, area postrema; fi, fimbria; MnPO, median preoptic area; MPA, medial preoptic area; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ; Sol, solitary nucleus; vhc, ventral hippocampal commissure; VMH, ventromedial hypothalamic nucleus. *p < 0.05, **p < 0.01, ***p 0.001 vs. the control by an ANOVA with Tukey's post-hoc test. pain and high osmolality detection by responding to cellular shape alterations [bib_ref] Central mechanisms of osmosensation and systemic osmoregulation, Bourque [/bib_ref]. Although TRPV1 is known to be expressed at somatosensory neuron nerve endings [bib_ref] The transient receptor potential vanilloid-1 channel in thermoregulation: A thermosensor it is..., Romanovsky [/bib_ref] , it is also highly expressed in adult brain CVOs [bib_ref] Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum..., Ciura [/bib_ref] [bib_ref] Integration of thermal and osmotic regulation of water homeostasis: The role of..., Sladek [/bib_ref]. Moreover, prominent expression of TRPV1 is detected at astrocytic and tanycytic cellular processes, which contact closely with fenestrated capillaries and form dense networks;. The TRPV1 deficiency of the OVLT results in decreased sensitivity to hyperosmolality [bib_ref] Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum..., Ciura [/bib_ref]. Fulllength TRPV1 responds to hyperosmolality but is less sensitive to hypoosmolality [bib_ref] Osmosensitivity of transient receptor potential vanilloid 1 is synergistically enhanced by distinct..., Nishihara [/bib_ref]. In contrast to these in vitro experiments, acute and chronic hyperosmolality produces a similar increase in the number of Fos-positive neurons in the OVLT, AVP secretion, and water intake in WT vs. TRPV1 KO mice [bib_ref] Mice lacking the transient receptor vanilloid potential 1 channel display normal thirst..., Taylor [/bib_ref] [bib_ref] Osmoregulatory thirst in mice lacking the transient receptor potential vanilloid type 1..., Kinsman [/bib_ref] [bib_ref] Hypernatremia-induced vasopressin secretion is not altered in TRPV1-/-rats, Tucker [/bib_ref]. These results indicate that TRPV1 is not responsible for in vivo body fluid homeostasis.
An alternative candidate for in vivo fluid homeostasis control is the Na x channel, which detects Na + levels [bib_ref] The subfornical organ, a specialized sodium channel, and the sensing of sodium..., Noda [/bib_ref] [bib_ref] Hydromineral neuroendocrinology: Mechanism of sensing sodium levels in the mammalian brain, Noda [/bib_ref] [bib_ref] Sodium sensing in the brain, Noda [/bib_ref]. The expression of the Na x channel occurs specifically at astrocytes and tanycytes in the CVOs such as the OVLT, SFO, ME, and NH [bib_ref] Nav2/NaG channel is involved in control of salt-intake behavior in the CNS, Watanabe [/bib_ref] [bib_ref] Sodium-level-sensitive sodium channel Na x is expressed in glial laminate processes in..., Watanabe [/bib_ref]. The Na x channels are activated depending on Na + concentration, but not osmolality, with a threshold of about 150 mM [bib_ref] Na x channel in CNS sodim-level sensing, Hiyama [/bib_ref]. WT mice show extensive water intake and aversion to saline intake, whereas Na x KO animals did not exhibit such behaviors [bib_ref] The subfornical organ is the primary locus of sodium-level sensing by Na(x)..., Hiyama [/bib_ref]. Abnormal behaviors, such as water intake and aversion to saline in Na x KO mice, are recovered by a site-directed infection of an adenoviral vector with the Na x gene into the SFO, indicating that the SFO is the primary brain center regulating water and salt intake behaviors [bib_ref] The subfornical organ is the primary locus of sodium-level sensing by Na(x)..., Hiyama [/bib_ref].
## Warm-sensitive transient receptor potential vanilloid 1-expressing glial cells in the circumventricular organs
Body thermoregulation is closely connected to body water homeostasis, and the CVOs act as integrators of thermal and osmotic signals in the brain [bib_ref] Integration of thermal and osmotic regulation of water homeostasis: The role of..., Sladek [/bib_ref]. The physiological benefits of this are; first, the CVOs lack the typical BBB to protect from blood-derived molecules such as ions and low and high molecular hydrophilic molecules [bib_ref] Different vascular permeability between the sensory and secretory circumventricular organs of adult..., Morita [/bib_ref] [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] , but they directly and rapidly detect ions, osmolality, and blood temperature. Second, the capillary density is higher in the CVOs than in other brain regions, enabling efficient detection of blood-derived signal. The peripheral and central administration of the TRPV1 agonist resiniferatoxin, induced Fos expression preferentially in the CVO astrocytes and tanycytes and neurons of the median preoptic nuclei and Sol;. Moreover, the peripheral and central administration of resiniferatoxin robustly induces STAT3 signaling in astrocytes of the CVOs and POA, but less in neurons;. A similar response to the peripheral and central administration of LPS [bib_ref] Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory..., Nakano [/bib_ref]. IL-6 activates STAT3 signaling in the brain [bib_ref] IL-6-regulated transcription factors, Akira [/bib_ref] and is necessary to induce fever [bib_ref] The role of interleukin-6 in lipopolysaccharide-induced fever by mechanisms independent of prostaglandin..., Nilsberth [/bib_ref]. The activation of TRPV1 stimulates c-Jun and p38 mitogen-activated protein kinases, releasing cytokines IL-6 and -8 in CD4 + cells [bib_ref] The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4?..., Bertin [/bib_ref]. The central administration of RTX causes a strong dose-dependent decrease in body temperature; [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref]. Peripheral administration of LPS induces less fever response in TRPV1 KO mice compared with WT animals [bib_ref] Attenuated fever response in mice lacking TRPV1, Iida [/bib_ref]. Thus, TRPV1 is necessary to maintain LPS-induced fever, as LPS sensitizes TRPV1 through TLR4 activation in the trigeminal sensory neurons [bib_ref] LPS sensitizes TRPV1 via activation of TLR4 in trigeminal sensory neurons, Diogenes [/bib_ref].
The deletion of TRPV1-expressing somatosensory neurons eliminates heat avoidance behavior in two-plate preference tests [bib_ref] The cellular code for mammalian thermosensation, Pogorzala [/bib_ref]. However, TRPV1 KO mice are dispensable for typical heat avoidance between 40 and 50 - C by two-plate preference tests [bib_ref] The cellular code for mammalian thermosensation, Pogorzala [/bib_ref] and normal warm sensation between 32 and 42 - C by goal-directed thermal perception task [bib_ref] The sensory coding of warm perception, Paricio-Montesinos [/bib_ref]. No significant difference is detected in colonic temperature between WT and TRPV1 KO mice when exposed to 37.0 - C [bib_ref] Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin..., Szelenyi [/bib_ref] , 39.0 - C [bib_ref] Thermoregulatory phenotype of the Trpv1 knockout mouse: Thermoeffector dysbalance with hyperkinesis, Garami [/bib_ref] , or 40 - C ; [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref]. This raises the question as to the function of TRPV1 upon acute ambient temperature increase. Recently, our research suggests that TRPV1 is crucial for thermoregulation by initiating heat loss behaviors under warm ambient temperatures less than 37 - C ; [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref]. TRPV1 KO mice showed significantly shorter durations for heat loss behaviors such as sleeping and body licking than WT mice upon exposure conditions between 30.0 and 35.0 - C [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref]. On the other hand, no significant difference in heat loss behavior time is observed between WT and TRPV1 KO mice upon exposure to 40 - C [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref] , indicating that exposure to heat greater than body temperature is beyond the cooling capacity of these heat loss behaviors. Thus, TRPV1 is necessary for thermoregulation under moderate warm ambient temperatures to control heat loss behaviors. The oral gavage of the TRPV1 agonist capsaicin quickly decreases core body temperature together with increased tail surface temperature in WT mice via activation of the CVOs and POA, whereas TRPV1 KO animals do not exhibit such changes [bib_ref] Oral gavage of capsaicin causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase..., Inagaki [/bib_ref]. Moreover, oral gavage of capsaicin induces a long-lasting increase in locomotor activity in a TRPV1-independent manner, as increased locomotor activity was seen in both WT and TRPV1 KO animals [bib_ref] Oral gavage of capsaicin causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase..., Inagaki [/bib_ref]. Capsaicin dilates blood vessels in the skin, and increases heat exchange and metabolic activity in humans [bib_ref] Recent advances in the study on capsaicinoids and capsinoids, Luo [/bib_ref] [bib_ref] Chemical and pharmacological aspects of capsaicin, Mde [/bib_ref] [bib_ref] Properties of capsaicin and its utility in veterinary and human medicine, Adaszek [/bib_ref].
Transient receptor potential vanilloid 1 is expressed in astrocytes and tanycytes of the CVOs, as previously mentioned [bib_ref] Integration of thermal and osmotic regulation of water homeostasis: The role of..., Sladek [/bib_ref]. Central and peripheral administration of RTX causes STAT3 signaling activation and Fos expression at astrocytes and tanycytes rather Frontiers in Neuroscience 13 frontiersin.org Prominent TRPV1 and TRPV1-dependent Fos expression at astrocytes in the CVOs of adult mouse brain and hypothermia by central activation of TRPV1. The expression of TRPV1 is prominent at GFAP + astrocytes in the CVOs (A-C). The i.c.v. infusion of the TRPV1 agonist resiniferatoxin at 500 ng/kg causes prominent Fos expression at astrocytes rather than neurons in the CVOs (D-F). AP, area postrema; GFAP, glial fibrillar acidic protein; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ. Scale bars = 50 µm. Quantitative analysis shows central infusion of resiniferatoxin induces significant Fos expression at astrocytes preferentially in the CVOs and at neurons in the CVOs and neighboring brain regions (G than neurons in the CVOs. In the CVOs, the fenestrated capillaries are surrounded by dense networks of astrocytic and tanycytic cellular processes. Neuronal somata are generally located at a moderate distance from fenestrated capillaries and are covered with glial cellular processes [bib_ref] New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular..., Miyata [/bib_ref] [bib_ref] Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of..., Morita [/bib_ref]. This unique spatial niche is necessary to protect neurons from molecules and ions that diffuse beyond the fenestrated capillaries. Hypoosmolality results in an influx Abnormal hyperthermia in TRPV1 KO mice upon exposure to moderately warm ambient temperature. Both WT and TRPV1 KO mice showed similar increases in core body temperature upon exposure to 40 - C (A). Upon exposure to 32.5 - C, TRPV1 KO mice exhibited hyperthermia, but hyperthermia was not seen in WT mice (B). *p < 0.05 between WT and TRPV1 KO mice with unpaired Student's t-test. Graphs are rearranged with permission from Springer Nature [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref].
of water into the brain parenchyma via glial cells, then brain water is shunted into astrocytes, which preserves neuronal cell size and prevents brain damage [bib_ref] Brain cell volume regulation in hyponatremia: Role of sex, age, vasopressin and..., Ayus [/bib_ref]. These results indicate that TRPV1-expressing astrocytes and tanycytes are "warm-sensitive glial cells."
Glia-neuronal communication in the circumventricular organs and transmission to other brain regions
Bidirectional communications between glial cells and neurons are essential for axonal conduction and synaptic transmission [bib_ref] New insights into neuron-glia communication, Fields [/bib_ref]. A transient increase in astrocytic Ca2 + levels results in a gliotransmitter release, which acts on neurons and vascular smooth muscle [bib_ref] Astrocyte calcium signaling: The third wave, Bazargani [/bib_ref]. Raising the question of how the fluid or thermal information sensed by astrocytes and tanycytes is then transferred to neurons. Coordinated activation of Na x and Na + /K + ATPase enhances glucose uptake and extensive lactate production in astrocytes and tanycytes in the SFO in response to elevated Na + levels [bib_ref] Glial Na x channels control lactate signaling to neurons for brain [Na..., Shimizu [/bib_ref]. Moreover, the Na + -dependent release of the glial transmitter lactate from astrocytes and tanycytes stimulates GABAergic neurons in the SFO to induce water intake behavior [bib_ref] Glial Na x channels control lactate signaling to neurons for brain [Na..., Shimizu [/bib_ref]. Endothelin-3 decreases the Na x sensitivity threshold of Na + from ∼ 150 to 135-145 mM and increases lactate release in the SFO [bib_ref] Endotheli-3 expression in the subfornical organ enhances the sensitivity of Na x..., Hiyama [/bib_ref]. Thus, thermal information detected by TRPV1 in astrocytes and tanycytes is also transferred to neurons by glial transmitters. Peripheral administration of a TRPV1 agonist induces hypothermia [bib_ref] The capsaicin receptor: A heat-activated ion channel in the pain pathway, Caterina [/bib_ref] , but a TRPV1 antagonist causes fever [bib_ref] Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin..., Szelenyi [/bib_ref]. While central administration of TRPV1 agonists induced hypothermia, TRPV1 antagonists had no effect [bib_ref] TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the..., Yoshida [/bib_ref] [bib_ref] TRPV1 is crucial for thermal homeostasis in the mouse by heat loss..., Park [/bib_ref]. These results indicate that peripheral TRPV1 is always activated, but central TRPV1 is inactivated. The glial-neuronal pathways of activated TRPV1 in the CVOs are well understood; however, the hypothermic brain center was recently reported. Opsin 5-, adcyap1-, or QRFP-expressing neurons in the lateral hypothalamic area around the third ventricle of the anterior part of the hypothalamus are shown to be hypothermic and hypometabolic neurons [bib_ref] Neurons that regulate mouse torpor, Hrvatin [/bib_ref] [bib_ref] A discrete neuroal circuit induces a hibernation-like state in rodents, Takahashi [/bib_ref] [bib_ref] Violet-light suppression of thermogenesisby opsin 5 hypothalamic neurons, Zhang [/bib_ref]. These hypothalamic neurons are closely located to the OVLT, indicating that glial TRPV1 activation information is transferred to these neurons, thereby causing hypothermia.
Thermal and osmotic information monitoring and integration in the OVLT and SFO are sent to the median preoptic area (MnPO) that functions as osmoregulation, thermoregulation, and sleep homeostasis [bib_ref] Integration of thermal and osmotic regulation of water homeostasis: The role of..., Sladek [/bib_ref]. In addition, the OVLT and SFO project to the supraoptic and paraventricular nuclei, which are the autonomic center, oxytocin, and vasopressin-synthesizing regions [bib_ref] Activity-related, dynamic neuron-glial interactions in the hypothalamo-neurohypophysial system, Miyata [/bib_ref] [bib_ref] Integration of thermal and osmotic regulation of water homeostasis: The role of..., Sladek [/bib_ref]. The SFO and OVLT respond to blood angiotensin II, relaxin, and hyperosmolarity to drive thirst-related neural pathways. However, amylin and leptin act at the AP to influence neural pathways inhibiting food intake [bib_ref] From sensory circumventricular organs to cerebral cortex: Neural pathways controlling thirst and..., Mckinley [/bib_ref]. Activation of ADCYAP1expressing neurons in the AP and Sol induces LPS-induced sickness responses such as reduced food and water intake, locomotor activity, and body temperature using the TRAP2 system. In contrast, inhibition of these neurons significantly weakens all responses except for effect on body temperature. Moreover, the activation of PHOX2Bexpressing neurons in nearly all AP cells and some NTS cells and DBH-expressing neurons in LPS-activated AP neurons decreases food and water intake and locomotor activity. These data indicate that ADCYAP1-expressing neurons in the AP and Sol are essential for endotoxin-induced sickness behaviors.
# Author contributions
The author confirms being the sole contributor of this work and has approved it for publication.
# Funding
This work was supported in part by the Scientific Research Grants from The Japan Society for the Promotion of Science (Nos. 19K06921 and 22K06456).
## Conflict of interest
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
## Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. |
“You would not be in a hurry to go back home”: patients’ willingness to participate in HIV/AIDS clinical trials at a clinical and research facility in Kampala, Uganda
Background: Few studies have examined factors associated with willingness of people living with HIV (PLHIV) to participate in HIV treatment clinical trials in Sub-Saharan Africa. We assessed the factors associated with participation of PLHIV in HIV treatment clinical trials research at a large urban clinical and research facility in Uganda. . Data were collected using structured questionnaires, focused group discussions with respondents categorised as either participated or never participated in clinical trials and key informant interviews with IDI staff. A generalized linear model with a logit link function was used for multivariate analyses while the qualitative data were summarized using a thematic approach.Results: We enrolled a total of 202 and analysed 151 participants, 77 (51%) of whom were male with mean age of 41 years. The majority 127 (84%) expressed willingness to participate in treatment clinical trials if given an opportunity. At bivariate analysis, willingness to participate was significantly associated with respondents' perception of a satisfactory compensation package (P-value < 0.002, 0.08-0.56), special status accorded (P-value < 0.001, 0.05-0.39) and belief that their health status would improve (P-value< 0.08, 0.03-0.58) while on the clinical trial. At multivariate analysis, a satisfactory compensation package (P-value< 0.030, 0.08-0.88) and special status accorded in clinical trials (P-value< 0.041, 0.01-0.91) remained significant. The qualitative data analysis confirmed these findings as participants valued the privilege of jumping the clinic waiting queues and spending less time in clinic, the wide range of free tests offered to trial participants, unrestricted access to senior physicians and regular communication from study team. Additionally, free meals offered during clinic visits meant that participants were not in a hurry to go back home. Barriers to participation included the perception that new drugs were being tested on them, fear of side effects like treatment failure and the uncertainty about privacy of their data.Conclusion:We found overwhelming willingness to participate in HIV treatment clinical trials. This was largely extrinsically influenced by the perceived material and health-related benefits. Investigators should pay attention to participants' concerns for benefits which may override the need to understand study procedures and risks.
# Background
The burden of HIV in Uganda remains high with an estimated prevalence of 6.2%. This corresponds to approximately 1.2 million people living with HIV in Uganda. In the general population, new annual HIV infections declined from 160,000 to 95,000 between 2010 and 2014. However, new infections remain unacceptably high, p. 1) with significant variability especially among key populations. While antiretroviral therapy is effective for long term suppression, research questions remain with respect to strategies for prevention of HIV transmission, strategies to retain patients in care, and optimal treatment regimens that are effective. Addressing these questions will require additional studies including welldesigned clinical trials.
Over the past decade, the number of HIV treatment clinical trials (CT) in Africa has increased. This increase has led to emergence of ethical concerns about people living with HIV participating in drug treatment clinical trials in Africa.
Studies conducted in high-income countries and in low-income countries on the willingness to participate in clinical trials have reported varied results. In the United Kingdom and United States, poor recruitment rates have been reported and attributed to nonapproachable clinicians, fear and mistrust of researchers, patient attitudes, stringent eligibility criteria and lack of knowledge about clinical research. Studies in sub-Saharan Africa showed that participants tend to exhibit a moderate willingness to participate in HIV vaccine trials. This is largely associated with perceived benefits, necessity, safety and understanding of clinical trials. The impression that there is a better understanding of clinical trials in Africa and a lack of knowledge about clinical research in high-income countries is rather contentious. It is possible that individuals in sub-Saharan Africa are more willing to participate in clinical trials because of compensation packages and research benefits and not because they understand clinical trials better than individuals in the high-income countries do. The barriers to participation in clinical trials include low literacy levels, stigma, discrimination and fears, convenience, and compensation and receipt of medical care.
Understanding why people volunteer to participate in treatment clinical trials will provide important information that can improve the recruitment process, inform public health policy and practice by aiming at improving the quality of clinical trials. A qualitative study that investigated how volunteers taking part in HIV clinical trials in central Uganda perceived informed consent, trial procedures, study information and their interaction with study staff, found that during the consent process, volunteers paid more attention to procedures requiring biological tests than to study design issues. The sustained participation and cooperation of patients is essential for the success of a clinical trial (Agnes.
This study aimed to understand whether people are intrinsically or extrinsically motivated to participate in HIV treatment clinical trials. We hypothesised that there is a relationship between perceived benefit/ compensation and willingness to participate in clinical trials.
We examined factors, perceptions and motivations associated with participation in drug treatment clinical trial research. We sought to determine the relationship between perceived benefit/ compensation and participants' willingness to participate in HIV/ AIDS treatment clinical trials at IDI and to establish other factors that could affect their willingness to participate in treatment trials.
# Methods
An intrinsic and extrinsic theory of motivation was utilised in this study.. Intrinsic motivation refers to doing something for its own sake, interest or for the pure enjoyment of a task. When clinical trial participants are active, curious, and eager to learn and engage in the studies, they display intrinsic motivation. Extrinsic motivation refers to doing something in order to attain some external goal or meet some externally imposed constraint.
## Design and sample quantitative sample
We conducted a cross-sectional survey using a mixed methods approach among patients receiving care at IDI. Two categories of respondents were identified, category one: those who were either enrolled in an on-going treatment clinical trial or had concluded participation within the last 3 years because of their recent experience; category two: those who had never participated in any treatment clinical trial.
Our operational definition of willingness to participate was "the expressed desire by a respondent to participate in a clinical trial, if given an opportunity". Respondents who had participated in clinical trials before the start of our study were classified as having expressed willingness to participate in clinical trials. We also assumed that they would still be interested in participating in CTs after 3 years. Respondents with no prior experience with clinical trials were asked if they were interested in participating in future CTs and those with a positive response were categorised as having expressed willingness to participate. However, only respondents with knowledge about clinical trials were assessed for willingness to participate which was calculated using the following formula: The operational definition of a satisfactory compensation package included incentives like compensation for time and transport reimbursement that were reasonable and acceptable to the study participants.
[formula] Willingness [/formula]
A sample size of 384 participants was estimated using Kish and Leslie formula. Quantitative research participants were selected from the IDI adult clinic. Systematic sampling was performed to obtain a true representative sample of the HIV population attending this clinic and every third person was approached using a detailed information sheet.
## Qualitative sample
A sample of 45 participants were selected for the qualitative arm of the study and they participated as either key informants or focus group discussants. The participants details are described in the results section. A total of 5 key informants were purposively selected to include health providers and a peer educator for their knowledge and involvement in HIV treatment clinical trials. Focus group participants were systematically selected from an electronic data base, and every third participant on the list was contacted until a total of 40 was realized.
## Setting
The study was conducted at the Infectious Diseases Institute (IDI) adult HIV clinic, Kampala, Uganda between July 2016 and January 2017. Established within Makerere University College of Health Sciences, the Institute was opened in 2002. IDIs adult HIV clinic currently provides care and treatment services to over 80,000 people living with HIV in urban and rural settings in Uganda and has completed 33 clinical trials. Study procedures, compensation and benefits vary across studies. In pharmacokinetic studies within the institute, every clinical trial participant is provided compensation in monetary terms equivalent to UGX 30,000-50,000 shillings (8-13USD) for time spent during a clinic visit and an additional 10,000-30,000 shillings (2-8 USD) as transport reimbursement. Additional non-monetary benefits include special status accorded like enhanced follow-ups by research teams, jumping patient queues, access to medical specialists, vital tests and free meals during clinic visits.
## Inclusion and exclusion criteria
Participants were eligible if they provided informed consent, were at least 18 years of age, had a confirmed HIV positive test and were attending IDI clinic for HIV care.
Participants were excluded if they were very sick, unable to provide informed consent and if they had participated in a clinical trial that ended more than 3 years prior to consenting to this study.
## Data collection qualitative data
The qualitative data were collected using focus group discussions (FGDs) and key informant interviews (KII). An FGD guide was used during the discussions to explore participants motivations and barriers to participating in HIV treatment clinical trial research. Four FGDs were held each comprising of 7-12 participants with either respondents who had participated in drug treatment clinical trials or those who had never participated in any clinical trials. Luganda, the most widely spoken language in Kampala was used to conduct the focus group discussions and English was used to conduct the key informant interviews in a private room on the IDI premises.
Five KIIs were conducted using an interview guide to obtain information from experienced IDI staff members to capture their views on the willingness of people living with HIV (PLHIV) to participate in HIV treatment clinical trials at the IDI. Additionally, the KIIs sought to understand how institutional factors influence PLHIV to participate in HIV treatment CTs. The first author and two research assistants conducted the interviews. The research assistants were trained in good clinical practice and on how to conduct FGDs and KIIs. The interview guides were piloted and revised prior to the full data collection process. Audio recorders were used to capture the FGDs and KII interviews to ensure all information was collected. The recorded interviews were transcribed in the language they were conducted and where necessary translated into English.
## Quantitative data
The Interviewer administered structured questionnaires which lasted approximately 30 min. The questionnaires were pre-tested to ensure question clarity and length. Four research assistants were trained on the research protocol, data collection tools and interviewing techniques before conducting any interviews. The interviews were conducted in English, and Luganda which is the most widely spoken language in the study area. The questionnaire included basic demographic information and experience with HIV treatment clinical trials. Awareness of clinical trials was assessed by asking, "Have you ever heard of clinical trials?" The participants who had never heard of clinical trials were excluded from the interview. Participants were then asked; From where did you hear about clinical trials? What is your understanding of clinical trials? What is your attitude to participating in clinical trials? How do you perceive participation in clinical trials?
# Ethical statement
Ethics approval was obtained from the Mulago Hospital Research Ethics Committee (MREC 991) in June 2016. Additional approvals were obtained from the Uganda National Council for Science and Technology (HS SS 5013) in July 2016. All participants signed an informed consent sheet and confidentiality was maintained by anonymizing participants' identifiers at all stages of data collection and reporting.
## Data manage2ment and statistical analyses
The data were entered into a computer using EPIDATA Version 3.02 and later exported to STATA for analysis. Descriptive statistics for background, demographics were summarized using frequencies, median, means and proportions. Univariate and bivariate analyses were conducted to investigate relationships between these data and awareness of clinical trials, perceptions of clinical trials, and willingness to participate in clinical trials. Associations between the participants' characteristics and willingness to participate in a clinical trial were tested using the chi-square test statistic (χ 2 test) for categorical variables, independent sample t-test and ANOVA for continuous variables. Multivariate analyses were conducted using a generalized linear model (GLM) with a logit link function.
# Qualitative data analysis
The FGDs and key informant interviews were transcribed by an experienced research assistant. Thematic analysis was performed manually. This involved the first author carrying out multiple readings of the scripts to understand the data and subsequently coding the data using pre-defined areas of analysis based on the objectives of study. However, the process was open to identifying new themes from the data, which were integrated into the pre-existing themes.
# Results
We consented 202 participants, excluded 51 who had no knowledge of clinical trials and analysed data of 151 participants..
## Characteristics of the participants quantitative
We analysed data for 151 adult participants, of whom 77(51%) were male. Only 127 (84%) met the operational definition of willingness to participate in clinical trials and 75(59%) of whom had ever participated in clinical trials. Of the respondents enrolled, 75(50%) had participated in previous trials and 76(50%) had not been previously enrolled in any clinical trials but expressed willingness to participate in such studies if given an opportunity. The mean age was 41.6 (SD 11.9). The majority subscribed to the Roman Catholic faith 56 (37%), 51 (33%) were married, 67(44%) had attained secondarylevel education and 104(69%) were employed. Most of the participants were self-employed 52(50%) and earning an average income of Uganda Shillings (120,000-300, 000UGX) (approx. 50-100 USD) per month.
## Factors associated with willingness to participate in clinical trials
# Quantitative results
For participants in pharmacokinetic trials, receiving all meals (breakfast, lunch, dinner) was perceived as being a satisfactory incentive. A transport refund of 10,000 UGX (2.7USD) was deemed as the threshold for desatisfaction which was a hindrance to CT participation. At bivariate analysis, we assessed the association between participants' age, sex, perception of benefits including if they viewed the compensation package as satisfactory and willingness to participate in drug treatment clinical trials. Gender, age, future benefit of clinical trials were not statistically significant and results are summarised in. The statistically significant factors for willingness to participate in clinical trials were: participants' perception of a satisfactory compensation package (P-value = 0.002, 0.08-0.56), belief that they would have special status accorded to them (Pvalue = 0.001, 0.05-0.39), and belief that they would have improved health status (P-value = 0.08,0.03-0.58) if they participated in treatment clinical trials.
At multivariate analysis, a threshold of 0.25 was set and variables with P-values < 0.25 from the Univariate analysis were considered. A generalised linear model (GLM) was then adjusted at multivariate analysis. Age and sex were considered fixed factors from literature. We established two significant variables: participant perception that the compensation package was satisfactory (P-value < 0.030, 0.08-0.88) and special status accorded in clinical trials (P-value < 0.041, 0.01-0.91). The odds of willingness to participate in clinical trials among participants who believed that the compensation package was not satisfactory was 0.27 times that of participants who felt they were satisfactorily compensated. Similarly, the odds of willingness to participate in clinical trials among those who believed that they would not have special status accorded to them was 0.11 times that of participants who thought they would have a special status in clinical trials. This means that perception of a satisfactory compensation package and special status accorded in clinical trials influenced willingness to participate, thereby proving our hypothesis.
# Qualitative results
We explored experiences of 45 participants, balanced by gender. A total of 40 participants took part in the FGDs and while 5 took part in key informant interviews; these included four health providers and a peer educator. Participation in the FGDs is presented in.
## Main themes identified
Content analysis identified four main themes from both the FGDs and KIIs. These are summarised in the table below:
Qualitative data suggests that a satisfactory compensation package and incentives were the most important motivation for participation. Two FGD participants observed:
They treated us really well, they would give us something to eat, and you would not be in a hurry to go home because you would know that you were going to get something to eat (R4 female FGD2).
Transport is very important because they would give me 20,000 Ug shs. Sometimes I may have The health providers appeared to be aware of the various forms of motivation, but were particularly concerned about whether the financial reimbursement did not amount to coercion:
We have seen that even the little bits of compensation [laughs] to a participant in research is making people more interested to participate and sometimes I begin to wonder if it's not coercion hmm [laughs] (KII 4, Female).
However, some of the FGD participants thought that the compensation they received during clinical trials was not satisfactory and expected more despite the wide range of benefits available, for example one participant argued:
For each time you came they would give you transport to go back they would give [UGX] 10,000. Each time they would call us, they would give breakfast for those that came early and others lunch. Apart from that, there was nothing else we would get. That is what they would give us and each time they would draw blood, they would give biscuits and splash (R11 female FGD 3).
Other participants positively perceived the benefits that accrued in non-monetary terms, that is, the auxiliary services provided to them during their participation in clinical trials. For example, one FGD respondent appreciated the apparent 'special' status accorded to clinical trial participants that ensured that they jumped the queues and were closely followed-up by the study team: They would even call us on the day we are supposed to return to remind us that on such and such a day, you are expected which doesn't happen the other side (out of the study). They really showed us care. Many times, you come and are worried about the queue, you worry about the time you will leave and yet the other side (while on the study) they would look for your file and bring it to you. They used to do everything for us (R8 male FGD 3).
Discrepancies were observed with respect to perception of time spent in the clinic. Respondents were willing to participate in drug treatment clinical trials because of shorter time spent at the clinic, especially because they jumped the patient waiting queues. However, the requirement for participants to make frequent visits to the clinic for monitoring was a barrier to their willingness to participate in the trials. The health providers appreciated this practical challenge:
If you're going to require a participant to return to the clinic thrice a month yet they are used to coming back once every 3 months, then they are not likely to take part in a clinical trial. If somebody is used to coming here and spending 30 min then after the clinical trial, they have to spend 4 h, or something then may not want to participate (KII 5, male).
There appeared to be gender variations regarding the importance of medical tests. While female participants appreciated the free comprehensive medical tests and examinations they were subjected to, the males found them many and seemed not to appreciate their importance, and this was perceived as a barrier to participation in clinical trials. Notably, the medical tests provided to participants enrolled in clinical trials would otherwise have been unaffordable to them. A female respondent described the services thus:
It really helped me because I did not have the capacity to pay for all those tests. They checked the heart, screened for cervical cancer, checked for diabetes, checked for hypertension because I wouldn't have been able to run all those tests (R4 female FGD3).
In contrast, a male FGD participant expressed concern about the many tests conducted, describing it as torture:
Personally, when I was told about the research the very first time, I felt uncomfortable. Now, my thinking is that my body is tortured checking for this and the other, do you understand? They check for this and the other and you are afraid. My wife told me if Two male participants who had never participated in clinical trials attributed their non-participation to lack of awareness of such opportunities, as they had never been approached to take part in any clinical trial.
"Now like myself, I personally have never been told about research and I reject it" (R7 male FGD1).
Furthermore, the second male FGD participant noted that even though he had never been approached to take part in clinical trials, he had previously heard about them and he was afraid of the uncertainty of likely risk.
Like I told you, I have never been approached but in the beginning, but my wife was involved in a research here. She was able to complete because theirs was for 2 years, God helped and there was no problem at all (R10 male, FGD4).
In addition, patients are more likely to participate in a treatment clinical trial if they believe that they will have an improved health status. One female participant mentioned the following when relating to improvement in her health status.
When I came, I was in a very poor state, I wanted to find out what the problem was. All the other hospitals I went to said I had a mental problem and that's why I joined (R10 female FGD3).
Furthermore, patients were willing to participate in clinical trials if they believed they were the ultimate beneficiaries of trials through the development of a wide variety of drugs or lead to reduced pill burden. Another male FGD participant observed how patients had benefited from a wide variety of drugs from clinical trials.
In the past, we used to take only one type of drug but now even at the dispensing window, each one is given a different type of drug (R1 male FGD2).
While drug treatment clinical trials were appreciated for the opportunity to access a wide variety of drugs, the myths and fears associated with new drugs like treatment failure and death led to reduced willingness to participate in clinical trials. During FGDs, for example, some participants were aware of the processes involved in drug development, including trials done on animals such as monkeys. They saw clinical trials as an extension of drug development processes, and worried about being used as test animals, with some equating the human beings who take part in drug trials to monkeys.
Among the white people they would try the drugs on monkeys but then now, you the black person when they start trying drugs on you, they perceive it so negatively. Some people out there say that if you go and participate in a clinical trial, they remain using you. (R8 male FGD4).
Patients who have negative beliefs about clinical trials were likely to negatively influence those contemplating participation in clinical trials. A female FGD participant explained:
I was in a line, we had come to pick our drugs but when one health worker came to explain to us about a research study, one lady said what; research! I don't like them (researches) then I asked her why she didn't like it and she said those drugs can bring you harm to your life and you may even die (R6 female FGD3).
It was not only patients who expressed this concern. The health providers too identified the negative consequences of myths associated with the development of new drugs on participants' willingness to get involved in clinical trials:
"Myths, myths about new drugs they are associated with so many risks and this is a barrier to participation" (KII 3, female).
The uncertainty about how their data will be used, and the fear that their identities maybe publicised, were other important issues discussed as barriers to willingness to participate in clinical trials. By saying "…. they [clinical trials] remain using you", participant R8 above neatly articulated this fear and a particular sense of vulnerability that perhaps researchers exploited study participants, who are often unable to determine what happens after enrolling in a clinical trial. In relation to information management, participants appeared to believe that research information somehow usually gets published in the newspapers. There were several people who held this view, but the account of one male participant suggests that it was a generalised belief and fear that researchers tend to discuss their participants in the newspapers.
When you talk about research, they [patients] have the thinking that they may take part and they may start writing about them and publish them in Newspapers. I also discussed with my wife and she said if you are the ones that they are going to research on, you will appear in Newspapers (R3 male FGD4).
The selection process of participants is key in influencing willingness to participate in clinical trials. Selection processes that are considered fair are more likely to attract a bigger number of participants. One FGD male participant seemed to be aware of this fact and attributed it to the fact that he had been a clinic patient for a long time and thus more knowledgeable of how things are run in the clinic:
What I know is that the way that they have been selecting people for research is fair. The time I have spent here, I have never seen things not moving well when we come to get drugs. I have never seen anything not going well so that gives me strength that the way things are done at IDI, they have a way of doing things well. That helps me not to get upset that sometimes I may be left behind because they look for their people and we remain (laughs). Because I see that the treatment we get here is equal (R7 male FGD4).
# Discussion
In this urban population of people living with HIV, we found that the willingness to participate in clinical trials was high. Quantitative data found that willingness to participate in drug treatment clinical trials was strongly associated with monetary benefits that accrued from participation and a belief that they received a special status during clinical trials. This means that perception of a satisfactory compensation package and special status accorded in treatment clinical trials influences willingness to participate in clinical trials.
Additionally, qualitative data suggests that provision of meals, compensation for time and transport reimbursement influenced participation in clinical trials. Participants who perceived the compensation package and incentives as satisfactory were not in a hurry to go back home when they visited the clinic. This suggests that participants were extrinsically motivated, as found reported in other studies. However, people who thought that the compensation package was not satisfactory were less likely to participate in clinical trials. Our findings contradict research done in Floridaand earlier in Uganda, which found that HIV research participation is not necessarily influenced by material benefits and [monetary] compensation. However, this could be attributed to the differences in the population and settings where the research was conducted.
Both the Uganda National Guidelines and various international guidelines, such as the Declaration of Helsinki and CIOMS, state that compensation of participants enrolling in research should not be considered a research benefit, and should not present undue inducement to potential research participants. However, Uganda National Council for Science and Technology has not put a cap on how much people should be compensated across different studies. It is estimated that over 80% of Ugandans live in abject poverty on less than one USD per dayand, therefore, any financial compensation for research participation could be seen as a substantial income, especially for HIV patients who tend to be poorer than the general population. Undue inducement is a critical concept to the discussion on participants' willingness to take part in HIV treatment clinical trials. This concept is controversial and can be conceived as acting under duress to make a decision that one would not otherwise have made. The danger of undue inducements can be a real cause of concern for research participants. In the context of engaging poor vulnerable participants in clinical trials in HIV, monetary incentives paid out as compensation to participants, as well as other benefits offered, can be contentious since it is highly possible that participants will accept to take part due to receiving money regardless of the risks associated with such involvement. A key question in this debate is whether research participants should be compensated differently based on their incometo minimise coercion or undue inducement but this too may not resolve the question of the most poor in resource limited settings, for whom any monetary compensation is far beyond their usual income. Researchers and ethics review boards will continue to confront this challenge, but in doing this, they must ensure that a balance between promoting the social good of research and respecting the dignity of human patients should be considered.
Both the quantitative and qualitative findings demonstrate that participants' motivation to participate in HIV treatment clinical trials was driven by a belief that they were receiving better treatment and that they were being treated by some of the best physicians with the latest medication. This created confidence and boosted their esteem. Importantly it conveyed a sense of special status to the participant, increasing their motivation to participate in treatment clinical trials. This can be described as a form of extrinsic motivation. These findings are consistent with a study that indicated that being seen by good physicians and taking the latest drugs strongly motivated participation in clinical trials research, and in particular, when a study drug is believed to possess health benefits. Traditionally, in research ethics, there is a well-established dichotomy between being enrolled in a clinical study and receiving routine medical care. This distinction is usually articulated to prevent therapeutic misconception. This term is used to describe situations where a research subject fails to appreciate the distinction between the imperative of clinical research and of ordinary treatment, and therefore, inaccurately attributes therapeutic intent to research procedures. This means that a participant enrolled in a study believes they are going to receive therapy and do not recognize that they are enrolled in a clinical research that may, for example, not provide any medical benefits, for instance if provided a placebo. Our study participants did not seem to appreciate this possibility; rather all of them appeared to believe that participating in a clinical trial came with many medical benefits. Such misconception can lead people to act against their own best interest and enrol in studies regardless of the potential risk. In a well-argued article, Gearhart examines this issue and noted that, indeed, there are cases where therapeutic misconception remains an ethical concern, such as when comparing an experimental treatment with placebo. However, he argues that the traditional division between pure research and routine medical care might be blurring, as in fact, the whole point of clinical research is to assess a potential health benefit, and therefore we should not be surprised when study participants ignore our disclaimers and assume there is, in fact, a good chance they will obtain a health benefit (p.g 1).
Qualitative findings revealed that tests carried out during clinical trials motivated participation in trials. Previous research conducted by Whyteamong people living with HIV in Uganda has described the value that lay people attach to the importance of tests in the routine monitoring of treatment experience. Tests such as an HIV test, viral load and CD4 count, according to Whyte et al's participants added new dimensions to bodily experiences; people related social situations and possibilities, for instance the hope to get a child, or even a partner. In our study, qualitative data found that participants appreciated the various tests conducted on them, although there appeared to be a gendered variation. While women particularly appreciated the tests and saw it as sign of special attention accorded to them by monitoring their health status for tests they could not have afforded on their own, some men did not seem to attach the same importance to the many tests and were concerned about the pricks and the amount of blood drawn. These qualitative findings are not conclusive. Future quantitative research should explore these issues.
Both the qualitative and quantitative results show that clinical trial participants appeared to receive special consideration from study staff who monitored their health status, provided regular communication, provided access to senior physicians and helped them jump the patient waiting queues. In this study, participants appeared to find great satisfaction with bypassing the queues and finding that clinicians were ready to review them. This meant that one spent less time during the clinic visits and were able to attend to other personal activities. Previous studies have shown similar findings and describe the importance of patients spending less time in the clinic. In addition, a sense of care and belonging was associated with receipt of regular communication on adherence and reminders about clinic appointments.
The qualitative findings revealed that in this HIV positive population, participants held with high regard issues of confidentiality and esteem. Patients were not comfortable taking part in clinical trials for fear that their privacy would be breached if they got published in Newspapers and their esteem affected if they were categorised as monkeys because CTs were used to test medicine on them which was a barrier to participation. This is a display of intrinsic motivation; the participants are concerned about their welfare regardless of incentives offered to them. Concerns about protecting the privacy of participant information, esteem, respect for persons which encompasses health information privacy and data anonymization are important motivators for willingness to participate in CTs.
Additionally, both quantitative and qualitative research findings demonstratethat a fair selection process for research participants coupled with information and awareness about clinical trials are important and have an influence willingness to participate in HIV treatment clinical trials. Similar research conducted on willingness to participate in clinical trials is in agreement with the our findings that showed that people who have a better understanding and knowledge of clinical trials are more likely to take part in treatment clinical trials.
Gender considerations were noted, while most of the men had never been approached, their spouses had prior experience with participation. This could be attributed to the busy schedules demonstrated by men who spend less time in the clinic.
The study results confirm what other scholars have documented in relation to extrinsically motivating participants in research. Our participants highly valued material and health rewards received during in clinical trials, as has been found elsewhere. However, by identifying barriers such as lack of privacy and fear of breach of confidentiality and the perception that clinical trial participants are equivalent to test animals, which affects their esteem, suggests that participants are not always extrinsically motivated. Cherry refers to the value of intrinsic motivation in sustaining participation in an activity, and warns against the costs associated with external rewards. Extrinsic motivation leads to higher enrolment of participants over a short period of time because of the rewards associated with participation. On the other hand, intrinsic motivation leads to an improvement in the retention rates in the long run. Participant understanding of the relevance of clinical trials to individuals and their contribution to the community, is an influencing factor for willingness to participate in clinical trials.
Three key limitations of our study were as follows; first is a small sample size which was not attained because despite the study site being a large clinical trial centre, participant enrolment into this study was not feasible as only specialized patients are currently only being reviewed at IDI. Nevertheless, the use of a mixed methods approach ensured that qualitative data provided in depth insights about willingness to participate in clinical trials and demonstrated that most participants were willing to participate in clinical trials. Secondly, the findings are limited to HIV positive population in treatment clinical trials. This limits generalizability of our results to HIV treatment clinical trials. However, findings in this study will help address the challenges involved in undertaking clinical trials in this population. Thirdly, compensation expectation comparisons were not clear for a once-off clinical trial involving a single dosage and one follow up visit to those on a 5 year study involving numerous visits over the course of 5 years. Never the less the study provides useful information on compensation for consideration in treatment clinical trials for single dosage and follow up visits.
# Conclusion
In conclusion, our study indicates that individuals are extrinsically motivated to participate in HIV treatment clinical trials by the perceived rewards such as a fair compensation package and additional benefits. Investigators should provide the rationale of conducting clinical trials to participants with a focus on intrinsic motivation as this will aid recruitment in the long run. Study investigators, researchers should leverage this willingness to facilitate enrolment in clinical trials and pay attention to how participants' concern for benefits may override the need to understand study procedures and risks. Further research should explore patient willingness across diverse settings. Future studies should compare compensation expectations for participants in a once off CT to those with numerous visits over a longer period. |
Pain control according to the periprostatic nerve block site in magnetic resonance imaging/transrectal targeted prostate biopsy
We analyzed the intensity of pain at each site of systemic prostate biopsy (SBx) and compared the intensity of pain among magnetic resonance (MRI)-targeted transrectal biopsies according to the periprostatic nerve block (PNB) site. We collected data from 229 consecutive patients who had undergone MRI-targeted biopsy. Patients were stratified into two groups according to the site of PNB (base versus base and apex PNB). Pain was quantified at the following time points: probe insertion, injection at the prostate base, injection at the prostate apex, MRI cognitive biopsy (CBx), MRI/ transrectal ultrasound fusion biopsy (FBx), SBx, and 15 min after biopsy. For all biopsy methods, the average pain were significantly higher in the base PNB group than in the base and apex PNB group (CBx, p < 0.001; FBx, p = 0.015; SBx, p < 0.001). In the base and apex PNB group, FBx was significantly more painful than SBx (p = 0.024). Overall, regardless of the PNB site, pain at the anterior sites was more than that at the posterior sites in FBx (p = 0.039). Base and apex PNB provided better overall pain control than base-only PNB in all biopsy methods. In the base and apex PNB group, FBx was more painful than CBx and SBx.Prebiopsy prostate magnetic resonance imaging (MRI) is recommended by the National Comprehensive Cancer Network (NCCN) to determine the need for performing prostate biopsy. Prostate MRI is more sensitive in detecting clinically significant prostate cancer than systemic prostate biopsy (SBx). Prebiopsy MRI is helpful in identifying lesions in not only the anterior prostate but also in the apex of the prostate; these lesions are not routinely assessed using SBx because of the associated challenges 1 . With the increasing applications of prebiopsy MRI, the MRI/transrectal ultrasound (TRUS) fusion targeted prostate biopsy (FBx) has been recommended by the NCCN owing to its improved accuracy 2 .NCCN also recommends the use of local anesthesia to reduce pain during prostate biopsy 2 . A combination of intrarectal lidocaine gel and periprostatic nerve block (PNB) is useful during SBx 3-8 . Base PNB is the most common method of PNB. Major neurovascular bundles of the prostate pass through the base PNB site and a large portion of the prostate is anesthetized by base PNB 9 . The apex PNB blocks the somatic nerve in the apex of the prostate, one of the most painful areas during biopsy 9 . The apex PNB is usually performed before local transperineal biopsy10,11. As the location of the target lesion in FBx is different from that in SBx, pain intensity in SBx should be predicted according to the biopsy site, and the optimal local anaesthesia method for FBx should be used. However, to our best knowledge, no previous study has analyzed the differences in pain intensity according to the biopsy site, and only a few have assessed the optimal local anaesthesia method for FBx. Therefore, we investigated pain intensity during various biopsies according to the biopsy site and compared the pain alleviation during various biopsy methods, including MRI cognitive targeted prostate biopsy (CBx), FBx, and SBx, according to the site of PNB.OPENResultsBaseline characteristics. Patient characteristics according to the PNB site are presented inTable 1. There were no differences in age, prostate-specific antigen (PSA) level, prostate volume, or history of prostate biopsy between the base PNB and base and apex PNB groups.Visual analog scale scores during the biopsy. The visual analog scale (VAS) scores according to the PNB site are presented inTable 2. There were no significant differences in VAS scores at different time points (probe insertion, injection at the prostate base, injection at the prostate apex, and 15 min after prostate biopsy) between the base PNB and base and apex PNB groups. The differences in VAS scores for each core biopsy of SBx between the base PNB and base and apex PNB groups are presented inFig. 1. In the base PNB group, pain at the . Baseline characteristics of groups according to the site of the PNB. Data are expressed as number (%), mean ± standard deviation, and median (IQR range). AUR acute urinary retention, ISUP International Society of Urological Pathology, IQR interquartile, PI-RADS prostate imaging-reporting and data system, PNB periprostatic nerve block, PSA prostate-specific antigen.
## Adverse events
Vasovagal syncope 1 (0. www.nature.com/scientificreports/ apex lesions was more than that at the order lesions (p < 0.001,. For all biopsy methods, patients in the base and apex PNB group reported lower VAS scores than those in the base PNB group [fig_ref] Table 2: Mean VAS scores during prostate biopsy in patients according to the site... [/fig_ref]. In the base and apex PNB group, FBx was painful than CBx and SBx. FBx was significantly more painful than SBx (3.21 vs. 2.88, p = 0.024) and marginally more painful than CBx (p = 0.104). Between CBx and SBx, there was no significant difference in VAS scores (p = 0.327). In the base PNB group, there was no difference in VAS scores among the biopsy methods.
We compared the pain intensity between the anterior and posterior sites [fig_ref] Table 3: Differences in mean VAS scores during various types of biopsies between the... [/fig_ref]. In FBx, anterior site biopsy was significantly more painful than posterior site biopsy in all patients regardless of the PNB site (p = 0.039). In the base PNB group, no differences in pain were found between the anterior and posterior sites (p = 0.069). Patients in the base and apex PNB group reported lesser pain in the anterior site biopsy than those in the base PNB group, and the difference was greater for FBx than for CBx.
## Complications.
One patient (0.9%) showed vasovagal syncope in the base PNB group, but recovered without any medical therapy. No major complications were observed in the base and apex PNB group .
# Discussion
Here, we confirmed the efficacy of additional PNB performed at the prostate apex during CBx and FBx, based on previous studies on local anaesthesia in SBx, by comparing the differences in pain intensity among various biopsy methods. FBx was significantly more painful than other biopsy methods in the base and apex PNB group. We compared the difference in the pain intensity during anterior and posterior site biopsies and observed significant differences during FBx and marginally significant differences during CBx. Our findings can help in the selection of the optimal local anaesthesia method according to the core biopsy site, biopsy method, and number of core biopsies.
Studies have compared the pain intensity between FBx and SBx and reported that SBx is relatively more painful than FBx, which contradicts our results [bib_ref] Comparison of pain levels in fusion prostate biopsy and standard TRUS-Guided biopsy, Demirtas [/bib_ref] [bib_ref] MRI-guided core needle biopsy of the prostate: Acceptance and side effects, Egbers [/bib_ref] [bib_ref] MRI-targeted or standard biopsy for prostate-cancer diagnosis, Kasivisvanathan [/bib_ref] www.nature.com/scientificreports/ for FBx and 2.0 (1.0-4.0) for SBx, but they included patients in whom only MRI was performed without biopsy in the FBx group [bib_ref] MRI-targeted or standard biopsy for prostate-cancer diagnosis, Kasivisvanathan [/bib_ref]. Because of these differences, VAS scores during FBx and SBx were underestimated in these previous studies compared to our study. In a previous literature review, VAS scores during SBx in the base PNB group ranged from 3.37 to 4.97 [bib_ref] Pelvic plexus block is more effective than periprostatic nerve block for pain..., Cantiello [/bib_ref] [bib_ref] Transrectal ultrasonography (TRUS)-guided pelvic plexus block to reduce pain during prostate biopsy:..., Jindal [/bib_ref] [bib_ref] Doppler ultrasonography-guided pelvic plexus block before systematic needle biopsy of the prostate:..., Akpinar [/bib_ref]. In our study, the mean VAS score (4.00) during SBx was similar to that in the aforementioned studies. We found that biopsy in the anterior site was more painful than that in the posterior site. The base PNB blocks the nerve originating from the presacral and hypogastric plexuses, and the apex PNB blocks the somatic nerve originating from the pudendal canal, but not all nerves. There are a few other nerve fibers on the anterior and superolateral of prostate [bib_ref] Pain during transrectal ultrasound-guided prostate biopsy and the role of periprostatic nerve..., Nazir [/bib_ref]. Moreover, prostate size is associated with pain during SBx, and the pain intensity is greater during SBx of an enlarged prostate due to the longer distance between the local anesthesia and biopsy sites [bib_ref] Local anesthesia for pain control during transrectal ultrasound-guided prostate biopsy: A systematic..., Yan [/bib_ref] [bib_ref] Complications of prostatic echo-guided transrectal biopsy and tolerance depending on the patient..., Rodriguez-Patron [/bib_ref]. The distance between the PNB and biopsy sites affects adequate pain control. Additional apex PNB leading to a lower intensity of overall pain is associated with the decreased distance between the PNB and biopsy sites. As the site for PNB is located posteriorly, the anterior site was associated with a greater pain intensity.
VAS scores during anterior site biopsy were lower in the base and apex PNB group than in the base PNB group. The additional apex PNB could have reduced the intensity of anterior site pain. Apex PNB anesthetizes the somatic branch of the inferior rectal nerve from the pudendal nerve and hence reduces the pain intensity below the dentate line, which is the site of needle puncture during apex or anterior prostate biopsy [bib_ref] A randomized controlled comparison between periprostatic nerve block and pelvic plexus block..., Kim [/bib_ref]. Although the difference in the pain intensity between the two groups was not significant in our study, more meaningful results could be obtained through subsequent studies with larger sample sizes.
Core biopsies in FBx or CBx are mostly performed in sites that are relatively far from the rectum, such as the anterior prostate, or sites that are at a greater angulation with the natural orientation of the rectum, such as the prostate margin, which are not performed routinely in SBx 20 . Therefore, the probe could impinge on the rectum, leading to greater pain during FBx. FBx was marginally more painful than CBx because the manipulation speed of the freehand is greater and the manipulation is smoother in CBx than in the fusion system of FBx. This is in line with the result of a previous study that probe manipulation could induce pain during biopsy [bib_ref] Comparison of pain levels in fusion prostate biopsy and standard TRUS-Guided biopsy, Demirtas [/bib_ref] [bib_ref] Magnetic resonance imaging-targeted biopsy may enhance the diagnostic accuracy of significant prostate..., Schoots [/bib_ref]. Considering that the prostate volume in Caucasian men is larger than that in Asian men, the angle of manipulation of the probe for biopsy of the apex or anterior site is larger, and the distance from the PNB site to the apex or anterior prostate is greater. Therefore, the pain may be more severe in Caucasian men [bib_ref] Comparison of the response to treatment between Asian and Caucasian men with..., Chung [/bib_ref].
Our results showed no significant difference in the pain intensity according to the biopsy methods in the base PNB group, but pain during FBx in the base and apex PNB group was the most severe. The overall pain was more with all methods in the base PNB group, but the base and apex PNB group exhibited relatively good overall pain control. Therefore, patients in this group were more sensitive to pain during probe manipulation. Considering that the pain tended to be more severe as the biopsy proceeded, FBx is considered more painful than SBx 9,23 .
The present study was planned to determine the efficacy of additional apex PNB in the recent era of MRItargeted biopsy. We found meaningful results on the difference in pain intensity according to the biopsy method with PNB, which have not been demonstrated previously. Nonetheless, our study is not without limitations. First, this is a single-center retrospective pilot study with a relatively small sample size. The patients were blinded as to the methods of nerve block given, but physician was not blinded, therefore the results are not free of bias. In addition, consensus on pain intensity and the possibility of complications due to additional apex injections is needed. Moreover, even if the average pain of each puncture is lower, it is difficult to directly compare the pain level of each method with different core counts. We performed three biopsy methods sequentially, and there was a limitation in directly comparing pain between each biopsy method. Finally, as we attempted to analyze the difference in pain intensity by matching the target site corresponding to the SBx site, obtaining meaningful results was difficult owing to the small sample size. We plan to validate our results through a well-controlled, prospective, double-blind, randomized, multi-center study.
Prostate biopsy methods have undergone innovative changes, such as performing CBx or FBx with SBx. To increase the cancer diagnosis rate, FBx or CBx is often performed with SBx. Therefore, the optimal method of local anaesthesia should be determined based on the procedure planned and the site of the procedure. Additional PNB administered in the prostate apex provides better overall pain control in CBx, FBx, and SBx.
# Methods
Ethic approval. This study was approved by the institutional ethics committee (Yonsei university health system, Seoul, Korea, 3-2019-0418), and all procedures were conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. The informed consent requirement was waived by ethics committee of Yonsei university health system because this study was based on retrospective, anonymous patient data and did not involve patient intervention or the use of human tissue samples.
## Patient selection.
We prospectively collected data from 229 consecutive patients who underwent MRItargeted transrectal biopsy between January 2019 and September 2020. Patients who were unable to receive transrectal ultrasound probe injection, had severe haemorrhoids (Grade ≥ III, n = 2), had undergone related surgery (n = 2), and were unable to communicate (n = 4) were excluded. According to prebiopsy history taking, there were no patients with neurologic disease such as paraplegia or hemiplegia, and no patients with chronic pain who took analgesics routinely. Finally, 221 (96.5%) patients were selected for analysis [fig_ref] Figure 2: Study cohort flow diagram [/fig_ref]. Data collection. Patient data pertaining to age, PSA level, prostate volume, history of prostate biopsy, prostate imaging-reporting and data system (PI-RADS) scores, pathology results, time required for PNB and biopsies, adverse events (vasovagal syncope, allergic reaction, acute urine retention, urinary retention because of blood clot, and fever), VAS (0 as no pain to 10 as worst pain) pain scores were collected. MRI protocol and analysis. MRI was performed using a 3.0 Tesla system (Intera Achieva 3.0 T, Phillips Medical System, Best, Netherlands) equipped with a phased array coil (six channels). The MRI protocol involved diffusion-weighted imaging and T2 weighted imaging. T2-weighted turbo spin-echo MRI was acquired in three planes (axial, sagittal, and coronal). MRI datasets were obtained for identical slice locations, with a slice thickness of 3 mm and no intersection gap. Two b-values (0-1400) were used, and the diffusion restriction was quantified via apparent diffusion coefficient mapping. Dynamic contrast-enhanced MRI was also performed. All prostate MRIs were evaluated by an experienced urologic-radiologist and graded according to the PI-RADS Version 2.1 [bib_ref] Prostate imaging reporting and data system version 2.1: 2019 update of prostate..., Turkbey [/bib_ref]. Patients with PI-RADS scores of 3-5 were enrolled.
Local anesthesia methods. All patients were instructed to lie in the left lateral decubitus position during the procedure. All biopsies were performed by an experienced urologist. After povidone iodine rectal preparation, 10 cc of 2% lidocaine gel was applied intrarectally (Instillagel®, Farco-Farma GmbH, Köln, Germany). After 5 min, a transrectal probe was inserted, the prostate volume was measured, and PNB was performed with a Chiba needle (A & A M.D. Inc., Seongnam, Korea). The site of local anesthesia (base PNB vs. base and apex PNB) was determined as follows: (1) Odd days: Patients in the base PNB group received PNB on both sides of the prostate base and 2.5 cc normal saline on both sides of the prostate apex; (2) Even days: Patients in the base and apex PNB group received PNB on both sides of the prostate base as well as the prostate apex. The prostate base injections were aimed at the major neurovascular bundle after confirming the triangular echogenic "Mount Everest sign" between the prostate base and the seminal vesicle on the parasagittal longitudinal view of TRUS [bib_ref] Guidelines for transrectal ultrasonography-guided prostate biopsy: korean society of urogenital radiology consensus..., Lee [/bib_ref]. The prostate apex injections were aimed at a smaller triangular echogenic area between the puborectalis muscles and the prostate apex. Each PNB was performed using 2.5 cc of 2% lidocaine [bib_ref] Pain during transrectal ultrasound-guided prostate biopsy and the role of periprostatic nerve..., Nazir [/bib_ref]. Patients in all groups received base injections before apex injections. www.nature.com/scientificreports/ Concurrent prostate biopsy techniques. We routinely check urine analysis and urine culture prior to biopsy decision-making. If there is pyuria or positive urine culture, sufficient antibiotics are used before the biopsy, and biopsy is performed after the follow-up urine analysis and negative confirmation of urine culture. All of the patients in this study received third-generation cephalosporin orally as prophylactic antibiotics for 2 days after the biopsy. All biopsies were performed using the BK 3000 ultrasound system (Analogic Corporation, Peabody, MA, USA) with a 7.5-12 MHz multiplanar probe, in the following order: CBx, FBx, and SBx. First, CBx was performed with two core biopsies per target. After performing CBx, FBx was performed using the MRI/TRUS fusion system (BioJet; GeoScan, Lakewood Ranch, FL, USA) with two core biopsies per target. Therefore, four core biopsies per target were obtained. After performing CBx and FBx, SBx was performed in the order of the right lateral base, right lateral mid, right lateral apex, right medial base, right medial mid, right medial apex, left lateral base, left lateral mid, left lateral apex, left medial base, left medial mid, and left medial apex. The VAS scores were assessed at various time points: probe insertion, injection at the prostate base, injection at the prostate apex, CBx, FBx, SBx, and 15 min after prostate biopsy. We checked the VAS scores for all injections and punctures. All biopsies were performed using guide channels, which were at 19° to the transducer axis of the side-fire probe (Analogic Corporation, Peabody, MA, USA), and an 18G, 20 cm disposable core biopsy instrument (Max-Core®, CR Bard Inc., Covington, GA, USA). Study endpoints. The primary endpoint was VAS score for each biopsy site and PNB method. The secondary endpoints were differences in pain intensity among the biopsy methods and between the anterior and posterior sites according to CBx and FBx.
Statistical analysis. The VAS scores for injection at the base and apex were defined as the average VAS scores for the right and left sides in base as well as apex injections. The VAS scores during CBx, FBx, and SBx were defined as the average VAS scores during individual core biopsies for the three types of biopsies.
Continuous variables are expressed as the mean ± standard deviation or median (interquartile range). Categorical variables are reported as number and frequency. The base PNB and base and apex PNB groups were compared using the independent t-test for continuous variables and the Chi-square test (Fisher's exact test) for two or more variables. The results are presented using a linear mixed model and mean profile graph. The correlation matrix structure of the linear mixed model that showed the relationship between the collected data at various time points was calculated by applying compound symmetry. Statistical analyses were performed using SAS (version 9.4; SAS Institute, Cary, NC, USA). Statistical significance was set at p < 0.05.
## Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
[fig] Figure 1: Demirtas et al. reported VAS scores during FBx and SBx as 2.0 (1.0-4.0) and 3.0 (1.0-5.0), respectively, but the scores were recorded 5 min after the procedure 12 . Egbers et al. reported VAS scores during FBx and SBx as 2.0 (0.0-7.0) and 3.0 (0.0-9.0), respectively, but the scores were recorded 1 week after the procedure via a telephone 13 . Kasivisvanathan et al. reported VAS scores of 1.Mean profile plot of VAS scores for each SBx. SBx systemic prostate biopsy, Lat lateral, Lt left, Med medial, Rt right, VAS visual analog scale. [/fig]
[fig] Figure 2: Study cohort flow diagram. MRI magnetic resonance image, PNB periprostatic nerve block, TRUS transrectal ultrasound. Scientific Reports | (2022) 12:772 | https://doi.org/10.1038/s41598-022-04795-x [/fig]
[table] Table 2: Mean VAS scores during prostate biopsy in patients according to the site of the PNB. Data are expressed as mean ± SD. a Mean and SD of VAS scores during prostate base injections. b Mean and SD of VAS scores during prostate apex injections. c Mean and SD of VAS scores for individual CBx. d Mean and SD of VAS scores for individual FBx. e Mean and SD of VAS scores for individual SBx. CBx magnetic resonance imaging cognitive target prostate biopsy, FBx magnetic resonance imaging/transrectal ultrasound fusion targeted prostate biopsy, SBx prostate systemic biopsy, PNB periprostatic nerve block, SD standard deviation, VAS visual analog scale. [/table]
[table] Table 3: Differences in mean VAS scores during various types of biopsies between the two groups according to biopsy sites. Data are expressed as mean ± SD. CBx magnetic resonance imaging cognitive target prostate biopsy, FBx magnetic resonance imaging/transrectal ultrasound fusion targeted prostate biopsy, PNB periprostatic nerve block, SD standard deviation, VAS visual analog scale. [/table]
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Efficacy and safety of extended depth of focus intraocular lenses in cataract surgery: a systematic review and meta-analysis
Background: This study aims to evaluate the efficacy and safety of extended depth of focus (EDOF) intraocular lenes (IOLs) in cataract surgery. Methods: All comparative clinical trials that involved bilaterally implanting EDOF IOLs in patients with cataract were retrieved from the literature database. We used random effects models to pool weighted mean differences (WMD) and risk ratio (RR) for continuous and dichotomous variables, respectively. Results: Nine studies with a total of 1336 eyes were identified. The subgroup analysis was conducted according to the type of IOLs used in the control group. Compared with monofocal IOLs, EDOF IOLs produced better uncorrected intermediate visual acuity (WMD: -0.17, 95% CI: − 0.26 to − 0.08, P = 0.0001) and uncorrected near visual acuity (WMD: -0.17, 95% CI: − 0.21 to − 0.12, P < 0.00001). EDOF IOLs resulted in reduced contrast sensitivity, more frequent halos, however, higher spectacle independence (RR: 2.81, 95% CI: 1.06 to 7.46, P = 0.04) than monofocal IOLs. Compared with trifocal IOLs, EDOF IOLs produced worse near visual acuity (MD: 0.10, 95% CI: 0.07 to 0.13, P < 0.0001). EDOF IOLs performed better than trifocal IOls in contrast sensitivity, and there were no significant difference in halos and spectacle independence. Serious postoperative complications were rare, with no adverse events were reported in most studies. Conclusions: Increasing the risk of contrast reduction and more frequent halos, EDOF IOLs provided better intermediate and near VAs than monofocal IOLs. At the expense of near vision, patients receiving EDOF IOLs have better contrast sensitivity than those receiving trifocal IOLs. Halo incidence and spectacle independence of EDOF IOLs were similar to those of trifocal IOLs.
# Background
Monofocal intraocular lenses (IOLs) are the most commonly implanted IOLs in cataract surgery [bib_ref] Multifocal versus monofocal intraocular lenses after cataract extraction, De Silva [/bib_ref]. With a single focal point, monofocal IOLs are effective in restoring satisfactory distance vision; however, most patients require spectacle correction for intermediate and near vision, even after surgery [bib_ref] Multifocal versus monofocal intraocular lenses after cataract extraction, De Silva [/bib_ref] [bib_ref] Cataract surgery and lens implantation, Li [/bib_ref]. Thus, multifocal IOLs were designed to meet the increasing demand from patients for spectacle independence [bib_ref] Multifocal intraocular lenses: types, outcomes, complications and how to solve them, Salerno [/bib_ref]. For providing far, intermediate and near vision simultaneously, multifocal IOLs possess two or more independent focal points, which result in contrast reduction and increased photic phenomena, thus reducing visual quality [bib_ref] Dissatisfaction after implantation of multifocal intraocular lenses, De Vries [/bib_ref].
More recently, a new-concept IOL was introduced based on extended depth of focus (EDOF) technology [bib_ref] Extended depth of focus IOLs: the next chapter in refractive technology?, Rocha [/bib_ref]. The basic principle behind EDOF IOLs is to create a single elongated focal point to enhance the depth of focus or range of vision [bib_ref] Extended depth of focus intraocular lenses for presbyopia, Akella [/bib_ref]. A proprietary diffractive echelette design is used in EDOF IOLs and forms a step structure. The height, spacing, and profile of the echelettes are optimized to achieve constructive interference of light from different lens zones, thus producing a novel light diffraction pattern. In addition, proprietary achromatic technology and negative spherical aberration correction improve the image quality [bib_ref] Clinically relevant optical properties of bifocal, trifocal, and extended depth of focus..., Gatinel [/bib_ref]. With technological advancement, EDOF IOLs showed good visual outcomes with less contrast reduction and fewer photic phenomena commonly associated with multifocal IOLs [bib_ref] Dissatisfaction after implantation of multifocal intraocular lenses, De Vries [/bib_ref] [bib_ref] Visual outcomes of patients bilaterally implanted with the extended range of vision..., Sachdev [/bib_ref]. However, according to some studies, EDOF lenses worked less efficiently for near vision than did trifocal IOLs [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref]. Currently, several types of EDOF IOLs are commercially available, including the Tecnis Symfony (Johnson and Johnson Vision), Mini WELL (Sifi Medtech), IC-8 (AcuFocus Inc) and Wichterle Intraocular Lens-Continuous Focus (Medicem). Until 2018, the Tecnis Symfony was the only United States Food and Drug Administration (FDA)-approved EDOF lens [bib_ref] Extended depth of focus intraocular lenses for presbyopia, Akella [/bib_ref].
Although many studies have been conducted to characterize the efficacy and safety of EDOF IOLs, the unique features, such as visual acuity, vision quality and complications of EDOF IOLs is less clear-cut. Thus, we performed a systematic review and meta-analysis of randomized and nonrandomized controlled studies (NRCSs) to compare the clinical performance of EDOF IOLs with that of monofocal and trifocal IOLs. Finally, our study used only Tecnis Symfony IOL as the representative of EDOF IOLs due the lack of studies on other EDOF lenses.
# Methods
## Search strategy
The PubMed, EMBASE, Web of Science, ClinicalTrials.gov and Cochrane Library databases (most recently updated in 2019 January) were searched using the keywords "extended depth of focus intraocular lens", "extended range of vision intraocular lens" and "cataract surgery". No language limitations were applied in the search strategy. In addition, the references of identified articles and reviews were checked and matching publications were included. Two reviewers (J. L. and Y. D.) independently conducted searches and scanned the abstracts, followed by full-text articles to determine whether the articles met the eligibility criteria. A third reviewer (Y. W.) was consulted when disagreement existed between J. L. and Y. D.
## Eligibility criteria
We included all clinical controlled studies (randomized or nonrandomized, from 2000 to 2019 January) comparing clinical outcomes of EDOF IOLs with those of control IOLs in patients undergoing cataract surgery. However, studies involving patients with previous refractive surgery, irregular or > 1.0 diopter (D) corneal astigmatism and coexisting pathology, such as amblyopia, keratoconus, corneal endothelial dystrophy, chronic or recurrent uveitis, acute ocular disease or external/internal infection, diabetes mellitus with retinal changes, glaucoma and choroidal hemorrhage were excluded. We also excluded studies with double implantation in the same eye, no bilateral implantation, double reporting, in vitro experiments and no aggregated results.
## Qualitative assessment and data extraction
The Jadad [bib_ref] Assessing the quality of reports of randomized clinical trials: is blinding necessary?, Jadad [/bib_ref] and Newcastle-Ottawa Scale (NOS) [bib_ref] Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality..., Stang [/bib_ref] were used to assess the quality of randomized controlled trials (RCTs) and NRCSs, respectively. The maximum NOS score is nine points, and a score over six points indicates good quality. Two reviewers (J. L. and Y. D.) independently extracted the characteristic data of included studies using a standard form; we tried to contact the author for sufficient information and original data when necessary. Discrepancies between two reviewers were resolved by a third reviewer (Y. W.).
## Outcome measures
Primary outcomes included binocular uncorrected distance visual acuity (UDVA), uncorrected intermediate visual acuity (UIVA), uncorrected near visual acuity (UNVA), defocus curves and contrast sensitivity. Visual acuity was evaluated using the high-contrast Early Treatment Diabetic Retinopathy Study chart in logMAR units under photopic conditions. Binocular defocus curves were done with best distance correction. Different levels of defocus were introduced in 0.50 D steps from + 1.00 to − 4.00 D. Contrast sensitivity under photopic and scotopic conditions for 1.5, 3.0, 6.0, 12.0, and 18.0 cycles per degree. The contrast sensitivity data was difficult to pool because of the considerable variety of the measurement methods. Thus, contrast sensitivity was instead reported descriptively. Halos, spectacle independence and postoperative complications were defined as the secondary outcomes. Spectacle independence was obtained from selfreported questionnaires and defined as the proportion of subjects who reported wearing glasses or contacts "none of the time" or "a little of the time" for overall vision.
# Statistical analysis
We used RevMan software (version 5.3, Cochrane Collaboration) to analyze the data. The weighted mean difference (WMD) and risk ratio (RR) with 95% confidence interval (CI) were calculated for continuous and dichotomous variables, respectively. A P-value < 0.05 was defined as statistically significant. Forest plots were used to present the results. In forest plots, only subtotals were analyzed because of the evident difference in design principles between monofocal and trifocal IOLs in control groups. Green boxes indicate the mean value, and the size of boxes indicates the weighting given to that estimate. The 95% CI for the estimate is shown as a horizontal line. The diamond represents the mean effect size. The center of the diamond represents the pooled point estimate, and the horizontal tips show the CI. We chose the random effects model for all data analyses because studies differed in trial design, patient ages, implanted IOLs, and the longest follow-up time. For multiarm studies, we combined groups to create a single pairwise comparison as recommended by the Cochrane Handbook for Systematic Reviews of Interventions. To verify the stability of the results, we performed sensitivity analysis by individually omitting the included studies. Publication bias was measured visually using funnel plots.
## Heterogeneity management
Statistical heterogeneity was tested by I 2 tests [bib_ref] Quantifying heterogeneity in a meta-analysis, Higgins [/bib_ref]. Findings were considered statistically significant if I 2 > 50%. Under the assumption that the type of IOLs would explain a portion of heterogeneity, subgroups were defined as monofocal IOLs and trifocal IOLs in control groups.
# Results
## Result of the search
The electronic searches identified a total of 124 records. [fig_ref] Figure 1: Flow chart showing the study selection process [/fig_ref] shows a flow diagram of the included and excluded studies. Two conference abstracts were excluded because the full text was unavailable [bib_ref] Comparison of the functional results of bifocal, trifocal and extended-depth of focus..., Attia [/bib_ref] [bib_ref] Comparison of 3 Presbyopic correcting intraocular lenses, Paul [/bib_ref]. We tried to contact the author but did not receive a reply. Of 10 studies potentially relevant for this meta-analysis, one study enrolling patients with preexisting corneal astigmatism of 1.00 D or worse was excluded [bib_ref] Comparative analysis of visual outcomes with 4 intraocular lenses: Monofocal, multifocal, and..., Pedrotti [/bib_ref]. Ultimately, 9 studies were included in our quantitative analysis [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref]. [fig_ref] Table 1: Characteristics and quality of included studies AMO Abbott Medical Optics, IOL intraocular... [/fig_ref] summarizes the characteristics and quality of the 9 studies that met all inclusion criteria [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref]. Of the 9 selected studies, 3 were RCTs and 6 were NRCSs with a total of 1336 eyes. The studies were performed in various countries, and all studies were published between 2016 and 2018. The RCT sponsored by Abbott Medical Optics (AMO) company, leaded to the U.S. FDA approval of Tecnis Symfony IOL in 2016. Tecnis Symfony ZXR00 was used in the EDOF IOL group, while monofocal IOLs (Tecnis ZCB00 and AcrySof SN60WF) and trifocal IOLs (PanOptix, FineVison and Lisa tri 839MP) were used in the control groups. The follow-up period ranged from 3 to 29 months. The Jadad method was used to assess the methodological quality of RCTs in 3 respects: randomization, blindness and dropouts. Two of three RCTs were scored higher than 3 points. All six NRCSs were of relatively low risk of bias, scoring higher than 6 points on the NOS.
## Study characteristics and quality
## Primary outcomes binocular uncorrected visual acuity
Seven [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref] , five [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] and five [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] studies reported binocular UDVA, UIVA and UNVA, respectively . One study did not report the standard deviation (SD) or other data to calculate the SD and thus was excluded from the analysis. We tried to contact the author but did not receive a reply. The subgroup analysis was conducted according to the type of IOLs used in the control group. Compared with monofocal IOLs, EDOF IOLs provided comparable UDVA (WMD: 0.01, 95% CI: − 0.06 to 0.08, P = 0.81), better UIVA (WMD: -0.17, 95% CI: − 0.26 to − 0.08, P = 0.0001) and better UNVA (WMD: -0.17, 95% CI: − 0.21 to − 0.12, P < 0.00001). Compared with trifocal IOLs, EDOF IOLs showed no significant differences in UDVA (WMD: -0.01, 95% CI: − 0.03 to 0.01, P = 0.34) or UIVA (WMD: -0.03, 95% CI: − 0.07 to 0.01, P = 0.12) and performed worse in UNVA (WMD: 0.10, 95% CI: 0.07 to 0.13, P < 0.0001). In sensitivity analysis, no single study significantly changed the pooled estimate, indicating that the results were stable.
## Defocus curves
Six studies [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref] reported binocular distance-corrected defocus curves. The binocular defocus curves based on 3 trails of 215 subjects for EDOF and monofocal IOLs and 4 trails of 159 subjects for EDOF and trifocal IOLs are shown in [fig_ref] Figure 3: Defocus curves [/fig_ref]. Monofocal, EDOF and trifocal IOLs sustained 0.2 logMAR or better mean VA through 1.0 D, 2.0 D and 3.0 D, respectively. VA was significantly better with EDOF IOLs than with monofocal IOLs in the defocus levels from − 1.0 to − 4.0 D. VA was significantly better in trifocal IOL group than in EDOF IOL group from − 2.5 to − 4.0 D [fig_ref] Table 2: Results of Meta-analysis for Defocus Curve [/fig_ref]. The sensitivity analysis showed that no single study significantly changed the pooled estimate, indicating the results of defocus curves were stable.
## Contrast sensitivity
Seven studies [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref] reported contrast sensitivity and the results are summarized in [fig_ref] Table 3: Summary of Contrast Sensitivity and Halos [/fig_ref]. The U.S. FDA clinical trial reported that the median contrast scores for the EDOF IOL group were reduced compared to the monofocal control group under both conditions and each spatial frequency. Pilger et al. reported that EDOF IOLs performed worse than did monofocal IOLs under scotopic conditions [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref]. Pedrotti et al. reported no significant difference in contrast sensitivity between EDOF and monofocal IOLs under both photopic and scotopic conditions [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref]. Mencucci et al. reported that EDOF performed significantly better than trifocal IOLs under both photopic and scotopic conditions [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref]. Escandón-García et al. reported that EDOF IOLs performed better than trifocal IOLs at a frequency of 1.5 cycles per degree under scotopic conditions [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref]. Two studies reported no difference in contrast sensitivity between EDOF and trifocal IOLs [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref].
## Secondary outcomes halos
Eight studies [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref] used questionnaires and Halo software to record halos. Because these studies used different questionnaires and measurements, conducting quantitative analyses of halos was inappropriate.
Instead, the results are descriptively summarized in [fig_ref] Table 3: Summary of Contrast Sensitivity and Halos [/fig_ref]. Two studies reported no significant difference in halos between EDOF and monofocal IOLs [bib_ref] Comparative analysis of the clinical outcomes with a Monofocal and an extended..., Pedrotti [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref]. The U.S. FDA clinical trial reported that EDOF IOLs resulted in more frequent halos than monofocal IOLs. Monaco et al. reported that both EDOF and trifocal IOLs resulted in more frequent halos than did monofocal IOLs [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref]. Five studies reported no difference in halos between EDOF and trifocal IOLs [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] [bib_ref] A comparative study of the visual outcomes between a new trifocal and..., Ruiz-Mesa [/bib_ref].
## Spectacle independence
Six studies [bib_ref] A comparative evaluation of a new generation of diffractive trifocal and extended..., Cochener [/bib_ref] [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Visual performance after bilateral implantation of 2 new presbyopia-correcting intraocular lenses: trifocal..., Monaco [/bib_ref] [bib_ref] Clinical outcome and higher order aberrations after bilateral implantation of an extended..., Pilger [/bib_ref] [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref] reported spectacle independence. There was a significant difference in the overall effect that favored higher spectacle independence with EDOF IOLs than with monofocal IOLs (RR: 2.81, 95% CI: 1.06 to 7.46, P = 0.04) [fig_ref] Figure 4: Forest plot of spectacle independence [/fig_ref]. The studies were characterized by high heterogeneity (I 2 = 83%, P = 0.003). There was no significant difference between EDOF and trifocal IOLs in the overall effect (RR: 0.96, 95% CI: 0.85 to 1.07, P = 0.45) [fig_ref] Figure 4: Forest plot of spectacle independence [/fig_ref]. No significant heterogeneity was found (I 2 = 0%, P = 0.61).. One study reported 0 and 5% of patients had posterior capsule opacification 12 months postoperatively in the EDOF IOL group and trifocal IOL group respectively [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref].
## Publication bias
The publication bias of the studies was determined by a funnel plot. The symmetrical funnel plot showed no significant publication bias in the publications [fig_ref] Figure 5: Funnel plot for publication bias test [/fig_ref].
# Discussion
The present meta-analysis compared the clinical performance of EDOF IOLs with those of monofocal and trifocal IOLs. According to the results, compared with monofocal IOLs, EDOF IOLs have benefits for intermediate and near vision, but also increase the risk of contrast reduction and more frequent halos. Although EDOF IOLs worked less efficiently for near vision than did trifocal IOLs, they maintained better contrast sensitivity and no differences were found in halo incidence and spectacle independence. All studies included in this meta-analysis involved bilateral implantation. Implantation of the same IOLs in both eyes avoids overestimating or underestimating the efficacy of the IOL caused by interference from the follow eye. Therefore, bilateral implantation is a more effective way to measure the effect of IOLs on quality of life [bib_ref] Multifocal versus monofocal intraocular lenses in cataract surgery: a systematic review, Leyland [/bib_ref].
Creating a single elongated focal point to enhance the range of vision, EDOF IOLs expectedly provided better uncorrected intermediate and near VA than that of monofocal IOLs [bib_ref] Extended depth of focus intraocular lenses for presbyopia, Akella [/bib_ref]. However, EDOF IOLs performed worse on near vision than did trifocal IOLs that splits light into distant, intermediate and near focal points. So the near vision of EDOF IOLs is somewhere between that of monofocal and trifocal IOLs. EDOF IOLs and trifocal IOLs performed similarly on distance and intermediate visions. To reflect vision-related quality of life more directly, uncorrected VAs, instead of corrected VAs were main vision outcomes in our meta-analysis [bib_ref] Impact of presbyopia on quality of life in a rural African setting, Patel [/bib_ref].
Binocular defocus curves also showed comparable distance and intermediate visions with EDOF and trifocal IOLs and better near vision with trifocal IOLs. Although EDOF IOLs improved the range of defocus with VAs of 0.2 logMAR or better by approximately 1 D than monofocal IOLs, trifocal IOLs had the longest range of defocus from 0 to − 3.0 D (VA above 0.2 logMAR). Therefore, EDOF IOLs had superior visual outcomes All monofocal IOLs involved in the current study were aspherical. Aspherical monofocal IOLs have been reported to provide higher contrast sensitivity than spherical IOLs and multifocal IOLs [bib_ref] Aberration and contrast sensitivity comparison of aspherical and monofocal and multifocal intraocular..., Zeng [/bib_ref]. Although the Symfony EDOF IOL employed achromatic and aspheric technologies to maintain visual quality [bib_ref] Extended depth of focus intraocular lenses for presbyopia, Akella [/bib_ref] , it caused a reduction in contrast sensitivity compared to aspherical monofocal IOLs. With EDOF IOLs, there is a tradeoff between the clarity of near vision and contrast sensitivity. However, the present study found that the contrast sensitivity of EDOF IOLs was higher than that of trifocal IOLs, especially under scotopic conditions [bib_ref] Comparative analysis of visual outcomes, reading skills, contrast sensitivity, and patient satisfaction..., Mencucci [/bib_ref] [bib_ref] Through-focus vision performance and light disturbances of 3 new intraocular lenses for..., Escandon-Garcia [/bib_ref]. In trifocal IOLs, the distribution of light to more than one focus results in contrast reduction postoperatively, one of the major limitations of multifocal IOLs [bib_ref] Dissatisfaction after implantation of multifocal intraocular lenses, De Vries [/bib_ref]. Depending on the difference in individual habits and lifestyle in real contexts, spectacle independence is a subjective parameter. Although EDOF IOLs worked less efficiently for near vision than did trifocal IOLs, there was no difference between EDOF and trifocal IOLs in self-reported spectacle independence. In addition, there was no difference in halo incidence between the two groups. This may be explained by the fact that most patients are capable of adapting and tend to become more tolerant of photic phenomena several months postoperatively [bib_ref] Patient acceptability of the Tecnis multifocal intraocular lens, Sood [/bib_ref].
Serious postoperative complications were rare and most of studies did not routinely include complications in their outcome measures. One study reported that trifocal IOLs induced more posterior capsule opacification than EDOF IOLs 12 months postoperatively [bib_ref] Comparison of visual outcomes after bilateral implantation of extended range of vision..., Ruiz-Mesa [/bib_ref]. More studies are needed to prove the safety of EDOF IOLs.
To our knowledge, this meta-analysis is the first to compare the clinical performance of EDOF IOLs in cataract surgery with that of monofocal and trifocal IOLs, respectively. However, this meta-analysis has several limitations. First, between-study heterogeneity was substantial. The included studies varied in length of follow-up, types of IOLs in the control group, study location and measurement methods. We chose the random model for all data analyses and tried to explain the heterogeneity by subgroup analyses and sensitivity analyses. The results were stable in sensitivity analyses by individually omitting the included studies. Second, only 3 of the included studies were RCTs, and the remaining studies were NRCSs that had a potential selection bias. Third, publication bias was suspected due to the exclusion of unpublished studies and conference abstracts. Last, limited number of studies reported postoperative complications. More clinical trails that record postoperative adverse effects are needed to assess the safety of EDOF IOLs.
# Conclusions
This systematic review revealed the unique features of EDOF IOLs when compared with other types of IOLs. Compared with monofocal IOLs, EDOF IOLs have benefits for intermediate and near vision but also increase the risk of contrast reduction and more frequent halos. Compared to trifocal IOLs, EDOF IOLs worked less efficiently for near vision; however, this limitation may be an acceptable compromise to patients, given the accompanying retained contrast sensitivity. In conclusion, EDOF IOLs are efficient at providing distance and intermediate visions and safe with rare serious postoperative complications. Nevertheless, more clinical trails with randomized and controlled study designs and adequate duration are needed to clarify the tradeoffs between EDOF IOLs and other presbyopia-correcting IOLs.
[fig] Figure 1: Flow chart showing the study selection process [/fig]
[fig] Figure 3: Defocus curves. a EDOF and monofocal IOLs. b EDOF and trifocal IOLs Liu et al. BMC Ophthalmology (2019) [/fig]
[fig] IOL: intraocular lens, MD mean difference, CI confidence interval, I 2 extent of inconsistency [/fig]
[fig] Figure 4: Forest plot of spectacle independence. a EDOF and monofocal IOLs. b EDOF and trifocal IOLs [/fig]
[fig] Figure 5: Funnel plot for publication bias test [/fig]
[table] Table 1: Characteristics and quality of included studies AMO Abbott Medical Optics, IOL intraocular lens, RCT randomized controlled trial, NRCS non-randomized controlled studyFig. 2 Forest plot of binocular uncorrected visual acuity. a UDVA. b UIVA. c UNVA [/table]
[table] Table 2: Results of Meta-analysis for Defocus Curve [/table]
[table] Table 3: Summary of Contrast Sensitivity and Halos [/table]
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Trends in CD4 counts in HIV-infected patients with HIV viral load monitoring while on combination antiretroviral treatment: results from The TREAT Asia HIV Observational Database
Background: The aim of this study was to examine the relationship between trends in CD4 counts (slope) and HIV viral load (VL) after initiation of combination antiretroviral treatment (cART) in Asian patients in The TREAT Asia HIV Observational Database (TAHOD).Methods: Treatment-naive HIV-infected patients who started cART with three or more and had three or more CD4 count and HIV VL tests were included. CD4 count slopes were expressed as changes of cells per microliter per year. Predictors of CD4 count slopes from 6 months after initiation were assessed by random-effects linear regression models.
Results: A total of 1676 patients (74% male) were included. The median time on cART was 4.2 years (IQR 2. . In the final model, CD4 count slope was associated with age, concurrent HIV VL and CD4 count, disease stage, hepatitis B or C co-infection, and time since cART initiation. CD4 count continues to increase with HIV VL up to 20 000 copies/mL during 6-12 months after cART initiation. However, the HIV VL has to be controlled below 5 000, 4 000 and 500 copies/mL for the CD4 count slope to remain above 20 cells/microliter per year during 12-18, 18-24, and beyond 24 months after cART initiation.
Conclusions: After cART initiation, CD4 counts continued to increase even when the concurrent HIV VL was detectable. However, HIV VL needed to be controlled at a lower level to maintain a positive CD4 count slope when cART continues. The effect on long-term outcomes through the possible development of HIV drug resistance remains uncertain.
# Background
Studies show that latent infection of CD4 cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy [bib_ref] Latent infection of CD4+ T cells provides a mechanism for lifelong persistence..., Finzi [/bib_ref]. To suppress viral replication so that the VL is below the level of detection with standard assays is thus one of the aims at the start of antiretroviral treatment. Maximal and durable suppression of HIV VL prevents or delays development of drug resistant mutations, preserves CD4 cells, and eventually results in better clinical outcomes. According to the US guidelines, if HIV VL suppression is not achieved, it is necessary to change to a new regimen, a second or third line regimen, with at least two active drugs.
HIV-infected patients in most developing countries have limited second and third line antiretroviral treatment options. In many countries in Asia, second-line combination antiretroviral treatment (cART) is not widely accessible [bib_ref] Prevention and treatment of human immunodeficiency virus/acquired immunodeficiency syndrome in resource-limited settings, Hogan [/bib_ref] [bib_ref] Second-line combination antiretroviral therapy in resource-limited settings: facing the challenges through clinical..., Boyd [/bib_ref] [bib_ref] Deferred modification of antiretroviral regimen following documented treatment failure in Asia: results..., Zhou [/bib_ref]. There remains some uncertainty about the short-term risks to patients receiving first line cART, in particular how their immune status might deteriorate if they persist with a virologically failing regimen. The Pursuing Later Treatment Options (PLATO) collaboration [bib_ref] Predictors of trend in CD4-positive T-cell count and mortality among HIV-1-infected individuals..., Ledergerber [/bib_ref] reported that in patients experiencing triple class failure, treatment regimens that maintain the VL below 10 000 copies/mL or at least provide 1.5 log10 copies/mL suppression below the off-treatment value do not seem to be associated with appreciable CD4-cell-count decline. More recently, Mocroft et al [bib_ref] Estimated average annual rate of change of CD4(+) T-cell counts in patients..., Mocroft [/bib_ref] also reported that CD4 did not significantly decrease even HIV VL exceeded 10 000 copies/mL in patients treated with regimen containing a boosted protease inhibitor. The issue of when to switch from first line regimens may therefore be difficult, especially for patients with modest, stable HIV VL who are clinically doing well [bib_ref] Second-line combination antiretroviral therapy in resource-limited settings: facing the challenges through clinical..., Boyd [/bib_ref] [bib_ref] Antiretroviral roll-out: the problem of second-line therapy, Boyd [/bib_ref].
The aims of this study were to examine the relationship between trends in CD4 count and VL after initiation of combination antiretroviral treatment in HIV-infected Asian patients, using data from The TREAT Asia HIV Observational Database (TAHOD).
# Methods
Established in 2003, TAHOD is a collaborative observational cohort study involving 18 sites in the Asia-Pacific region (See acknowledgement). Detailed methods are published elsewhere. Briefly, each site recruited approximately 200-300 HIV-infected patients, including both patients on or not initiating antiretroviral treatment. Recruitment was based on a consecutive series of patients regularly attending a given site from a particular start-up time. Ethical approval for the study was obtained from the University of New South Wales Ethics Committee and respective local ethics committee.
The following data were collected: patient demographics and baseline data, CD4 and CD8 count, HIV VL level, prior and new AIDS defining illness (ADI), date and cause of death, prior and current prescribed HAART, and reason for treatment change. Data are collected according to a common protocol. Upon recruitment, all available data prior to entry to TAHOD (considered as retrospective data) are extracted from patient case notes. Prospective data are updated sixmonthly at each clinic and transferred to data management centre for aggregation and analyses. TAHOD sites are encouraged to contact patients who were not seen in the clinics in the previous 12 months.
TAHOD patients were included in this analysis if they were treatment naïve and initiated with triple or more combination antiretroviral treatment, and had three or more concurrent CD4 and HIV VL test pairs (within 28 days if not tested on the same day) during follow-up.
Both retrospective and prospective data were included in the analysis.
Trends in CD4 count (slope) was calculated by linear regression with the values at time T, before T, and after T, and was expressed as changes of cells per microliter (μL) per year. The HIV VL was related to the CD4 count slope at time T. Previous studies reported a two-phase CD4 count response, demonstrated as a rapid increase (a high CD4 count slope) in the first several months after treatment initiation and followed by slower increase (a smaller slope compared to the initial several months) [bib_ref] Rapid restoration of CD4 T cell subsets in subjects receiving antiretroviral therapy..., Kaufmann [/bib_ref] [bib_ref] CD4+ cell count 6 years after commencement of highly active antiretroviral therapy..., Moore [/bib_ref] [bib_ref] Long-term patterns in CD4 response are determined by an interaction between baseline..., Egger [/bib_ref]. Preliminary analyses in eligible TAHOD patients showed that the mean CD4 count slope was significantly higher in the first 6 months after cART initiation than in the period afterwards (179 vs. 44 cells/μL per year, p < 0.001). The CD4 slopes were therefore calculated from CD4 counts measured 6 months after cART initiation.
Predictors of the CD4 slope at time T was assessed by random-effect linear regression models which take account of within and between patient variability. Covariates included sex, age (per 10 years), disease stage (CDC category A, tuberculosis with or without other AIDS defining illness, other non-tuberculosis AIDS defining illness), hepatitis coinfection (hepatitis B or C antibody positive), haemoglobin level, time since cART initiation, initial cART regimen non-nucleoside/nucleotide reverse transcriptase inhibitor (NNRTI)-based, nonboosted PI, or boosted PI, and treatment containing abacavir. Disease stage, CD4 count and HIV VL were fitted in the model as time-dependent variable. We did not include CD4 count and HIV VL at baseline for the following three reasons: first, a large proportion of patients did not have the tests at treatment initiation (approximately 25% of patients had no CD4 count and 45% HIV VL, [fig_ref] Table 1: Patient characteristics at baseline in patients selected in the analysis and patients... [/fig_ref] ; second, the model aimed to help clinicians in this region to assess the status of immune system with the clinical information at hand (e.g., age, hepatitis status, current CD4 count, time since treatment initiation, etc) where the baseline information on CD4 count and HIV VL may not be readily available; and third, when we included baseline CD4 and HIV VL in a sensitivity analyses based on the subset of patients with baseline data available, the results remained comparable with the model without the baseline CD4 count and HIV VL. The multivariate models were built using a forward-step approach, the final model included covariates that remained significant at the 0.20 level. Nonsignificant variables were also presented and adjusted for in the final multivariate models.
To take into consideration of the treatment interruption and switch, the following sensitivity analyses were performed: 1. restricting the records measured during initial NNRTI-based regimen; 2. excluding the records measured when patients were off-treatment for more than 30 days for various reasons. Finally, sensitivity analysis was also performed by restricting the records in patients contributing at least 4 or more concurrent CD4 and HIV VL tests.
Data management and statistical analyses were performed using SAS for Windows (SAS Institute Inc., Cary, NC, USA), and Stata (StataCorp, STATA 10.1 for Windows, College Station, Texas 77845 USA).
# Results
There were 4699 patients with data collected in TAHOD as at September 2009. Approximately 75% of patients had a clinic visit in the 12 months before September 2009, and 214 patients died since entry to TAHOD (mortality 1.36 per 100 person years). Among the 4699 TAHOD patients, 612 were not currently receiving antiretroviral treatment, 31 were receiving mono or dual therapy, and 4056 had initiated cART with three or more drugs. 1676 naïve patients initiated cART, and had three or more concurrent CD4 and HIV VL data pairs available beyond 6 months after cART initiation. [fig_ref] Table 1: Patient characteristics at baseline in patients selected in the analysis and patients... [/fig_ref] shows the patient characteristics at cART initiation in patients included in the analysis and in all TAHOD patients who initiated cART with three or more drugs. The characteristics of the patients included in the analysis are generally comparable to those of the whole TAHOD patients, except that the patients included were less likely to be anemic.
At cART initiation, the median age of the patients included in the analysis was 36 years (interquartile range, IQR, 30-42), median CD4 count 140 cells/μL (IQR 42-230), median HIV VL 5.00 log10 copies/mL (IQR 4.33-5.56), 12% had hepatitis B or C co-infection, and 36% were diagnosed with an AIDS defining illness (ADI). The median time on cART was 4.2 years (IQR 2.5 -5.8). The median time between each CD4 and HIV VL tests was 165 days (IQR 106 -223). The initial cART was predominantly an NNRTI-based regimen (63% of 1676 patients in the analysis, with either nevirapine or efavirenz, plus two NRTI drugs, mostly stavudine or zidovudine, plus lamivudine). Approximately 15% of the patients started a non-boosted PI (mostly indinavir, nelfinavir or atanazavir) regimen with two NRTI drugs and 20% started with a ritonavir-boosted PI (mostly lopinavir, atanazavir or saquinavir). The annual rate of a drug class change or change of at least two or more drugs was approximately 20%. After cART initiation, viral logical suppression (HIV VL < 400 copies/ mL) was achieved in 83% of patients at 6 month and 82% in 12 months. [fig_ref] Table 2: Random-effect linear regression analyses of trend of CD4 count [/fig_ref] shows the random-effect linear regression analysis of the CD4 count slope. Concurrent haemoglobin level, initial cART containing NNRTI or boosted PI were not significantly associated with the study endpoint in both univariate and multivariate analyses. Initial cART containing abacavir was significant in the univariate analysis. In the final multivariate model, CD4 count In we gave the formula obtained from [fig_ref] Table 2: Random-effect linear regression analyses of trend of CD4 count [/fig_ref] and an example to estimate the CD4 slope. The formula needs the following information: current age, concurrent CD4 count and HIV VL, hepatitis coinfection, disease stage, and time since cART initiation.
The models shows that, after cART initiation, mean CD4 counts continued to increase even when the concurrent HIV VL was detectable. In addition, the model also shows that to maintain a positive CD4 count slope, the HIV VL needed to be suppressed to a lower level in later periods of cART. To illustrate, the estimated CD4 slopes from the model in two patients with specific baseline characteristics are shown in [fig_ref] Table 3: Estimated CD4 slope [/fig_ref] and in [fig_ref] Figure 2: Estimated CD4 slope [/fig_ref]. The bold cells are when the estimated CD4 count slope falls between -20 and +20 cell/μL per year, which we considered as indicative of borderline CD4 count decreases. In the case of the first patient (aged 30 years, no hepatitis coinfection and AIDS defining illness, and concurrent CD4 count 200 cells/μL), the CD4 count continues to increase with HIV VL up to 20 000 copies/mL during 6-12 months after cART initiation. However, the HIV VL has to be controlled below 5 000, 4 000 and 500 copies/mL for the CD4 count slope to remain on a safe level above 20 cells/μL/year during 12-18, 18-24, and beyond 24 months after cART initiation. Hepatitis co-infection had a significant effect on the CD4 count slope. In one scenario, shown in [fig_ref] Table 3: Estimated CD4 slope [/fig_ref] of a 30-year old patient with concurrent CD4 count 200 cells/μL, no AIDS defining illness and no hepatitis coinfection, CD4 counts continues to increase with HIV VL up to 5 000 copies/mL during 12-18 months after cART. If this patient was hepatitis co-infected, the CD4 count starts to fall when the HIV VL increases up to 3 000 copies/mL.
The analyses were repeated in three subgroups as sensitivity analysis [fig_ref] Table 4: Sensitivity analyses of the CD4 count slope [/fig_ref]. The results are comparable to the final model.
# Discussion
In a subset of TAHOD patients who were treatment naïve and initiated with three or more combination antiretroviral treatment and had concurrent CD4 count and HIV VL tests, the CD4 count slope was associated with age, concurrent CD4 count and HIV VL, disease stage, hepatitis coinfection and time since cART initiation. After cART initiation, CD4 counts continued to increase even when the concurrent HIV VL was detectable. However, HIV VL needed to be controlled at a lower level to maintain a positive CD4 count slope when cART continues at later stages, particularly from 6 months to more than 24 months after cART initiation. The inverse relationship between age and CD4 restoration has been reported in previous studies. In these studies younger age was associated with more rapid CD4 recovery and was associated with preserved thymic function [bib_ref] Predictors of trend in CD4-positive T-cell count and mortality among HIV-1-infected individuals..., Ledergerber [/bib_ref] [bib_ref] CD4+ cell count 6 years after commencement of highly active antiretroviral therapy..., Moore [/bib_ref] [bib_ref] Long-term patterns in CD4 response are determined by an interaction between baseline..., Egger [/bib_ref] [bib_ref] Influence of age on CD4 cell recovery in human immunodeficiency virus-infected patients..., Viard [/bib_ref]. The increase in CD4 slope after TB diagnosis, compared to CDC category A illness, might seem counterintuitive. This might be simply due to the increased total lymphocytes during active infections. The increase could also be the short-term response due to the increased adherence to cART [bib_ref] Motivational interviewing and cognitive-behavioral intervention to improve HIV medication adherence among hazardous..., Parsons [/bib_ref] [bib_ref] Peer support and pager messaging to promote antiretroviral modifying therapy in Seattle:..., Simoni [/bib_ref] and introduction of treatment for TB or other ADI [bib_ref] CD4+ lymphocyte count in African patients co-infected with HIV and tuberculosis, Martin [/bib_ref] [bib_ref] Human immunodeficiency virus-1 RNA levels and CD4 lymphocyte counts, during treatment for..., Morris [/bib_ref].
Studies have shown that neither HBV nor HCV coinfection influence virological response to cART [bib_ref] Arminio Monforte A: Coinfection with hepatitis viruses and outcome of initial antiretroviral..., De Luca [/bib_ref] [bib_ref] Influence of hepatitis C virus infection on HIV-1 disease progression and response..., Rockstroh [/bib_ref] [bib_ref] Hepatitis B and C virus coinfection in The TREAT Asia HIV Observational..., Zhou [/bib_ref]. However, in terms of immunological response, the results were mixed [bib_ref] Arminio Monforte A: Coinfection with hepatitis viruses and outcome of initial antiretroviral..., De Luca [/bib_ref] [bib_ref] Influence of hepatitis C virus infection on HIV-1 disease progression and response..., Rockstroh [/bib_ref] [bib_ref] Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with..., Greub [/bib_ref] [bib_ref] Hepatitis C and progression of HIV disease, Sulkowski [/bib_ref] [bib_ref] Hepatitis B and HIV: prevalence, AIDS progression, response to highly active antiretroviral..., Konopnicki [/bib_ref]. Law et al observed in HIV-infected patients with HBV or HCV an initially delayed CD4 count recovery at week four after HAART treatment, but at week 48 the CD4 count increase was similar to the patients only infected with HIV [bib_ref] Impact of viral hepatitis coinfection on response to antiretroviral therapy and HIV..., Law [/bib_ref]. These studies examine the absolute CD4 count rather than the trend since cART initiation. A decrease in CD4 count slope of less than 20 cells might not be clinically significant in the early phase of cART, but from our estimates [fig_ref] Table 3: Estimated CD4 slope [/fig_ref] , it does have a significant impact on whether the CD4 count slope decreased after longer durations of cART. For example, the patient with no hepatitis co-infection would continue to have a CD4 count increase over 20 cells/μL more than 24 months after cART initiation even the concurrent HIV VL is above 500 copies/mL. If this patient is co-infected with hepatitis and on cART for more than 24 months, the CD4 count slope is below 20 cells/μL even the concurrent HIV VL is 500 copies/mL. The PLATO study [bib_ref] Predictors of trend in CD4-positive T-cell count and mortality among HIV-1-infected individuals..., Ledergerber [/bib_ref] reported that in patients experiencing triple class failure, treatment regimens that maintain the VL below 10 000 copies/mL or at least provide 1.5 log10 copies/mL suppression below the off-treatment value do not seem to be associated with appreciable CD4-cell-count decline. In a combined analysis between Asian and Australian patients infected HIV, Egger et al [bib_ref] Long-term patterns in CD4 response are determined by an interaction between baseline..., Egger [/bib_ref] reported a three-way interaction between the time since cART, baseline CD4 and post-cART HIV VL and estimated that for patients with intermittent HIV viral suppression (below 400 copies/mL), the mean absolute CD4 count begins to decrease or plateau after 4 years of cART. These studies and our findings show that after cART initiation, mean CD4 count slope can continued to increase even when the concurrent HIV VL is detectable. While Egger et al introduced the effect of time in the equation of long term patterns of CD4 response, the results from this analysis further added that the concurrent HIV VL level is a significant factor in determining the trend of CD4 after cART.
Using data from EuroSIDA, Mocroft et al [bib_ref] Estimated average annual rate of change of CD4(+) T-cell counts in patients..., Mocroft [/bib_ref] reported that CD4 did not significantly decrease even HIV VL exceeded 10 000 copies/mL in patients treated with regimen containing a boosted protease inhibitor. Drug class and cART containing abacavir was also included in the analysis, however, none remained significant in the final model. This might be due to three reasons: first, the paper by Mocroft et al analysed data from Euro-SIDA where the predominant cART regimen was PIbased (46% non-boosted, 23% boosted PI). TAHOD recruits patients from the Asia Pacific region, with NNRTI-based regimen as the most common initial cART (63%, 15% non-boosted and 20% boosted PI). In addition, abacavir was not frequently used in TAHOD; second, the patients who received PI-or NNRTI-based cART as initial regimen might be different between EuroSIDA and TAHOD, which could result in a different recovery pattern of the immune system; three, as suggested by Mocroft et al, larger studies with increased power are needed. Nonetheless, our study provided complementary evidence in patients from Asia Pacific region that CD4 counts continues to increase even when the concurrent HIV VL was detectable.
Similar to other studies [bib_ref] Rapid restoration of CD4 T cell subsets in subjects receiving antiretroviral therapy..., Kaufmann [/bib_ref] [bib_ref] CD4+ cell count 6 years after commencement of highly active antiretroviral therapy..., Moore [/bib_ref] , our data showed a two-phase CD4 count response with a high CD4 count slope in the first six months after treatment initiation followed by a lower slope. The only factor in the final multivariate model [fig_ref] Table 2: Random-effect linear regression analyses of trend of CD4 count [/fig_ref] that could be modified and had a significant impact on CD4 count slope was the concurrent HIV VL, which is a 40 CD4 cells decrease for every 1 log10 HIV VL increase. From our estimation [fig_ref] Table 3: Estimated CD4 slope [/fig_ref] , the CD4 count continues to increase with HIV VL up to 20 000 copies/mL during 6-12 months after cART initiation. However, the HIV VL has to be controlled below 5 000, 4 000 and 500 copies/mL for the CD4 count slope to reach a safe level above 20 cells/μL/year during 12-18, 18-24, and beyond 24 months after cART initiation.
In many countries in Asia, second-line cART is not widely accessible [bib_ref] Prevention and treatment of human immunodeficiency virus/acquired immunodeficiency syndrome in resource-limited settings, Hogan [/bib_ref] [bib_ref] Second-line combination antiretroviral therapy in resource-limited settings: facing the challenges through clinical..., Boyd [/bib_ref] [bib_ref] Deferred modification of antiretroviral regimen following documented treatment failure in Asia: results..., Zhou [/bib_ref]. Several studies reported sustainable CD4 count increases in patients with virological failure but remained on the same failing cART [bib_ref] Determinants of sustainable CD4 lymphocyte count increases in response to antiretroviral therapy, Staszewski [/bib_ref] [bib_ref] Duration and predictors of CD4 T-cell gains in patients who continue combination..., Deeks [/bib_ref] [bib_ref] Changes in viral load in people with virological failure who remain on..., Cozzi-Lepri [/bib_ref]. Our results suggest that patients with detectable but modest VL may continue their failing cART regimen without increasing their immune deficiency and the risk of poor clinical outcomes over the short term. This is in agreement with the US treatment guideline, which recommended adherence assessment, repeated HIV VL tests to rule out "blips" [bib_ref] Intermittent HIV-1 viremia (Blips) and drug resistance in patients receiving HAART, Nettles [/bib_ref] , and genotypic tests to detect drug resistant mutations before considering treatment switch. The recent 2009 revision of the WHO antiretroviral therapy guidelinesrecommended adherence assessment, repeated HIV VL test, and switch only when HIV VL remains more than 5 000 copies/ mL. If HIV VL monitoring is available, switch to second-line cART should be done as soon as possible when treatment failure is established. However, in many countries in Asia, especially those developing countries, frequent HIV VL monitoring and genotypic tests are beyond the limited resource for HIV treatment and care [bib_ref] Measures of site resourcing predict virologic suppression, immunologic response and HIV disease..., Oyomopito [/bib_ref]. If CD4 count is the only way for monitoring treatment response, the result of this analysis showed that a patient can have a considerable duration of virological failure without meeting CD4 criteria recommended by WHO for switch of ART to second line. In addition, the effect of delaying switching treatment on longer term outcomes through the possible development of HIVdrug resistance that could compromise the efficacy of later cART regimens remains uncertain.
# Limitations
Several limitations should be considered in interpreting the results in this paper. First, TAHOD participating sites are generally urban referral centres, and each site recruits 200-300 patients who are considered by local clinicians to have a reasonably good prospect of longterm follow-up. Hence TAHOD patients, and their treatment, are not representative of all HIV-infected patients in the Asia and Pacific region. Second, we do not have data on adherence and treatment against TB and other ADI. Finally, a more thorough analysis would include the survival outcome. However, because of the limited number and follow-up of patients who were failing virologically, this analysis is currently underpowered. Further analyses will be considered with longer duration of follow-up.
# Conclusion
The analyses suggest that after cART initiation, mean CD4 slope can continue to increase even when the concurrent HIV VL is detectable. HIV VL needed to be controlled at a lower level to maintain a positive CD4 slope beyond 2 years of cART. However, the effect on longer term outcomes through the possible development of HIV-drug resistance remains uncertain.
[fig] Figure 2: Estimated CD4 slope (cells/μL/year) by duration of treatment and HIV VL. [/fig]
[table] Table 1: Patient characteristics at baseline in patients selected in the analysis and patients starting 3 or more cART in TAHOD [/table]
[table] Table 2: Random-effect linear regression analyses of trend of CD4 count (slope, cells/μL per year) [/table]
[table] Table 3: Estimated CD4 slope (cells/μL/year) by duration of treatment and HIV VL. Patient 1, 30 years old, no hepatitis coinfection, no AIDS defining illness, and current CD4 200 cells/μL 20 cells/μL per year Patient 2, 30 years old, coinfected with hepatitis, no AIDS defining illness, and current CD4 200 cells/μL [/table]
[table] Table 4: Sensitivity analyses of the CD4 count slope. [/table]
|
A Case of Anti-glutamic Acid Decarboxylase-65 Antibody Positive Stiff Person Syndrome Presenting Initially as Acute Peripheral Vestibulopathy, Leading to Delayed Diagnosis After Multiple Hospitalizations
# Introduction
First described by Woltman and Moersch in 1956 when they reported 14 patients, stiff person syndrome (SPS) is an extremely uncommon neuro-immunological condition characterized by axial muscle rigidity which is progressive in nature and stiffness along with episodes of painful muscle spasms. Found to affect females more than males, it is categorized into four main variants --classic, partial, paraneoplastic, and progressive encephalomyelitis with myoclonus and rigidity. Treatment involves the use of GABAergic drugs such as benzodiazepines, along with corticosteroids, plasmapheresis, and intravenous immune globulin. The pathophysiology is not completely understood, but is widely believed to be associated with the involvement of anti-glutamic acid decarboxylase (anti-GAD), which are cytoplasmic enzymes that take part in synthesizing GABA in the spinal cord and brain. The GAD-65 isoform is associated with SPS, along with cerebellar ataxia, limbic encephalitis, and type 1 diabetes mellitus. Approximately three out of five SPS patients are positive for anti-GAD2 1 1 1 antibodies, however, amphiphysin remains the dominant auto-antigen in paraneoplastic SPS. Owing to the rarity of the condition, roughly 60% of cases are detected only because of the presence of anti-GAD-65 in the blood. In this case report, we present an atypical case of SPS which, due to symptoms of vertigo and dizziness, was confused with peripheral vestibulopathy, leading to a delay in treatment.
## Case presentation
A 58-year-old previously healthy female patient presented to hospital with complaints of vertigo and imbalance for one week, along with back pain. She appeared anxious and her vital signs were within normal limits. Her neurological examination was essentially unremarkable except for bilateral gaze evoked nystagmus, more on the right side, and gait ataxia. Over the course of a four-day hospitalization, she underwent MRI of the brain and echocardiogram which were normal. While in hospital, she also had an episode of hyperventilation and blank staring look, which was attributed to panic episode at that time. A working diagnosis of acute peripheral vestibulopathy was made and she was treated symptomatically with betahistine and anxiolytics.
Her symptoms of vertigo and back pain along with nausea and headache persisted after being discharged. She also had blurry vision, gait ataxia, and neck stiffness. Twelve days after discharge, she was re-admitted to another hospital and investigated extensively. MRI brain and MRI spine were normal except for exaggerated lumbar lordosis. Creatine phosphokinase (CPK) was found to be 695 (normal 26-192 U/L). She was treated symptomatically with analgesics, anti-emetics, anti-depressants, gabapentin, and propranolol over a one-week hospitalization. At this stage, the diagnosis was still unclear but a few conditions like subacute cerebellar ataxia, paraneoplastic syndrome, and essential oil toxicity were being considered.
She was brought to us a week after this episode. Her back pain and difficulty walking had persisted. She also had diffuse headache with giddiness along with back spasms and left leg weakness. On examination, she was wheelchair bound, conscious, and co-operative. She was unable to stand and ambulate without assistance and had severe tenderness in her neck and back muscles, along with tightness and stiffness in the trapezius. On examination, her deep tendon reflexes were brisk and power was 4/5 in all four limbs. Sensations were intact and cerebellar function was normal. Notably, she was always keeping her left leg in a flexed position and complained of pain on moving the left hip. She was admitted again to be investigated. Whole body MRI revealed marrow edema within left acetabulum and right femoral head showing heterogenous postcontrast enhancement with surrounding enhancing soft tissue suggestive of neoplastic etiology, concerning for metastasis/marrow infiltrative disorder. Subsequent bone biopsy was largely normal, revealing only scanty tissue showing bony trabeculae. Anti-nuclear antibody (ANA) and urine Bence Jones protein were negative. CPK level was 262. Anti-glutamic acid decarboxylase (GAD 65) antibodies were markedly elevated at >2000I U/mL (normal <10 IU/mL). This along with the constellation of characteristic symptoms established the diagnosis of SPS. Treatment with intravenous immune globulin (IVIG), diazepam, baclofen, steroids, and mycophenolate were started. She complained of intractable vomiting on the second day of treatment, so all oral feeds were stopped. Esophagogastroduodenoscopy revealed large hiatal hernia and gastric ulcers, which were treated accordingly. Post this, IVIG was restarted. Her symptoms improved dramatically, with progressive increase in muscle strength and mobility and decrease in hip pain and spasticity. She was soon able to ambulate without support and was discharged on oral medications.
# Discussion
Stiff person syndrome's uncommon occurrence and its elusive signs make the diagnosis of this condition difficult. Patients generally present insidiously, with episodic stiffness and pain of the axial musculature gradually progressing to the proximal muscles. Hyperlordosis of the lumbar spine is pathognomonic. This case report demonstrates how, due to the rarity of the condition, the diagnosis is often overlooked, leading to delayed diagnosis and treatment. Our patient also initially presented with atypical cerebellar symptoms instead of the classical pain and stiffness, leading to multiple hospitalizations and extensive workup before the diagnosis was made.
A very typical but often underappreciated feature of this disease is marked anxiety, excessive startle, and specific phobias with the latter being a rather common feature as compared to the others mentioned. The symptoms of ataxia and vertigo, and anxiety coupled with the hyperventilation and staring spells made the diagnosis a challenge during the first visit, leading to missed or delayed diagnosis of SPS. This delay in diagnosis led to deterioration of the patient's condition, repetitive hospital admission, and loss of precious time and resources.
Our case report highlights the need for practicing neurologists to have a reasonably high index of suspicion for diagnosing SPS, a rare but progressively debilitating autoimmune disease of the central nervous system, which is easily treatable but often overlooked. |
Effects of Robotic Interactive Gait Training Combined with Virtual Reality and Augmented Reality on Balance, Gross Motor Function, Gait Kinetic, and Kinematic Characteristics in Angelman Syndrome: A Case Report
# Introduction
Angelman syndrome (AS) is a genetic neurological disorder resulting in cognitive and neuromuscular impairments, such as lack of safety awareness and attention, as well as lack of balance and locomotor control. This may predispose patients to a high risk of fatal fall injuries [bib_ref] Angelman syndrome-Insights into a rare neurogenetic disorder, Buiting [/bib_ref] [bib_ref] Angelman syndrome: Consensus for diagnostic criteria, Williams [/bib_ref] [bib_ref] Refining the Behavioral Phenotype of Angelman Syndrome: Examining Differences in Motivation for..., Heald [/bib_ref]. In particular, such impaired balance and locomotor control are complicated by force and kinematic coordination, which affect single-limb stance balance or stability and also compromise spatiotemporal variables during gait . Specifically, an abrupt or uncoordinated waddling gait pattern, with reduced stride length, variable cadence and gait speed, and increased double-support and stance-phase duration were commonly observed in the gait of children with AS [bib_ref] Angelman syndrome-Insights into a rare neurogenetic disorder, Buiting [/bib_ref] [bib_ref] Neurologic manifestations of Angelman syndrome, Thibert [/bib_ref]. Furthermore, the lack of motivation and interest or easy distraction during treatment activities are identified as barriers to rehabilitation. However, conventional physical therapy has difficulty in increasing the motivation and interest levels of children with AS [bib_ref] Refining the Behavioral Phenotype of Angelman Syndrome: Examining Differences in Motivation for..., Heald [/bib_ref]. Therefore, an effective and sustainable intervention is needed while considering balance and locomotor coordination, and factors affecting personal motivation and interest level [bib_ref] Physical therapists' perceptions of factors influencing the acquisition of motor abilities of..., Bartlett [/bib_ref].
To mitigate balance and locomotor control impairments in AS, conventional physical therapy, including a body-weight-supported treadmill (BWST) exercise, was used but
# Materials and methods
## Case description
The participant was a 15-year-old male with AS. The study was approved by the Yonsei University Mirae campus Institutional Review Board (1041849-202108-BM-126-02). Initially, the participant was wheelchair-bound and referred to our center to improve ambulation and cognitive and psychological (aggressive and impulsive) behaviors. At the start of the intervention, he mostly used a wheelchair. The participant had been receiving conventional physical therapy and sensory integration therapy prior to participation in the present case study. However, according to the caregiver interview, the patient experienced balance and falling dysfunction. The primary goal at the time of examination was to improve confidence in reducing fall likelihood and improving balance while walking to ease the burden on his family. The participant's parents provided informed consent prior to beginning the study. Demographic and clinical characteristics are shown in [fig_ref] Table 1: Clinical characteristics of the adolescent [/fig_ref].
## Robotic interactive gait-training system
The RIGT system was designed to facilitate earlier ambulation by allowing the user to adjust body-weight-bearing control and real-time visual biofeedback for torque and stiffness. Kinematics for the hip, knee, and ankle joints provide accurate ankle-knee-hip joint movement and facilitate earlier ambulation by providing adjusted body-weightbearing control and real-time visual biofeedback for torque and stiffness, and kinematics for the hip, knee, and ankle joints. The device consists of a suspension harness for body-Children 2022, 9, 544 3 of 9 weight support, a motorized treadmill, and a hip-knee-ankle coordinated exoskeleton. The treadmill speed and torque, assistance force, and resistive force can be adjusted to accommodate the patient's level of locomotor performance as the training progresses [fig_ref] Figure 1: Robot interactive gait training Walkbot [/fig_ref]. stiffness. Kinematics for the hip, knee, and ankle joints provide accurate ankle-kn joint movement and facilitate earlier ambulation by providing adjusted body-w bearing control and real-time visual biofeedback for torque and stiffness, and kine for the hip, knee, and ankle joints. The device consists of a suspension harness fo weight support, a motorized treadmill, and a hip-knee-ankle coordinated exosk The treadmill speed and torque, assistance force, and resistive force can be adju accommodate the patient's level of locomotor performance as the training progres [fig_ref] Figure 1: Robot interactive gait training Walkbot [/fig_ref]. The RIGT system provides weight-bearing control and real-time audiovisua back for torque, active, and resistive force, and kinematics for the hip and knee join provides VR games and AR scenes to maximize the user motivation and concen [bib_ref] Effects of innovative hip-kneeankle interlimb coordinated robot training on ambulation, cardiopulmonary function,..., Park [/bib_ref] [bib_ref] Effects of Walkbot gait training on kinematics, kinetics, and clinical gait function..., Hwang [/bib_ref]
## Experimental task and procedures
RIGT was performed two times per week for 10 weeks (20 sessions) for 30 m session on the Walkbot (P&S mechanics, Seoul, Korea) system, excluding the t The RIGT system provides weight-bearing control and real-time audiovisual feedback for torque, active, and resistive force, and kinematics for the hip and knee joints, and provides VR games and AR scenes to maximize the user motivation and concentration [bib_ref] Effects of innovative hip-kneeankle interlimb coordinated robot training on ambulation, cardiopulmonary function,..., Park [/bib_ref] [bib_ref] Effects of Walkbot gait training on kinematics, kinetics, and clinical gait function..., Hwang [/bib_ref].
Children 2022, 9, x FOR PEER REVIEW 3 of 9 stiffness. Kinematics for the hip, knee, and ankle joints provide accurate ankle-knee-hip joint movement and facilitate earlier ambulation by providing adjusted body-weightbearing control and real-time visual biofeedback for torque and stiffness, and kinematics for the hip, knee, and ankle joints. The device consists of a suspension harness for bodyweight support, a motorized treadmill, and a hip-knee-ankle coordinated exoskeleton. The treadmill speed and torque, assistance force, and resistive force can be adjusted to accommodate the patient's level of locomotor performance as the training progresses [fig_ref] Figure 1: Robot interactive gait training Walkbot [/fig_ref].
## Figure 1.
Robot interactive gait training Walkbot.
The RIGT system provides weight-bearing control and real-time audiovisual feedback for torque, active, and resistive force, and kinematics for the hip and knee joints, and provides VR games and AR scenes to maximize the user motivation and concentration [bib_ref] Effects of innovative hip-kneeankle interlimb coordinated robot training on ambulation, cardiopulmonary function,..., Park [/bib_ref] [bib_ref] Effects of Walkbot gait training on kinematics, kinetics, and clinical gait function..., Hwang [/bib_ref]
## Experimental task and procedures
RIGT was performed two times per week for 10 weeks (20 sessions) for 30 min per session on the Walkbot (P&S mechanics, Seoul, Korea) system, excluding the time re-
## Experimental task and procedures
RIGT was performed two times per week for 10 weeks (20 sessions) for 30 min per session on the Walkbot (P&S mechanics, Seoul, Korea) system, excluding the time required for set-up. Time for breaks was provided when requested by the participant; however, the treatment time was maintained for at least 30 min. The participant's anthropometric data, including age, height, weight, hip-knee length, knee-ankle length, and foot length were initially measured and used for RIGT training. He was equipped with harness and exoskeletal attachments for the thigh, knee, and foot, which were adapted on the basis of anthropometric characteristics. Gait parameter data, including velocity (1.1 km/h), 0% body-weight suspension, and duration and frequency of walking sessions (30 min per day per week) were documented. The participant successfully completed 20 sessions of intervention training [bib_ref] The effect of robot-assisted gait training on locomotor function and functional capability..., Jin [/bib_ref]. RIGT speed and torque (passive and active) were adjusted to accommodate the participant's locomotor performance as the training progressed. The session incorporated VR-AR games (e.g., a virtual side-scrolling game-Jordon jumping and taking the coins) and AR scenes (e.g., three-dimensional walking to explore a king's castle) to maximize patient motivation and concentration [bib_ref] Effects of innovative hip-kneeankle interlimb coordinated robot training on ambulation, cardiopulmonary function,..., Park [/bib_ref] [bib_ref] Comparative effectiveness of robot-interactive gait training with and without ankle robotic control..., Park [/bib_ref] [bib_ref] Test-retest reliability and construct validity of the tinetti performance-oriented mobility assessment in..., Canbek [/bib_ref] [bib_ref] Reliability and validity of the gross motor function classification system for cerebral..., Bodkin [/bib_ref] [bib_ref] Validity, responsiveness, minimal detectable change, and minimal clinically important change of Pediatric..., Chen [/bib_ref] [bib_ref] The Short FES-I: A shortened version of the falls efficacy scale-international to..., Kempen [/bib_ref] , while decreasing anxiety and depression. Clinical outcome and biomechanical measures were obtained.
## Clinical outcome measurements 2.4.1. tinetti performance-oriented mobility assessment
The performance-oriented mobility assessment (POMA) test was used to measure dynamic and static balance and gait [bib_ref] Clinimetric properties of the performance-oriented mobility assessment, Faber [/bib_ref]. The POMA version used in this clinical evaluation consisted of eight balance items and eight gait items. Balance test items included assessment of sitting balance, rising from a chair and sitting down again, standing balance (eyes open and eyes closed), and turning balance; a maximum score of 12 points can be achieved [bib_ref] POMA: A tangible user interface to improve social and cognitive skills of..., Mahmud [/bib_ref]. The gait test items included assessment of gait initiation, step length, step height, step length symmetry and continuity, path direction, and trunk sway; a maximum score of 16 points can be achieved. The total score ranges from 0 to 28 points [bib_ref] Is the Tinetti Performance Oriented Mobility Assessment (POMA) a feasible and valid..., Tinetti [/bib_ref].
## Gross motor function measures
This GMFM test was specifically developed for clinical and research purposes and is used to assess five domains of gross motor function in children with disabilities [bib_ref] The gross motor function measure: A means to evaluate the effects of..., Russell [/bib_ref]. The five areas include A (lying and rolling), B (sitting), C (crawling and kneeling), D (standing), and E (walking, running, and jumping). It contains 66 categories. Each item has a score of 3, with 17 items for area A, 20 for B, 14 for C, 13 for D, and 24 for E. The score obtained in each domain divided by the possible score ×100 is the score for each region, and the addition of each domain score divided by 5 becomes the total score.
## Pediatric balance scale
The pediatric balance scale (PBS) is used to assess functional sitting and standing balance ability in children with neuromuscular motor impairments [bib_ref] The performance of children developing typically on the pediatric balance scale, Franjoine [/bib_ref]. Fourteen test items are included as follows: moving from a seated to standing position, moving from a standing position to sitting position, transfer, standing without support, sitting without support, standing with eyes closed, standing with feet together, standing with one foot in front of the other, standing on one foot, rotating 360 degrees, turning to look back, picking up an object off the floor, placing alternate foot on step or footrest, and reaching forward with an extended arm. The test score ranges from 0 ("low function") to 4 ("highest function"), with a total possible score of 56 points [bib_ref] Pediatric balance scale: A modified version of the berg balance scale for..., Franjoine [/bib_ref].
## Short fall efficacy scale
The short falls efficacy scale (sFES) questionnaire is used to measure fear of falling in patients with neuromuscular motor impairments during a range of activities of daily living included in the activity domain of the international classification of functioning, disability and health (ICF) model. The sFES comprises seven items. Each activity of daily living is scored on a 4-point scale as follows: 1 ("not at all concerned"), 2 ("somewhat concerned"), 3 ("fairly concerned"), and 4 ("very concerned"). Total possible scores range from 7 ("low concern") to 28 ("high concern") [bib_ref] The Short FES-I: A shortened version of the falls efficacy scale-international to..., Kempen [/bib_ref] [bib_ref] Assessment of fear of falling in older adults: The falls efficacy scale-international..., Greenberg [/bib_ref].
## Biomechanical measurement
Biomechanical measurement was used to determine joint movement and force information during treadmill gait before and after Walkbot robotic gait training. The participant underwent biomechanical measurements, including hip and knee joint kinematics (angular displacement) and kinetics (torque, active, and resistive force). The RIGT system is used in conjunction with gait evaluation mechanics software (GEMS), including kinematic and kinetic computing software, which can calculate angular displacement, moment or torque of the hip, knee, and ankle joints in real time. The GEMS was determined using the kinematic and kinetic computing software (P&S Mechanics, Seoul, Korea) of the RIGT system, which calculates joint angular displacement, active and resistive hip, knee, and ankle joint forces, and torque. Kinematic measurements encompassed the joint angle, angular velocity, and acceleration, which were then used to calculate the moment or torque associated with the active and resistive forces of the body segment acting on the ankle, knee, and hip joints of the participant during walking. Kinetic measurements included the active and resistive forces and torques of the body segment acting on the hip joint during RIGT. With the thigh lever arm acting on the RIGT system, the recorded force data can be converted into hip joint torques acting between the RIGT system and the participant's leg. The hip-knee-ankle joint torque data were collected by the servomotors mounted in the robotic system, in which the corresponding encoders modulate the hip, knee, and ankle joint kinetics. Specifically, active force is defined as a positive directional rotation force that occurs in line with the target movement direction, whereas the resistive force is defined as a negative directional rotation force that acts against the target movement direction [bib_ref] Validity and feasibility of intelligent Walkbot system, Jung [/bib_ref] [bib_ref] Force/torque sensorless impedance control for indirect driven robot-aided gait rehabilitation system, Van Tran [/bib_ref]. The RIGT system is modulated by six servomotors for the bilateral hip, knee, and ankle joints, which enable the safe, coordinated control of locomotor kinematics and kinetics using encoders and a full dynamic model of the exoskeletal system. Each actuated servomotor has a built-in encoder to instantaneously detect the joint angle, angular velocity, and acceleration, and is used for torque computation. Foot load sensors are used to monitor the pressure distribution of each foot when the exoskeleton's feet are in contact with the treadmill surface, thus providing information on the center of the pressure trajectories during the stance phase. All foot sensors are hardwired to a network circuit of electronic input/output module boards, which are then linked to a central command and operating system. The validity and feasibility of intelligent RIGT systems are well established [bib_ref] Validity and feasibility of intelligent Walkbot system, Jung [/bib_ref] [bib_ref] Concurrent validity and test-retest reliability of the walkbot-K system for robotic gait..., Lee [/bib_ref] [fig_ref] Table 2: Clinical outcome measurements [/fig_ref].
# Results
## Clincial outcome measurements
The POMA score increased from 12 (pre-robotic walking) to 15 (post-robotic walking), which was a 25% improvement in gait and balance ability. The GMFM score increased from 43 (pre-robotic training) to 48 (post-robotic training), which was an 11.62% increase in gross motor function. The PBS score increased from 1 (pre-robotic walking) to 32 (post-robotic walking), indicating a 3100% improvement in functional balance ability. The sFES score increased from 16 (pre-robotic walking) to 19 (post-robotic walking), indicating a 18.75% improvement in fear of falling [fig_ref] Table 3: Biomechanics measurements [/fig_ref].
## Biomechanics measurements
Both knee and hip joint torque values increased approximately 20% as a function of robotic training, supporting clinical improvement in the active muscle strength required for gait function. The resistive hip joint torque decreased; however, the kinematic analysis revealed a significant increase in maximal hip flexion angle after Walkbot training.
# Discussion
The present case study is the first clinical evidence highlighting the positive effects of RIGT combined with VR-AR on gait ability, as determined by POMA score, gross motor function by GMFM score, balance by PBS score, knee and hip joint kinetics, and kinematics during gait in an adolescent patient with AS. As hypothesized, all outcome measures related to balance, gait, and biomechanical characteristics (hip and knee active torque, active and resistive force, and joint angular displacement kinematics) were enhanced after 20 sessions of intensive RIGT combined with VR-AR. To the best of our knowledge, no previously published studies are available for comparison with our RIGT case study.
Clinical balance analysis demonstrated that RIGT effectively increased POMA (25%) and PBS (3100%) in an adolescent patient with AS. This finding was consistent with that of previous gait and balance ability results [bib_ref] Cardiorespiratory fitness, balance and walking improvements in an adolescent with cerebral palsy..., Cesar [/bib_ref] [bib_ref] Effects of robotic rehabilitation on walking and balance in pediatric patients with..., Yazıcı [/bib_ref] [bib_ref] Clinical application of a robotic ankle training program for cerebral palsy compared..., Sukal-Moulton [/bib_ref]. [bib_ref] Cardiorespiratory fitness, balance and walking improvements in an adolescent with cerebral palsy..., Cesar [/bib_ref] reported that eight weeks of gait training with a motor-assisted elliptical device improved PBS (17.0%) and timed up-and-go (16.4%) in 13 children with cerebral palsy and autism [bib_ref] Cardiorespiratory fitness, balance and walking improvements in an adolescent with cerebral palsy..., Cesar [/bib_ref]. found that robotic gait training improved balance (2.5%) more than standard physical therapy after 12 weeks in 24 children with hemiparetic cerebral palsy (CP) [bib_ref] Effects of robotic rehabilitation on walking and balance in pediatric patients with..., Yazıcı [/bib_ref]. Sukal-Moulton et al. found an increase in PBS (8.5%) after 6 weeks with the application of a robotic ankle training system in 28 children with CP [bib_ref] Clinical application of a robotic ankle training program for cerebral palsy compared..., Sukal-Moulton [/bib_ref]. A possible explanation for this is that the RIGT provides an ample number of repetitions, with accurate proprioceptive and kinesthetic pressure sense on the hip, knee, and ankle joints, resulting in neuroplasticity and associated functional balance motor recovery [bib_ref] Comparative effectiveness of robot-interactive gait training with and without ankle robotic control..., Park [/bib_ref].
Analysis of gross motor function measures demonstrated that RIGT effectively increased GMFM (11.6%) in an adolescent with AS. The present findings on GMFM were consistent with those of previous robotic-gait-training studies [bib_ref] The effect of robot-assisted gait training on locomotor function and functional capability..., Jin [/bib_ref] [bib_ref] Effect of robotic-assisted gait rehabilitation on dynamic equilibrium control in the gait..., Wallard [/bib_ref]. Recently, Jin et al.
(2020) showed more enhanced GMFM-88 (34.7%) after 6 weeks of RIGT application in 20 children with CP who were able to ambulate with assistance [bib_ref] The effect of robot-assisted gait training on locomotor function and functional capability..., Jin [/bib_ref]. [bib_ref] Effect of robotic-assisted gait rehabilitation on dynamic equilibrium control in the gait..., Wallard [/bib_ref] reported that 20 sessions of robotic-assisted gait training (RAGT) using the hip-knee modulated RAGT (Lokomat pediatric) improved GMFM (13.4%) in 30 children with CP [bib_ref] Effect of robotic-assisted gait rehabilitation on dynamic equilibrium control in the gait..., Wallard [/bib_ref]. A possible explanation could be that cortical reorganization enhanced by task-specific training is related to the intensity and frequency of RIGT training [bib_ref] Brain plasticity and stroke rehabilitation: The Willis lecture, Johansson [/bib_ref]. Impaired selective motor control occurs when abnormal flexor or extensor synergies interfere with ankle joint movements, resulting in impaired gait and gross motor movement. It has been suggested that adolescents with CP consistently present with a lack of isolated knee extensor (e.g., quadriceps) control and ankle plantarflexion (e.g., gastrocnemius) control. This finding suggests that RIGT may have facilitated ankle proprioception and kinesthesia, which plays an important role in "reference correction" during locomotor re-training. A possible underlying rationale is that corrective ankle guidance during RIGT-enhanced ankle joint movement sensation and awareness is required for selective, coordinated neuromotor control of active tibialis anterior facilitation and reciprocal inhibition of GCM during gait training. Furthermore, kinetic and kinematic biomechanical analyses demonstrated an improvement of approximately 20-25.2% in the hip and knee joint force and angular movement performance test during gait, as a function of RIGT in the present case. It is possible that the Walkbot RAGT accurately guided the interlimb hip-knee-ankle movement in a coordinated fashion to improve the participant's gait pattern while improving the motivation and interest with AR and VR games, which corroborates previous results [bib_ref] Effects of innovative hip-kneeankle interlimb coordinated robot training on ambulation, cardiopulmonary function,..., Park [/bib_ref] [bib_ref] The effect of robot-assisted gait training on locomotor function and functional capability..., Jin [/bib_ref].
Some research limitations should be considered for future studies. One major limitation in the present case study was the limited sample size. Although we demonstrated positive therapeutic effects of RIGT combined with VR-AR in a participant with AS, caution should be exercised when interpreting our results. Please note that we were unable to implement a clinical study with a large sample size, as AS is an extremely rare genetic condition in children. Another limitation was that our robotic system was a stationary exoskeletal robotic-gait-training device, which allowed safe and repetitive gait training. However, utilizing a wearable robot may better assist community-based ambulation training. Nevertheless, a wearable system is not safe for children with cognitive impairments and may compromise safety in children with AS.
# Conclusions
The present case study demonstrated that RIGT combined with VR and AR can improve gait ability, as demonstrated by POMA score, gross motor function by GMFM score, balance by PBS score, knee and hip joint kinetics, and kinematics during gait in an adolescent patient with AS. Our clinical and biomechanical evidence provide important clinical insights to improve the effectiveness of current neurorehabilitation approaches for treating patients with AS, in balance and locomotor control dysfunction and reduction in the fear of falling. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
# Data availability statement:
The data presented in this study are available on request from the corresponding author.
[fig] Figure 1: Robot interactive gait training Walkbot. [/fig]
[fig] Figure 2: (A). Side-scrolling virtual reality game in Walkbot. (B). 3D walking-exploring th castle augmented reality in Walkbot. [/fig]
[fig] Author: Contributions: Conceptualization, C.P. and J.H.Y.; methodology, C.P. and S.H.; software, S.H. and C.P.; validation, S.H., C.P. and J.H.Y.; formal analysis, S.H., C.P. and J.H.Y.; investigation, C.P.; resources, S.H.; data curation, S.H. and C.P.; writing-original draft preparation, C.P. and J.H.Y.; writing-review and editing, S.H., C.P. and J.H.Y.; visualization, S.H.; supervision, J.H.Y.; project administration, S.H., C.P. and J.H.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was approved by the Yonsei University Mirae campus Institutional Review Board (1041849-202108-BM-126-02). [/fig]
[table] Table 1: Clinical characteristics of the adolescent. [/table]
[table] Table 2: Clinical outcome measurements. [/table]
[table] Table 3: Biomechanics measurements. [/table]
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Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development
Premature infants are highly vulnerable to aberrant gastrointestinal tract colonization, a process that may lead to diseases like necrotizing enterocolitis. Thus, spread of potential pathogens among hospitalized infants is of great concern. Here, we reconstructed hundreds of high-quality genomes of microorganisms that colonized co-hospitalized premature infants, assessed their metabolic potential, and tracked them over time to evaluate bacterial strain dispersal among infants. We compared microbial communities in infants who did and did not develop necrotizing enterocolitis. Surprisingly, while potentially pathogenic bacteria of the same species colonized many infants, our genome-resolved analysis revealed that strains colonizing each baby were typically distinct. In particular, no strain was common to all infants who developed necrotizing enterocolitis. The paucity of shared gut colonizers suggests the existence of significant barriers to the spread of bacteria among infants. Importantly, we demonstrate that strain-resolved comprehensive community analysis can be accomplished on potentially medically relevant time scales.
# Introduction
Infection by potentially pathogenic and antibiotic-resistant bacterial strains is a major source of disease in hospitalized patients. However, the spread of bacteria among patients is hard to track because most methods cannot distinguish between closely related strains. Strain transmission is especially important during colonization of newborns, a process that is critical for proper development [bib_ref] The intestinal microbiome in early life: health and disease, Arrieta [/bib_ref]. Premature infants, in particular, are highly susceptible to aberrant colonization, as their microbiome is often disrupted by antibiotic treatments [bib_ref] Early empiric antibiotic use in preterm infants is associated with lower bacterial..., Greenwood [/bib_ref] and since the source of colonists likely includes the hospital environment.
Necrotizing enterocolitis (NEC) is a common and life-threatening gastrointestinal disease that primarily affects hospitalized premature infants. Recent data indicate that ∼7% of infants born weighing <1.5 kg develop NEC [bib_ref] Necrotizing enterocolitis, Neu [/bib_ref]. Various observations support a microbial role in NEC, including the high incidence of pneumatosis intestinalis (gas in the bowel wall) in affected infants and resolution of symptoms in a majority of patients after antibiotic therapy and bowel rest [bib_ref] Redefining the role of intestinal microbes in the pathogenesis of necrotizing enterocolitis, Morowitz [/bib_ref] [bib_ref] The intestinal microbiome and necrotizing enterocolitis, Carlisle [/bib_ref]. NEC is characterized by intestinal inflammation and commonly progresses to necrosis, sepsis, and death. Risk factors may include feeding with artificial infant formula, blood transfusion, infant genetics, and overall health status [bib_ref] Association of necrotizing enterocolitis with elective packed red blood cell transfusions in..., Mally [/bib_ref] [bib_ref] Necrotizing enterocolitis: a multifactorial disease with no cure, Schnabl [/bib_ref] [bib_ref] Necrotizing enterocolitis, Neu [/bib_ref] [bib_ref] Packed red blood cell transfusion is an independent risk factor for necrotizing..., Wan-Huen [/bib_ref]. Such factors might be expected to give rise to a fairly constant disease incidence rate. However, NEC is commonly reported to occur in outbreaks [bib_ref] Nosocomial necrotising enterocolitis outbreaks: epidemiology and control measures, Boccia [/bib_ref] [bib_ref] Epidemiology of necrotizing enterocolitis temporal clustering in two neonatology practices, Meinzen-Derr [/bib_ref] , suggesting involvement of a contagious microorganism. A review of 17 published outbreaks of NEC did not identify a reproducible pattern of bacterial infection [bib_ref] Nosocomial necrotising enterocolitis outbreaks: epidemiology and control measures, Boccia [/bib_ref].
Cultivation-based approaches to identify and track medically relevant organisms can be labor intensive, biased, and inefficient. Yet, sequencing of the genomes of these cultured organisms can distinguish between strains with divergent phenotypes such as antibiotic susceptibility and virulence [bib_ref] Transforming clinical microbiology with bacterial genome sequencing, Didelot [/bib_ref] , and has enabled analysis of pathogen dispersal [bib_ref] The origin of the Haitian cholera outbreak strain, Chin [/bib_ref] [bib_ref] Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak, Köser [/bib_ref] [bib_ref] Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing, Snitkin [/bib_ref] [bib_ref] Emergence and global spread of epidemic healthcare-associated Clostridium difficile, He [/bib_ref]. An alternative approach uses 16S rRNA gene sequencing to identify organisms without cultivation, but the taxonomic resolution is limited, and distinct strains cannot be differentiated or tracked. Nonetheless, the method has been used to compare gut bacterial populations in fecal samples from infants with and without NEC. The results have been inconclusive. Some studies have identified no differences between cases and controls [bib_ref] Intestinal microbial profiles in extremely preterm infants with and without necrotizing enterocolitis, Normann [/bib_ref] , while others have reported positive, but divergent findings [bib_ref] 16S rRNA gene-based analysis of fecal microbiota from preterm infants with and..., Wang [/bib_ref] [bib_ref] Intestinal microbial ecology in premature infants assessed with non-culture-based techniques, Mshvildadze [/bib_ref] [bib_ref] Fecal microbiota in premature infants prior to necrotizing enterocolitis, Mai [/bib_ref].
In contrast, whole community DNA sequencing methods (metagenomics) can profile microbial communities with strain resolution and probe the metabolic potential of community members [bib_ref] Community structure and metabolism through reconstruction of microbial genomes from the environment, Tyson [/bib_ref] [bib_ref] Metagenomic analysis of the human distal gut microbiome, Gill [/bib_ref] [bib_ref] Experimental and analytical tools for studying the human microbiome, Kuczynski [/bib_ref]. Applied to series of samples, the approach can document shifts in community structure and identify responses to medical treatments, increasing age, altered diet, and changing health status. Unlike 16S rRNA gene surveys, metagenomics does not rely on previously established information (e.g. conserved sequences that guide PCR-based rRNA-based detection) and is less likely to miss community members eLife digest The spread of potentially harmful bacteria is a major source of disease in patients staying in hospitals. Premature babies-born before 37 weeks of pregnancy-can be particularly vulnerable to these infections because their organs may not yet be fully developed. Also, young babies do not have the fully established populations of beneficial microbes that help to protect us from dangerous bacteria.
Necrotizing enterocolitis-a life-threatening disease that can cause portions of the bowel to die-is mostly seen in extremely premature babies. Although it is not known what causes this serious condition, research has suggested that a contagious microbe may be responsible.
The development of methods that can sequence DNA from whole communities of microbes, known as metagenomics, allows researchers to identify the presence of individual strains of bacteria within these communities. This makes it possible to compare and contrast the strains of bacteria present in both diseased and healthy individuals, to help identify the bacteria responsible for a disease.
Here, Raveh-Sadka et al. used a metagenomics approach to study the communities of microbes present in premature babies in a hospital unit during an outbreak of necrotizing enterocolitis. The study found that very few bacterial strains were present in more than one baby, suggesting that bacterial strains are not readily transferred between the babies while they are in the hospital. Furthermore, Raveh-Sadka et al. reveal that no single bacterial strain was shared among all the babies who developed necrotizing enterocolitis.
These findings indicate that necrotizing enterocolitis is not caused by a single strain of bacterium. Instead, if bacteria do contribute to the disease, it maybe that it is caused by a variety of potentially harmful bacteria colonizing the gut at the cost of beneficial bacteria. In future, better understanding of the barriers that limit the transfer of bacteria between premature babies could help inform efforts to reduce the spread of infections between patients in hospitals. (e.g. organisms with unusual rRNA sequences, phage, and plasmids). Compared to cultivationbased methods, the metagenomic approach provides a relatively unbiased view of community composition and thus may be particularly helpful when an unknown microorganism is the cause of a disease [bib_ref] Microbial genomics and infectious diseases, Relman [/bib_ref]. A year ago, the power of such an approach was demonstrated in a retrospective analysis of banked samples from patients affected during a 2011 outbreak of a severe diarrheal illness caused by Shiga-toxigenic Escherichia coli [bib_ref] A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of..., Loman [/bib_ref]. More recently, shotgun sequencing of bacterial DNA present within cerebrospinal fluid enabled the diagnosis and treatment of leptospirosis in a critically ill child with meningitis [bib_ref] Actionable diagnosis of Neuroleptospirosis by next-generation sequencing, Wilson [/bib_ref].
Among gastrointestinal diseases with a possible microbial origin, NEC is somewhat unique as it is relatively common and because samples that provide information about gut consortia can be collected prior to the development of symptoms. This is because infants at risk for NEC are typically hospitalized for weeks to months in the neonatal intensive care unit (NICU) and onset of the disease occurs over a defined time period. Only one small study that we are aware of has analyzed metagenomic sequence data from infants with and without NEC, but assembly of the sequences was not attempted.
Recently, a group of infants developed NEC over a short time period in the NICU of Magee-Womens Hospital of the University of Pittsburgh Medical Center. Here, we investigated the degree to which specific microbial strains were shared among co-hospitalized infants and whether the disease could be attributed to a single infectious agent. Because the analysis required confirmation that the same strain was present in multiple infants, we deployed a genome-resolved sequencing-based approach. Our analyses included consideration of the fastest evolving features of genomes (e.g. prophage and the CRISPR/Cas loci) to maximize strain resolution. We also investigated strain-level metabolic potential and evaluated population heterogeneity for one abundant and widespread species. Genome-resolved approaches are typically slow and bioinformatics intensive because the data sizes are massive, simultaneous reconstruction of genomes for multiple community members is complex, and comparative and metabolic analyses for hundreds of genomes are challenging. We applied a new analysis system to resolve data into genomes and analyze the metabolic potential. To the best of our knowledge, this study is the first to provide comprehensive, genome-resolved analysis of gut bacterial communities in co-hospitalized patients. The core methods are fast enough to make them useful in some clinical settings, and we anticipate that analysis time can be substantially decreased with future developments.
# Results and discussion
Clinical information regarding study subjects and the NEC cluster When it became apparent that the incidence of NEC was increasing, we selected five infants who had developed NEC and five controls for comprehensive microbial community analysis. Ultimately, during the summer of 2014, nine infants were diagnosed with NEC (Bell's stage II or III). The total number of NEC cases was 10, as one of the affected infants developed recurrent NEC. This incidence rate was 2.5 times higher than average for this NICU.
For affected infants #2 (who developed NEC twice), #3, and #8, multiple fecal samples collected prior to the onset of symptoms were available. Two additional infants, #9 (not premature) and #10, were enrolled after diagnosis and treatment. The other infants who developed NEC were not enrolled in our study. Four infants (#1, #4, #6, and #7) did not develop NEC. Infant #5 was not diagnosed with NEC but had a single bloody stool on day of life (DOL) 20 and was treated with antibiotics for a suspected urinary tract infection. All infants were hospitalized concurrently within the same NICU (i.e. synchronous controls), and several were matched also according to gestational age. The selection of samples for sequencing was aimed to provide dense sampling around diagnosed NEC cases, from both the diagnosed infants as well as co-hospitalized infants who did not develop NEC (see sampling schedule in [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] and additional medical details in Supplementary file 1). Bacterial load in each sample was quantified by ddPCR (see 'Materials and methods' section). The estimated load was in general agreement with previous measurements in full-term infants of similar postnatal ages [bib_ref] Human milk glycomics and gut microbial genomics in infant feces show a..., De Leoz [/bib_ref]. Notably, the variation in the number of microbes per gram feces did not exceed a 100-fold across all samples. Infants who developed NEC did not show a consistent trend of change in bacterial load prior to or following diagnosis [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref]. Assembly and rapid binning yields hundreds of near-complete genomes DNA was extracted from up to nine samples per infant and sequenced using an Illumina HiSeq2500 at the University of Illinois. Overall, we analyzed 55 samples from the 10 infants [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] and Supplementary file 2). Between 2.22 and 7.35 Gbp of trimmed data from each sample was assembled independently. This enabled at least 4× coverage for genomes of organisms that comprised more than ∼0.2% to ∼0.6% of each community. For the 10 datasets, we assembled 181.2 Gbp of read sequence information. In total, 1.35 Gbp of genome sequence was generated on scaffolds >1000 bp (see 'Materials and methods' section and Supplementary files 2 and 3).
The genome reconstruction strategy involved a user assigning scaffolds to organisms using online binning tools (see 'Materials and methods' section). Genome bins were defined based on a combination of a phylogenetic profile, GC content, and coverage. These bins were then verified independently using emergent self organizing maps (ESOMs) that clustered either tetranucleotide composition or time series abundance pattern information. Genome completeness and purity were evaluated based on the inventory of ribosomal proteins and 51 genes expected to be in single copy in any genome (see 'Materials and methods' section for details).
A total of 509 bacterial genomes (including multiple genomes for the same organism in different samples) were recovered, with average read coverage of between 2 and 1148. Between 1 and 23 bacterial genome bins were detected per sample, and overall 260 near-complete genomes were reconstructed (see 'Materials and methods' section). Scaffolds identified as putative phage or plasmids based on their encoded genes were assigned to 328 bins (Supplementary file 3). Overall, between 86% and 98% of reads generated for each sample was assigned to a genome bin (Supplementary file 2).
Diverse bacterial strains in the NICU are rarely shared by co-hospitalized babies In order to assess the extent of strain dispersal among the hospitalized infants, genome bins with >0.5 Mbp of sequence were compared by aligning the single copy genes sequences. When these were too fragmented for conclusive results, entire genome bins were aligned. Genome bins that were >98% identical across >90% of bin length were considered indistinguishable. Manual curation of assemblies was performed in some cases to eliminate disagreements due to scaffolding errors that are introduced occasionally during assembly (see 'Materials and methods' section).
Remarkably, very few bacterial strains occurred in more than one infant and no strain was shared by all infants who developed NEC [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. In contrast, and as could be expected, identical genotypes were almost always detected in samples from the same infant, providing reassurance regarding the validity of our methods [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref].
Specifically, of the 149 strains compared, only four were shared by two or more infants, and only three of these were identified in infants who developed NEC. A Klebsiella oxytoca strain was present in infants #1 and #6, neither of whom developed NEC. Clostridium sporogenes was present in infants #3 and #5, but occurred at very low abundance in infant #3. Two strains were more widely distributed: a Clostridium butyricum strain was detected in infants who did (infants #3, #8) and did not (infants #1, #5, #6) develop NEC but was missing from infant #2, who developed NEC. C. butyricum has no predicted type III or type VI secretion system genes and no identified toxin-producing genes (Supplementary file 4). Thus, this strain seems unlikely to be a pathogen or the cause of NEC. Lastly, a Clostridium paraputrificum strain with a moderate predicted pathogenicity potential (Supplementary file 4) was shared by infants #2, #5, and #8, and also occurred in one sample from infant #6, although the predominant strain in this infant (who did not develop NEC) was different [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. C. paraputrificum was not detected in infant #3 (NEC case). Both C. paraputrificum and C. butyricum have been previously suggested as potential causative agents in NEC [bib_ref] Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a..., Waligora-Dupriet [/bib_ref]. Interestingly, although the colonizing strains were almost always distinct, infants often shared bacteria of the same genus or species. At high abundance in multiple infants, including two who developed NEC, were members of the genus Veillonella. However, multiple distinct strains and species were present across infants . A Veillonella strain was very abundant in infant #2 prior to development of NEC (Supplementary file 3) but disappeared after the first antibiotic treatment, to be replaced by different Veillonella species [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. In the other infants who developed NEC, Veillonella was either absent (infant #8) or present as a different strain altogether (infant #3). The results likely rule out a Veillonella strain as a single, shared agent of NEC.
Another organism found in multiple infants, often at high abundance [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref] and Supplementary file 3), was Enterococcus faecalis. This organism is common in fecal samples from both premature and term infants [bib_ref] Pyrosequencing-based molecular monitoring of the intestinal bacterial colonization in preterm infants, Chang [/bib_ref] [bib_ref] Microbial succession in the gut: directional trends of taxonomic and functional change..., Vallès [/bib_ref].
Interestingly, it appears that the host-E. faecalis relationship, as it pertains to the infant gut, is nuanced. Although E. faecalis has repeatedly been identified as a source of neonatal infection [bib_ref] Lateonset sepsis in very low birth weight neonates: the experience of the..., Stoll [/bib_ref] , it also has been studied as a potential probiotic [bib_ref] Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut, Nueno-Palop [/bib_ref] , with beneficial properties related to modulation of innate White boxes indicate that the strain was absent; shading intensity increases with increased organism abundance. Note the persistence of specific genotypes within infants and the almost complete lack of overlap in strains between infants. The few strains shared between infants are highlighted in red. Colors associated with organism names indicate the broader organism classification: green are Firmicutes, orange are Gammaproteobacteria, red are Epsilonproteobacteria, pink are Betaproteobacteria, and blue are Actinobacteria. Red lines indicate antibiotic administration associated with necrotizing enterocolitis diagnoses, blue lines indicate antibiotic administration for other reasons. DOI: 10.7554/eLife.05477.004 immunity [bib_ref] Enterococcus faecalis from healthy infants modulates inflammation through MAPK signaling pathways, Wang [/bib_ref]. Furthermore, links between mobile genomic elements and enterococcal virulence are well described [bib_ref] Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant..., Gilmore [/bib_ref]. These considerations suggest that strain-level variation in E. faecalis is significant and potentially clinically relevant.
For E. faecalis, we reconstructed 30 nearcomplete genomes (Supplementary file 3) for multiple strains [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. Alignment of the longest of the single copy genes tracked, the ∼2500 bp DNA gyrase subunit A (gyrA) gene, illustrates five distinct sequence types for this gene alone . Strains recovered from infants #2 and #7 and also strains recovered from infants #3 and #5 (early samples) could not be distinguished by this locus. Notably, reconstructed 16S rRNA gene sequences were identical in these strains, illustrating that the limited taxonomic resolution of this locus prevents its use in studies of strain dispersal.
A genomic region of interest for strain-level studies is the CRISPR/Cas locus. This locus can be one of the fastest evolving regions of bacterial genomes and thus can potentially provide highresolution insight into strain distinction, as well as shared ancestry [bib_ref] Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses, Tyson [/bib_ref]. All 30 well-sampled E. faecalis genomes encode a CRISPR spacer-repeat array that lacks proximal Cas proteins and some genomes (in infants #3, #5, #8) encode an additional locus with proximal Cas genes . Given that Cas proteins are required for CRISPR-Cas function, strains in infants #2, #6, #7, and #9 that lack Cas proteins altogether, have lost CRISPR-Cas-based phage immunity.
This pattern of loci with and without Cas proteins has been reported previously in E. faecalis. Different Cas1 sequences (types a and b) and a different repeat sequence were identified in E. faecalis from infants #3, #5 before antibiotic treatment, and #8, compared to the strain in infant #5 after antibiotic treatment . The repeatspacer arrays in the loci with Cas1 type a are identical in the genotypes of E. faecalis in infant #3 and in early samples from infant #5 [fig_ref] Figure 5: Comparison of CRISPR loci in Enterococcus faecalis genomes [/fig_ref] , reinforcing the very high similarity of these populations deduced from single copy gene sequence comparisons [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. As often happens in CRISPR loci [bib_ref] Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses, Tyson [/bib_ref] , a block comprising six spacers and flanking repeats has been excised in the strain from infant #8 and three novel spacers have been added at the growing tip, versus two in infants #3 and #5 [fig_ref] Figure 5: Comparison of CRISPR loci in Enterococcus faecalis genomes [/fig_ref]. Shared spacers at the older end (distant from the Cas) imply that the strains in infants #3, #5, and #8 had a recent common ancestor.
The Cas-less CRISPR array is flanked by DNA-related and antibiotic resistance-related genes, and differs in length considerably among strains. The repeat for the locus without Cas proteins is identical . A phylogenetic tree (RAXML; black dots indicate bootstrap values of ≥80%) for predicted RuBisCO Form IV (RuBisCO-like) proteins involved in methionine salvage. This protein was chosen for analysis because it is well studied and is not one of the 51 single copy (and generally highly conserved) genes used in other analyses. Colored dots identify the infant, while the number indicates the sample of origin. Red boxes highlight infants who developed necrotizing enterocolitis (NEC). Although Veillonella were prominent in many samples, sequence analysis revealed many distinct strains/species over the study cohort. Strain shifts occurred following antibiotic administration (e.g. in infant #2), but identical sequences were often detected in series of samples from the same infant. Note infants affected by NEC do not share the same strains/species. DOI: 10.7554/eLife.05477.005
to that of the loci with type a Cas1. All first repeats are defective, but the polymorphisms are only shared by strains in infants #3, #5, and #8. However, the repeat-spacer array distinguishes the genotype in infants #3 and #5 from that in infant #8 [fig_ref] Figure 5: Comparison of CRISPR loci in Enterococcus faecalis genomes [/fig_ref]. The loci in the strains in infants #2 and #7 are probably the same (the sequence from infant #7 is not shown due to very partial recovery).
Both the single copy gene and CRISPR-Cas analysis suggested that E. faecalis in infants #2 and #7 are very closely related. Similarly, the strains in infant #3 and in early samples from infant #5 are almost identical (a single SNP in the surveyed gene set distinguished the sequences). To gain better understanding of the type and extent of genomic differences between the recovered E. faecalis genomes, and specifically of these closely related genome pairs, we mapped reads from eight samples, representative of the eight different genotypes reported in [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref] , to a 1 Mbp E. faecalis scaffold recovered from infant #9, sample 1 (one third of the recovered genome). Multiple alignment of the consensus sequences from mapping of each sample provided a view of sequence variability across strains ; a similar alignment for C. paraputrificum strains is shown in -figure supplement 1). The analysis revealed many SNP locations and small indels that were spread across the entire length of the sequence, as well as a small number of longer (20-30 Kbp) indel regions. These regions included among other things a sucrose metabolism operon, mobile elements, and genes related to Fe-S protein biogenesis.
The high degree of similarity between the closely related genotypes (infants #2 and #7, infants #3, and early samples of infant #5) is evident from inspection of the multiple alignment . While, on average, sequence pairs were ∼90% identical across the scaffold length, those of infants #2 and #7 and those of infants #3 and #5 (early samples) showed identity of 99.3% and 98.7%, respectively. However, a more comprehensive comparison of these genome pairs revealed differences in the genotypes in both cases. Interestingly, a ∼1 Kbp region distinguishes strains in infants #3 and #5 , whereas a prophage insertion separates those in infants #2 and #7 . Both of these regions were missing from the genome recovered from infant #9, sample 1 and thus could not be discovered by read mapping to that genome. These differences are very subtle, and especially in the case of the prophage, might have arisen after colonization.
While the above analysis characterized differences in the major strains detected in the hospitalized infants, we further investigated whether E. faecalis strains from one infant occur at low or even trace levels in other infants. This was done by analysis of sequence polymorphisms in reads that map to the gyrA gene in each assembly. While in all infants colonized by E. faecalis, some polymorphic locations could be identified, in most infants, polymorphisms matching strains of other infants were undetectable (infants #2, #6, #8, #9). For the abundant population in sample 2 from infant #9, analysis of >20,000 reads indicated that the maximum abundance level of a strain detected in another infant was <0.01% (Supplementary file 5). However, in infant #3, ∼0.12% of reads have sequences consistent with derivation from the genotype in infants #2 and #7, which are indistinguishable at this locus . The gyrA analysis indicated that this genome is even more prominent in earlycollected samples from infant #5 (3-9% of reads; Supplementary file 5). Thus, compared to other E. faecalis strains, the population in infants #2 and #7 is relatively widely distributed.
The evidence from all infants is that multiple strains and species are present in the NICU. The intriguing pattern of mostly infant-specific E. faecalis could have arisen due to a very small number of colonizing E. faecalis cells. However, investigation of population-level sequence variation revealed . Alignment view of genome-wide differences in Enterococcus faecalis strains. Consensus sequence for the alignments (shown at the top of each alignment) represents the calculated order of the most frequent nucleotide residues. Alignments were done in Geneious v7.1.7 [bib_ref] Geneious Basic: an integrated and extendable desktop software platform for the organization..., Kearse [/bib_ref] , using MAFFT v7.017 [bib_ref] MAFFT: a novel method for rapid multiple sequence alignment based on fast..., Katoh [/bib_ref] with default parameters. Samples are ordered by similarity. For each sample, SNPs and indel locations relative to the multiple alignment are marked by black lines or boxes. (A) Reads from eight samples, from which different Enterococcus faecalis strains were recovered, were mapped to a 1 Mbp E. faecalis scaffold (scaffold 0) recovered from infant #9, sample 1. Shown is a multiple alignment of the consensus sequences derived for each sample from these mappings. Multiple SNPs and short indels are detected throughout the sequence. Several larger indels are also detected. (B) Enlarged view of a region in A showing a large indel locus. This view distinguishes sets of extremely closely related strains (i.e. strains in infants #7 and #2; strains in infants #3 and #5 [early samples]) from more distant strains. (C) Pairwise alignment of consensus sequences derived from read mapping to an E. faecalis scaffold (scaffold 2962) recovered from infant #5, sample 2 distinguishes closely related strains in infants #3 and #5 (early samples). (D) Pairwise alignment of consensus sequences derived from read mapping to an E. faecalis scaffold (scaffold 17) recovered from infant #7, sample 3 distinguishes closely related strains in infants #2 and #7. The region missing in the assembly from the other infants corresponds to a mobile element. DOI: 10.7554/eLife.05477.010 The following figure supplement is available for figure 6: some reads with shared polymorphisms that are not characteristic of the strains in the other infants. This, and the detection of the dominant strain from infants #2 and #7 in other infants indicate that multiple E. faecalis inoculation events occur during colonization.
The lack of overlap in strain genotype could indicate the existence of infant-specific strain sources (e.g. mothers), and barriers that prevent spread of those populations to other infants in the NICU. Even if dispersal occurs, the founding population may preclude establishment of later introduced populations. Alternatively, strains could be dispersing freely, and strain dominance could reflect strong selection in the gastrointestinal tract (possibly imposed by microbial community context and/or human genetics) leading to the establishment of a single, most adapted strain. Another model worth considering would involve stochastic acquisition of a strain from a set of populations that is so large that it is improbable that any two infants would initially acquire the same strain. As there is ongoing input of strains over the colonization period, the observation of one (usually) highly dominant population still suggests some barrier to establishment of other populations.
## Phylum-level community composition does not distinguish nec cases from other infants
Previous studies that were done at lower resolution than achieved in the current study have pointed to a high abundance of Proteobacteria as a factor in NEC development [bib_ref] 16S rRNA gene-based analysis of fecal microbiota from preterm infants with and..., Wang [/bib_ref] [bib_ref] Fecal microbiota in premature infants prior to necrotizing enterocolitis, Mai [/bib_ref] [bib_ref] Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis, Torrazza [/bib_ref]. To see if that pattern applied in the current study, we collapsed our organism identifications to the phylum level [fig_ref] Figure 7: Stacked bar plot of community composition across samples and infants after organism... [/fig_ref]. Proteobacteria are abundant in most infants, but Proteobacteria representation in the communities did not distinguish those infants who developed NEC from those that did not. In fact, the relative abundance of Proteobacteria declines in infant #2 prior to both NEC diagnoses. Abundances are generally low in infant #3, and consistently high in infant #8.
Community functions do not distinguish infants who developed NEC from other infants, but reveal many potential pathogens Given that no single organism could be associated with all NEC cases, we considered the possibility that overall metabolic imbalance was a contributing factor. Owing to the high quality and completeness of many of the recovered genomes, we are able with this type of data, to go beyond strain identification and search for unique characteristics in the predicted gene content of colonizers of NEC-affected infants. However, clustering analyses of the detailed profiles of genomically encoded metabolic capacities and inspection of individual patterns failed to distinguish the capacities of microbial communities in infants who did and did not develop NEC. Of course, other considerations, such as differences in gene expression levels, may play an important role in NEC, but cannot be studied with DNA sequence information.
While no metabolic imbalance was found, many pathogens were detected in fecal samples of infants who developed NEC. For example, Enterobacter cloacae is abundant in infant #2 and is predicted to have many toxin and type VI secretion system genes and an extensive antibiotic resistance repertoire . The resistance genes may explain why E. cloacae remained abundant after antibiotic treatment (sequence analyses indicate that the same genotype persisted through the treatments; [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref] and [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref]. ESOMs used to validate the binning also provide an overview of the community composition, and in the case of infant #2, also highlight the almost complete dominance of E. cloacae in response to antibiotics . Note that these maps do not reflect organism abundances, although very small areas can indicate genomes that were partially sampled due to low sequence coverage (for abundance information see Supplementary file 3 and [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref].
Many other potential pathogens are present prior to the NEC diagnoses in infant #2 (see and Supplementary file 4). Of interest due to their predicted gene complement are an Actinomyces strain that dominated the community prior to the first event and Clostridium difficile, which occurs in both samples (at low abundance) collected immediately prior to onset of NEC. C. difficile has been implicated as a cause of NEC [bib_ref] An outbreak of Clostridium difficile necrotizing enterocolitis: a case for oral vancomycin..., Han [/bib_ref] [bib_ref] An epidemic outbreak of necrotizing enterocolitis due to Clostridium difficile in term..., Pérez-González [/bib_ref] , although its role is controversial [bib_ref] Nosocomial necrotising enterocolitis outbreaks: epidemiology and control measures, Boccia [/bib_ref] [bib_ref] Intestinal microbial ecology in premature infants assessed with non-culture-based techniques, Mshvildadze [/bib_ref]. Notably, the genome of the organism in infant #2 encodes tcdABCDE genes characteristic of toxigenic strains [bib_ref] Evolutionary history of the Clostridium difficile pathogenicity locus, Dingle [/bib_ref] and Clostridial binary toxin B/anthrax toxin PA family proteins are affiliated with this organism. Also of potential significance in infant #2, from the perspective of its genetic repertoire, were Clostridium perfringens, Streptococcus salivarius, Enterococcus faecium, and E. faecalis .
Prominent in the communities of infant #3 prior to NEC diagnosis were Veillonella parvula (a strain predicted to have minimal pathogenicity, see Supplementary file 4, and unique to this infant; , E. faecalis, K. oxytoca, and a Citrobacter related to strain KTE32 [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref]. The same strains of E. faecalis and Citrobacter persist through treatment, likely reflecting their large repertoire of antibiotic resistance genes . Citrobacter and Klebsiella have many toxin and type VI secretion system genes, and thus may have contributed to disease in this infant. Pseudomonas aeruginosa was only detected after antibiotic treatment, and also has many predicted type III and toxin genes, as well as type VI secretion system genes (Supplementary file 4). Other potentially significant bacteria were strains of C. sporogenes and Paenibacillus. Interestingly, the communities in infant #3 included Bifidobacterium (MSTE12-related), an organism that is often considered to be a beneficial commensal and not frequently observed in premature infants [bib_ref] Conditions of bifidobacterial colonization in preterm infants: a prospective analysis, Butel [/bib_ref].
Infant #8 developed NEC 1 day after collection of the last sample. The communities in the two pairs of samples from different times on the same day contain generally similar organisms, but rapid abundance shifts occur, consistent with general observations over whole day periods. Especially prominent in samples from infant #8 were a Klebsiella pneumoniae-related strain [fig_ref] Figure 2: An overview of the distribution of 144 of the 149 tracked strains... [/fig_ref] and an E. cloacae [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] strain. C. perfringens has a notable inventory of predicted pathogenicity-related genes. E. coli, present in the three samples collected prior to diagnosis, may have contributed to intestinal inflammation, given that it has a large inventory of type III and type VI secretion system genes and many toxin-encoding genes .
Samples from infants #9 and #10 (both of whom developed NEC) were collected only after diagnosis. Notable in the post-treatment communities from infant #9 were E. faecalis, Candida parapsilosis (see below), and some Staphylococcus and Streptococcus. A variety of Lactobacilli and E. coli were prominent in infant #10.
In infants who developed NEC, the prominence of many potentially pathogenic organisms is striking. Although our results do not suggest that a single organism (abundant or not) caused NEC in the studied infants, bacteria that may have contributed to NEC were present. Infants who were not diagnosed with NEC were likewise colonized by a wide variety of potentially pathogenic bacteria and some strains were even shared by NEC cases and controls. If gut colonization by pathogenic bacteria is a significant factor in the development of NEC, other health and/or environmental attributes may ultimately determine which infants become sick. Due to the small number of cases studied to date and the large number of potentially important variables, a reliable model that predicts NEC development without over-fitting cannot be constructed at this point. However, accumulation of additional data may enable the construction of such a model in the future.
## Potential roles for plasmids, phage, and bacterial-phage interaction
The gastrointestinal tract can host a complex mixture of mobile elements, including phage, viruses, plasmids, and conjugative transposons, that can transfer virulence and antibiotic resistance factors [bib_ref] Human intestinal bacteria as reservoirs for antibiotic resistance genes, Salyers [/bib_ref] [bib_ref] Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in..., Schjørring [/bib_ref] [bib_ref] The human gut virome: interindividual variation and dynamic response to diet, Minot [/bib_ref]. To consider the possibility that a mobile element, moving around the NICU (potentially independently of the host bacterium), was the common factor leading to NEC, we compared all sequences from all samples that were binned as plasmid-like, phage or phage-like, or of unknown origin. We commonly found essentially identical sequences in different samples from the same infant and a few identical plasmids and phage were detected in different infants (e.g. one complete, circular plasmid from infant #6 that is affiliated with a Clostridium species, based on sequence similarity , also occurs in infants #5 and #8). However, no mobile elements were shared by all sick infants, or by all infants diagnosed with NEC (see 'Materials and methods' section).
We leveraged the fact that some bacteria have CRISPR loci to determine whether bacteria colonizing the gastrointestinal tract of newborns have CRISPR-Cas-conferred immunity to co-occurring phage. This is important because phage sensitivity could explain rapid shifts in organism abundance. Our analyses focused on E. faecalis because it was abundant and widely distributed over the infant cohort. In no case did we identify an E. faecalis CRISPR spacer with a perfect match to any phage that coexisted in the same community . However, we detected imperfect matches between E. faecalis CRISPR spacers and phage in the same sample, and some spacers matched perfectly to phage in other samples from the same infant and to phage in another infant. One spacer in the CRISPR locus of E. faecalis from infant #3 targets a prophage integrated into the genome of E. faecalis from infant #5 . These observations indicate recent exposure of E. faecalis to phage populations related to those that coexisted in the NICU during the study period and suggest phage sensitivity of bacterial populations in this early gut colonization period.
Notable in samples 2 and 3 from infant #1 were Enterobacteriales phage, the genomes of which were 95× and 30× more abundant than the genome of the probable Klebsiella host . Interestingly, ddPCR shows that the period of phage proliferation corresponded to an increase in overall cell numbers by a factor of 10 over ∼5 days [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref]. K. oxytoca must account for this increase in bacterial cell numbers because it is essentially the only species in early-sampled communities. We infer that phage predation moderated the Klebsiella bloom and probably facilitated the subsequent establishment of the more complex community in later-collected samples.
## Do the nec incident statistics support the existence of an infecting pathogen?
Our data provided a unique opportunity to study at high resolution possible factors that may have been responsible for the cluster of diagnosed NEC cases in the summer of 2014. We were able to eliminate the possibility that a single bacterial strain was the causative agent in all cases, and also did not find any support for a causative role of specific mobile elements, or particular metabolic functions. In light of these findings, we turned to statistical characterization of the disease cluster. Surprisingly, despite the frequent reference to disease outbreaks in the literature, the statistical significance of disease clusters is rarely studied [bib_ref] Outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care..., Turcios-Ruiz [/bib_ref] [bib_ref] Epidemiology of necrotizing enterocolitis temporal clustering in two neonatology practices, Meinzen-Derr [/bib_ref]. Here, we analyzed 67 months of monthly counts of NEC diagnoses in the NICU of Magee-Womens Hospital to determine whether the apparent outbreak in the summer of 2014 was a statistically significant anomaly [fig_ref] Figure 9: Statistical evaluation of the clustering of necrotizing enterocolitis cases during 2009-2014 [/fig_ref]. Monthly statistics are collected for other purposes and are based on different criteria for NEC, as outlined by the Vermont Oxford Network (VON). Infant #3, and another infant not enrolled in our study, were excluded due to lack of pneumatosis or pneumoperitoneum on X-rays.
No seasonal or otherwise periodic patterns in NEC diagnoses were observed, and no correlation between the number of NEC cases and daily average of hospitalized infants in the NICU was detected. Data were modeled using Poisson and negative binomial (NB) distributions and maximum likelihood estimates of the corresponding parameters were extracted (Poisson: λ = 1.90, NB: r = 5.81, p = 0.75). Data were somewhat over-dispersed relative to the Poisson distribution [fig_ref] Figure 9: Statistical evaluation of the clustering of necrotizing enterocolitis cases during 2009-2014 [/fig_ref] and fit the negative binomial distribution modestly better (R 2 = 0.85 for Poisson model, R 2 = 0.95 for NB model), in line with a potential dependency between diagnosed cases.
While the eight cases from summer 2014 that met VON criteria are undoubtedly at the high end of the scale, they could not be established as statistically significant, assuming these underlying Poisson or NB distributions. Inclusion of additional sick infants who do not meet the VON criteria could change the conclusion, but unfortunately monthly statistics for all diagnosed cases were unavailable. Results were essentially unchanged when data were normalized to the average daily NICU occupancy. Future study of clusters of NEC events should be tested for their statistical significance to evaluate whether consideration of a single infective agent is appropriate.
## Genome recovery from metagenomic datasets
The approach used here can, in a cost-and time-effective manner, generate very good draft genomes. For example, two near-complete Veillonella genomes (>4 Mbp) were assembled into 12 and 15 pieces, four E. faecalis genomes were reconstructed into 27-43 pieces, two Actinomyces into 21 and 24 pieces, one Negativicoccus succinicivorans genome into 12 pieces, and one Citrobacter strain genome into 23 pieces. Notably, some genomes reconstructed in this study represent organisms with no closely related sequenced relatives. For example, we achieved many near-complete genomes for bacteria related to Tissierella sp. LBN 295 (for which only four partial gene sequences are available in NCBI). This bacterium is more distantly related to, but currently profiled as related to, Clostridium ultunense. We also reconstructed a draft genome for an organism that we infer is related to Peptococcus niger. Both the Tissierella and P. niger genomes have been further curated (see 'Materials and methods' section). Also, we reconstructed hundred of genomes that, although similar to previously sequenced genomes, are different in potentially important ways (e.g. in antibiotic resistance potential and pathogenicity factors).
Interestingly, we reconstructed an ∼12.7 Mbp draft genome of C. parapsilosis, a microbial eukaryote (fungus) that was highly abundant in the gut of infant #9 after treatment for NEC [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref]. The genome shares >99% identity with the genome of the CDC317 isolate. Alignment of our genome with the CDC317 genome verified the overall accuracy of our assembly, a notable finding given that very few microbial eukaryote genomes have been recovered previously from metagenomic data [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref].
The ability of our approach to uncover organisms of clear medical relevance (and not detected otherwise) is also illustrated for infant #1, who developed early onset sepsis. Although present at very low abundance (∼0.2%), we identified an organism whose 16S rRNA gene sequence shares 99% identity with Streptococcus agalactiae (group B streptococcus, GBS), in samples 4 and 5 [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] and Supplementary file 3). The infant's mother tested positive on her GBS surveillance swab and the placenta was found by pathologists to contain trace amounts of GBS. However, the newborn blood culture was negative for GBS. Likely, this organism caused the episode of early onset sepsis in this infant. The ability to profile the genome for clinically relevant traits such as pathogenicity and antibiotic resistance is illustrated for K. oxytoca from infant #1 [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref]. The low predicted pathogenicity potential of C. butyricum is shown for contrast [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] and Supplementary file 4).
## Genome-resolved metagenomics on a clinically relevant timescale
The time between collection of the last fecal sample and receipt of DNA sequence information from all 55 samples was 10 days. For a group of three samples, as might be collected in a clinical setting, the time required for DNA extraction is a couple of hours and for sequencing, about 2 days (the sequencing speed is method dependent). Sequence assembly, annotation, and data import into our metagenomics analysis system required 6-8 hr/sample and the time required for a first round binning that largely defined community composition and metabolic potential (generated automatically) was ∼10 to ∼60 min/sample. ESOM-based bin confirmation (which may not be needed for some applications) requires 1-2 hr/sample. Thus, new bioinformatics approaches tested here enable comprehensive genome-based analyses on a timescale that approaches that required for some clinical applications, for example involving patients with long-term health issues. The analysis time will decrease as sequencing technologies continue to improve and with automation of binning steps. This will make strain-resolved analysis clinically relevant for a wider range of applications, potentially including acute illnesses like NEC.
# Conclusions
We applied newly developed methods to rapidly and extensively resolve into genomes, sequence data from gastrointestinal tract-associated microbial communities from premature infants. All microbial communities in all infants sampled prior to onset of NEC harbored organisms with significant pathogenicity potential. However, we found no evidence for one common, abundant (or even minor) genomically distinct infective agent. If bacteria contribute to NEC, the effect more likely is due to exposure to a variety of potentially dangerous hospital-associated bacteria. A major finding is that the dominant population of each bacterium acquired by each infant was generally genotypically distinct. Extremely closely related organisms were only identified in a handful of cases, and occurred in both sick and healthy infants. Yet, the fact that they were identified at all, and the detection of shared minor strains of E. faecalis in a few cases, confirms that dispersal can occur among infants in the NICU. Overall, we suspect the existence of significant barriers that limit establishment of strains during the early stages of colonization of the premature infant gastrointestinal tract.
# Materials and methods
## Medical information
Fecal samples for enrolled infants were collected as available. From the pool of all available samples, we selected for sequencing 55 samples from which sufficient amounts of DNA were extracted. Our selection of samples from infants who did and did not develop NEC was aimed to provide dense sampling around dates leading to and following cases of NEC diagnosis in the NICU. The sampling schedule is shown in [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] , and additional medical details for all infants are provided in Supplementary file 1.
# Ethics statement
The study was performed with approval from the University of Pittsburgh Institutional Review Board under protocol number PRO10090089, and written parental consent was obtained on behalf of the neonates.
# Metagenomic analysis details
Sequencing reads of 160 bp in length were processed with Sickle (Joshi and Fass, 2011) (v1.33; available at https://github.com/najoshi/sickle) to trim both ends to remove low quality base calls. After trimming, reads were assembled with idba_ud [bib_ref] IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with..., Peng [/bib_ref] (v1.1.1; available at http://i.cs.hku.hk/∼alse/hkubrg/projects/idba_ud/) using default settings. Resulting scaffolds >1000 bp were annotated. We used prodigal ; available at https://github.com/hyattpd/Prodigal/releases/tag/v2.60) to predict genes using default settings for metagenomics gene prediction. Protein sequences were searched against KEGG [bib_ref] Data, information, knowledge and principle: back to metabolism in KEGG, Kanehisa [/bib_ref] (KEGG FTP Release 2014-07-07; available at http://www.kegg.jp/kegg/ download/), UniRef100 (release 2014_07; available at ftp://ftp.uniprot.org/pub/databases/ uniprot/previous_releases/release-2014_07/), and UniProt [bib_ref] UniProt archive, Leinonen [/bib_ref] (same as UniRef) using USEARCH [bib_ref] Search and clustering orders of magnitude faster than BLAST, Edgar [/bib_ref] (v7.0.1001; available at http://www.drive5.com/). Additionally, reciprocal best-blast hits were determined. All matches with bit scores greater than 60 were saved, and reciprocal best hits with a bit score greater than 300 were also cataloged. We identified 16S rRNA sequences using Infernal (v1.1; available at http://infernal. janelia.org/) using default settings. The rRNA genes were predicted using Infernal [bib_ref] Infernal 1.1: 100-fold faster RNA homology searches, Nawrocki [/bib_ref] and tRNAs using tRNAscan_SE ; available at http://lowelab.ucsc.edu/tRNAscan-SE/). Scaffolds, gene predictions, and all associated annotations were uploaded to ggKbase.berkeley.edu for binning and analysis (http://ggkbase. berkeley.edu/project_groups/necevent_samples).
We estimated detection sensitivity for bacterial populations using the data size per sample and assuming a genome size of ∼3 Mbp. For example, the sample with the least amount of data was from infant #4, sample 4 (2 Gbp). This amount of data would allow detection (4× coverage) of an organism with a 3 Mbp genome that comprised 0.6% of the sample.
Evaluation of genome completeness relied in part on the number of expected single copy genes that were identified per bin. A bin was classified as very good if the genome size was not vastly different from that of genomes of closely related organisms and most single copy genes were present in one, and only one copy. The accuracy of our genome completeness statistics was somewhat affected by genes that were split by scaffold ends. Partial genes (<50% of the gene) were not counted.
## Phylogenetic profile
As the accuracy of binning depended in part on the quality of the phylogenetic profile, we tested two approaches. First, we inventoried the best matches of proteins on each scaffold by comparison to the UniRef100 database. As this did not provide sufficient taxonomic resolution, we adopted a second approach in which the profiles were established by comparison to the much larger UniProt database. In both cases, the phylogenic classification required that ≥50% of predicted proteins on a fragment had shared affiliation at some taxonomic level. If ≥50% of predicted proteins had best matches to the same species in the database, that scaffold was profiled as that species. If ≥50% had best matches to the same genus (but not the same species), the profile assigned was of that genus. This process continued, until each scaffold had been assigned a profile at some taxonomic level. Some scaffolds were assigned the profile 'unknown' because ≤50% of predicted proteins had hits to the same domain (these scaffolds were often from viruses and plasmids).
## Binning
Binning was carried out via an online interface within ggKbase (http://ggkbase.berkeley.edu/). When using this interface, the user selects a group of genome fragments (scaffolds) based on a specific phylogenetic profile, and/or scaffold coverage and/or GC information. The amount of sequence information, the number of expected single copy genes, the number of ribosomal proteins, and bin coverage statistics are displayed for the selection. For human microbiome research, usually the choice of scaffolds is first based on phylogenetic profile and is then fine-tuned by selection of a specific subset of scaffolds based on their coverage and/or GC content. If the bin size and single copy gene inventory are appropriate, the group of scaffolds is then binned. Following one round of binning (10-60 min/sample), typically ∼1 Mb of sequence information per sample was left unassigned to any organism or phage/plasmid.
Typically, the identity of genomically sampled organisms was determined based on overall sequence similarity to previously known genomes. In many cases we reconstructed partial or complete 16S rRNA genes and used this sequence information to inform organism classifications, although the presence of these genes in multiple copies often resulted in misbinning of small scaffolds encoding this gene (see notes in Supplementary file 3). An advantage of the presence of multiple copies of the 16S rRNA gene per genome is that it can allow us to detect populations that are otherwise at such low abundance that they would be invisible based on their overall genome coverage. The 16S rRNA scaffolds were the only parts of some very low abundance genomes detected for this reason.
The correctness of the assignment of scaffolds to genomes was verified with emergent self organizing maps (ESOMs), a clustering toolthat was applied to scaffold tetranucleotide frequency information [bib_ref] Community-wide analysis of microbial genome sequence signatures, Dick [/bib_ref]. In most cases, data points assigned to the same bin clustered into clearly defined and generally strongly bounded regions of ESOMs, supporting the accuracy of the binning method. Some bin adjustments were made based on the ESOM analyses.
When the approach described above was insufficient to resolve bins for closely related species/ strains (e.g. Enterobacteriales in infant #8), we constructed ESOMs using patterns of abundance of the organisms over the time series of samples from an infant [bib_ref] Time series community genomics analysis reveals rapid shifts in bacterial species, strains,..., Sharon [/bib_ref] , in combination with GC content. This led from minor to substantial improvements in bin purity and completeness.
Up to eight near-complete genomes (≥94% of expected single copy genes identified) were reconstructed per sample, and 221 near-complete genomes were reconstructed over the study. This accounting under-represents the completeness of the analysis because the presence of multiple highly related Enterobacteriales genotypes in many samples resulted in partial and fragmented recovery of specific conserved ribosomal proteins. When including Enterobacteriales genomes of the expected size but lacking these specific ribosomal proteins, 260 near-complete genomes were reconstructed.
Rank abundance curves were constructed based on coverage. For this analysis, coverage values were normalized to account for differences in data size per sample.
# Comparative genomic analysis
Strain comparison was done for genomes with >0.5 Mbp of recovered sequences, and was mostly based on alignment of sequences for 51 predicted single copy genes, many of which were ribosomal proteins. For cases with inconclusive results, mostly due to highly fragmented or very partial genomes in which many of these genes were missing, entire genome bins were aligned. In a few cases, mostly when verification of very small differences was required, manual curation of results based on inspection of read mapping to the regions in question was performed to detect local mis-assemblies.
## Alignment of single copy genes
To avoid detection of false differences based on local scaffolding errors, predicted single copy genes with one or more base pairs that were not covered by at least one perfectly matching read, or, in which >50% of mapped reads did not agree with the assembled sequence, were removed from analysis. Split genes were also removed from analysis, to avoid errors introduced at the scaffolding step.
Pairs of genomes with the same species assignment, and with at least 20 single copy genes that passed filtering were compared to each other by aligning the assembled gene sequences using nucmer [bib_ref] Fast algorithms for large-scale genome alignment and comparison, Delcher [/bib_ref]. Genome pairs that shared at least five single copy genes that passed filtering and for which all shared single copy genes were identical across their length were considered to be the same strain.
## Alignment of genome bins
Genome bins were aligned using nucmer [bib_ref] Fast algorithms for large-scale genome alignment and comparison, Delcher [/bib_ref]. The number of base pairs in alignments with over 98% identity was tallied (a higher identity threshold was not used in order to take into account occasional local scaffolding errors). If over 90% of bin length (for the shorter genome bin) was aligned using this threshold, the genomes were considered indistinguishable.
## Strain comparison via read mapping
For E. faecalis and C. paraputrificum [fig_ref] Figure 1: Overview of the sampling of infants affected by necrotizing enterocolitis [/fig_ref] , strains were compared by mapping reads from different samples to specific scaffolds. Multiple alignment of the consensus sequences resulting from each mapping provided a detailed view of SNPs and indel regions while avoiding false differences resulting from partial assemblies or from assembly and scaffolding errors. Mapping was done using bowtie2 and multiple alignment was done using default parameters for the MAFFT algorithm [bib_ref] MAFFT: a novel method for rapid multiple sequence alignment based on fast..., Katoh [/bib_ref] implemented in Geneious v7.1.7 [bib_ref] Geneious Basic: an integrated and extendable desktop software platform for the organization..., Kearse [/bib_ref].
## Detailed whole genome comparisons
In a few cases (comparison of E. faecalis genomes in infants #3 and #5, and in infants #2 and #7 as well as comparison of C. parapsilosis to the CDC317 isolate), a more detailed whole genome comparison was performed in order to locate sequence regions that were not shared between strains.
Mauve genome alignment software [bib_ref] progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement, Darling [/bib_ref] was used to perform pairwise comparisons of strains of the same species assembled from different babies. Genomes were first ordered relative to a reference genome from the same species (E. faecalis 62, gi 323478858; C. parapsilosis CDC317, gi 218176216) and then compared to each other. Stretches of DNA in one of the genomes that could not be aligned to the other genome (termed 'islands') were extracted. Stringent postprocessing steps were taken to filter out islands that could have resulted from missing or problematic segments in the assembly rather than actual differences in genomic sequence. Islands whose sequence was dominated by Ns (assembly gaps) were removed from further analysis. Islands that were very close (<100 bp) to scaffold edges or islands whose flanking regions mapped to two different scaffolds in the other genome, could have resulted from missing assemblies, and were therefore disregarded. To verify suspected islands, reads from samples of both babies were mapped to the island sequence and manually inspected.
## Single copy gene and crispr locus analyses
Geneious software v7.1.7 [bib_ref] Geneious Basic: an integrated and extendable desktop software platform for the organization..., Kearse [/bib_ref] was used to align individual single copy gene sequences and for manual curation of the CRISPR loci. We used the online CRISPR spacer and repeat finder tools to recover spacer and repeat sequences (http://crispr.u-psud.fr).
## Comparison of phage, plasmid, and mobile elements
Scaffolds longer than 5000 bp that were unbinned or were binned as plasmid, phage, or mobile elements, were extracted and aligned to each other (using nucmer [bib_ref] Fast algorithms for large-scale genome alignment and comparison, Delcher [/bib_ref]. Scaffolds that were 99% identical across 90% of their length were considered closely related.
## Genome completeness and metabolic profiling
An overview of the metabolic potential associated with genomes reconstructed in this study was established by searching the functional predictions for specific annotation terms. The number of genes that have the selected annotation terms is displayed in a table format in which rows are genomes and columns list the number of genes in each category (see Supplementary file 4). The search and exclusion terms for each functional category can be found via the ggKbase list function.
## Genome curation methods and results
Genomes of C. parapsilosis (infant #9), a species related to C. ultunense (infant #3), a Clostridiales from infant #6, a V. parvula-related strain (infant #3), an Actinomyces species (infant #4), and a N. succinicivorans strain (infant #5) were chosen for curation because they were significant and/or of very good draft quality. The curation used programs for correcting mis-assemblies and improving assemblies , which were identified through read mapping as follows. First, all reads were mapped to the genomes using bowtie2 (http://www.nature.com/nmeth/journal/ v9/n4/full/nmeth.1923.html) with the -sensitive option. Next, short deletions (which we found to be common in idba-ud assemblies) in the assembled sequences were identified based on the read mappings. Last, all regions on the genomes with exceptionally low coverage were checked by collecting reads that map to those regions and their mate pairs and re-assembling them. Improvement of assemblies was achieved through read-mapping based identification of scaffolds that could be elongated or connected to other scaffolds. Both elongations and connections were achieved through local assembly of reads that were mapped to the analyzed regions and their mate pairs. For the Candida genome, our pipeline corrected 106 mis-assemblies (about one mis-assembly for every 120 Kbp) and reduced the number of scaffolds from 401 to 348.
## Droplet digital pcr methods
To quantify bacterial load in infant fecal samples, ddPCR was performed on the Bio-Rad QX200 platform using EvaGreen-based chemistry (Bio-Rad, Hercules, CA). A conserved, approximately 150 bp region flanking the V7 region of the 16S rRNA gene was targeted, as it has been successfully used in other probe-based qPCR assays in the past (1048f: GTGSTGCAYGGYYGTCGTCA, 1194r: ACGTCRTCCMCNCCTTCCTC [bib_ref] Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis..., Ramirez-Farias [/bib_ref] [bib_ref] The impact of different DNA extraction kits and laboratories upon the assessment..., Kennedy [/bib_ref]. Sample gDNA was diluted to 1:1000 and used as template in a PCR reaction consisting of 0.25 μl of 10 μM forward and reverse primer, 12.5 μl of 2× ddPCR EvaGreen Supermix (Bio-Rad), and 12 μl of template, totaling 25 μl. This PCR mix was used to create droplets following the manufacture's instructions. Thermocycling parameters were: (1) 95˚C for 10 min, (2) 95˚C for 30 s, (3) 61˚C for 30 s, (4) 72˚C for 30 s, (5) 40 cycles (go to steps 2-4 ×39), (6) 98˚C for 10 min, and (7) 12˚C forever. All ramp rates were at 2.5˚C/s. Each reaction was done in triplicate. Analysis of the ddPCR data was conducted with the QuantaSoft software package (Bio-Rad) and negative/positive thresholds set manually (just above the negative population). To calculate cell density, the copies/μl output from QuantaSoft was normalized by grams of fecal mass used for each gDNA extraction reaction. To broadly correct for copy number, the assumption of four copies per bacteria was used [bib_ref] Human occupancy as a source of indoor airborne bacteria, Hospodsky [/bib_ref].
## Data dissemination
The sequence information can be accessed via NCBI, accession # SRP052967. All metagenomic data associated with this study can be accessed via the ggKbase NECEvent project: http://ggkbase. berkeley.edu/project_groups/necevent_samples. Note that this is a 'live data' repository, so that errors found after publication will be corrected and more highly curated assemblies may be available. A snapshot of the published dataset is also available for download.
## Ethics
Human subjects: The study was performed with approval from the University of Pittsburgh Institutional Review Board under protocol number PRO10090089, and written parental consent was obtained on behalf of the neonates.
## Additional files
Supplementary files · Supplementary file 1. Clinical characteristics of infants in this study. Necrotizing enterocolitis (NEC) was defined as definite NEC (Bell's stage II or III). CS: caesarean section; V: vaginal delivery; BM: breast milk; combination indicates a combination of breast milk and infant formula. DOI: 10.7554/eLife.05477.020 · Supplementary file 2. Overview of samples, the day of life on which each sample was collected, the original internal database sample number, the amount of data that was generated per sample after trimming to remove low quality bases, the amount of data that went into genome bins, percentage of all data that went into assemblies that ended up in bins, the amount of data that remained unbinned, the number of genome bins per sample, and the number of features (genes) per sample. Red lines indicate timing of necrotizing enterocolitis diagnosis. For two infants, samples were only available after diagnosis. indicators for all moderately to well sampled genomes from all infants. Note that the single copy gene inventory underestimates genome completeness for Gammaproteobacteria when multiple species were present (see main text). Each line represents a genome bin. The bin name provides information about the sample of origin: the first digit is the infant number, the second is the sample number (e.g. 1_2 is the second sample from infant #1) and the organism type. Lists to the right profile the electron transport chain in which the presence of a terminal oxidase, in combination with a TCA cycle, indicate aerobic metabolism. Information about pathways involved in fermentation processes, nitrogen metabolism, the cell surface and secretion, motility, toxicity and pathogenicity, mobile elements, and CRISPR-based virus defense and antibiotic resistance is also shown. Note that Peptoclostridium difficile and Clostridium difficile are equivalent.
## Major dataset
The following dataset was generated:
[fig] Figure 2: An overview of the distribution of 144 of the 149 tracked strains in the 55 samples from 10 infants (five rare organisms were not included for space reasons). [/fig]
[fig] Figure 4 007, Figure supplement 2: Strain differences in recovered Enterococcus faecalis genomes. (A) Alignment of the ∼2500 Enterococcus faecalis gyrA nucleotide sequences from all infants to that from infant #3, sample 1 revealing five distinct types (gray bars are scaffolds; SNPs are vertical black lines). Shown below are a tiny subset of reads from infant #3, sample 4 with SNPs that match nucleotides in the gyrA sequences from E. faecalis in another infant; all SNPs are consistent with a strain very similar to that in infants #2 and #7 (although derivation of some reads from other strains cannot be ruled out). (B) Phylogenetic representation illustrating two distinct Cas1 sequence types. (C) Inventory of 51 single copy genes showing that the 30 E. faecalis genomes are near-complete and providing information about encoded CRISPR and Cas. DOI: 10.7554/eLife.05477.006 The following figure supplements are available for figure 4: Figure supplement 1. Alignments showing single nucleotide polymorphisms (vertical colored lines on gray bars that represent the sequences) in the Histidyl-tRNA synthetase genes that distinguish from Enterobacter cloacae strains across samples and infants. DOI: 10.7554/eLife.05477.Aspartyl-tRNA synthetase from Klebsiella pneumoniae strains in samples from infants #4, #5, #6, #7, and #8. DOI: 10.7554/eLife.05477.008 [/fig]
[fig] Figure 5: Comparison of CRISPR loci in Enterococcus faecalis genomes. (A) The CRISPR-Cas loci in infants #3, #5 (early strain), and #8 and (B) the CRISPR locus lacking adjacent Cas proteins. The first defective repeats are shown in blue, other repeats are in green. The CRISPR loci are expanded below. In A, two versus three spacers have been added to the young end of the loci (left side, adjacent to Cas) in infants #3, #5 versus #8, respectively. In B, scaffolds encoding the loci are shown as horizontal gray bars (polymorphisms in the multi-sequence alignment are small vertical tic marks). The same color indicates shared sequences. Blue boxes to the left indicate that the genome encodes Cas proteins. Both loci (A and B) are identical in infants #3 and #5. DOI: 10.7554/eLife.05477.009 [/fig]
[fig] Figure supplement 1: Alignment view of genome-wide differences in Clostridium paraputrificum strains. DOI: 10.7554/eLife.05477.011 [/fig]
[fig] Figure 7: Stacked bar plot of community composition across samples and infants after organism identifications were collapsed to the phylum level to allow comparison to prior studies. Red lines indicate necrotizing enterocolitis diagnoses. DOI: 10.7554/eLife.05477.012 [/fig]
[fig] Figure 9: Statistical evaluation of the clustering of necrotizing enterocolitis cases during 2009-2014. (A) The number of diagnosed necrotizing enterocolitis (NEC) cases meeting the stringent Vermont Oxford Network (VON) criteria over 67 months. Gray shading highlights the studied period. (B) Observed frequency of each value of monthly NEC cases in collected data (blue); expected frequency from a Poisson (red) and negative binomial (NB; green) distributions that were fit to the observed data using maximum likelihood parameter estimation (Poisson: λ = 1.90, NB: r = 5.81, p = 0.75). DOI: 10.7554/eLife.05477.017 [/fig]
[fig] ·: Supplementary file 3. Overview of the bins from each sample and each infant. UNK indicates a bin of unclassified sequences. Coloring of the bin names generally corresponds to colors used in emergent self organizing map and rank abundance curves in the figures. SCG is the number of single copy genes identified per bin out of 51 expected genes. DOI: 10.7554/eLife.05477.022 Supplementary file 4. Overview of predicted metabolic potential and genome completeness [/fig]
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Wound care in the geriatric client
r e v I e wDovepress open access to scientific and medical research Open Access Full Text Article submit your manuscript | www.dovepress.com Dovepress wound care in the geriatric client Abstract: With our aging population, chronic diseases that compromise skin integrity such as diabetes, peripheral vascular disease (venous hypertension, arterial insufficiency) are becoming increasingly common. Skin breakdown with ulcer and chronic wound formation is a frequent consequence of these diseases. Types of ulcers include pressure ulcers, vascular ulcers (arterial and venous hypertension), and neuropathic ulcers. Treatment of these ulcers involves recognizing the four stages of healing: coagulation, inflammation, proliferation, and maturation. Chronic wounds are frequently stalled in the inflammatory stage. Moving past the inflammation stage requires considering the bacterial burden, necrotic tissue, and moisture balance of the wound being treated. Bacterial overgrowth or infection needs to be treated with topical or systemic agents. In most cases, necrotic tissue needs to be debrided and moisture balance needs to be addressed by wetting dry tissue and drying wet tissue. Special dressings have been developed to accomplish these tasks. They include films, hydrocolloids, hydrogel dressings, foams, hydrofibers, composite and alginate dressings.
# Introduction
The demographic reality in the United States and other developed countries is that the population is aging and the medical community must provide care for an everincreasing number of elderly individuals. As with other disease processes common in the geriatric population, the incidence of chronic ulcers related to diabetes, peripheral vascular disease, and mobility issues occurs with increasing frequency. The purpose of this article is to address the etiology, assessment, and treatment options for the most frequently encountered types of chronic skin breakdown. Although the original goal may be complete wound healing, palliation and achieving a manageable, chronic wound may be an acceptable outcome.
Approximately 70% of all pressure ulcers occur in the geriatric population. Their skin has a decrease in water content, tensile strength, and junctional integrity between the dermis and the epidermis. Atrophy of the apocrine and sebaceous glands causes drying of the skin. A loss of subcutaneous tissue, vascularity, and diminishing stability of small blood vessels add other factors to compromise skin integrity. [bib_ref] Issues and dilemmas in prevention and treatment of pressure ulcers: a review, Thomas [/bib_ref] [bib_ref] Facilitating positive outcomes in older adults with wounds, Stotts [/bib_ref] [bib_ref] Skin fails too: acute, chronic, and end-stage skin failure, Langemo [/bib_ref] The most common wounds to be discussed are pressure ulcers (decubitus ulcers or bedsores), vascular ulcers (arterial and venous), and neuropathic ulcers. Assessment tools and techniques, appropriate classification systems, dressing choices, and treatment modalities will be addressed. A brief discussion of some wound care products as they relate to chronic wound care will be presented.
## Dovepress
## Types of chronic wounds pressure ulcers
Pressure ulcers are, as their name implies, caused primarily by unrelieved pressure. They usually occur over bony prominences such as the sacrum or the heel, but can occur on any part of the body subjected to pressure. The damage causing a pressure ulcer can occur in a matter of hours. Shear forces and friction may also contribute to the formation of pressure ulcers. Pressure ulcers are described as stages relating to the depth of tissue damage incurred. In February 2007, the National Pressure Ulcer Advisory Panel (NPUAP) released updated definitions of these stages in order to clarify misconceptions and reduce confusion that had been a frequent complaint with the original definitions put forth by These stages are used to describe pressure ulcers only . Other wound classification systems include partial or full thickness wounds. Partial thickness wounds involve only the epidermis and dermis. Full thickness wounds involve deeper tissues and structures. Diabetic foot ulcers also have unique classification systems to be described later.
## Pressure ulcer prevention
Prevention of pressure ulcers is of paramount importance, and begins with a complete history and physical to determine risk factors. [bib_ref] Best practice recommendations for the prevention and treatment of pressure ulcers: update, Keast [/bib_ref] However, not all pressure ulcers are avoidable. As noted by Langemo and Brown, skin, as other organ systems can also fail. [bib_ref] Skin fails too: acute, chronic, and end-stage skin failure, Langemo [/bib_ref] Even with appropriate treatment, Stage IV ulcers may require surgical intervention such as surgical flaps, deep debridement or possibly amputation. Healing Stage IV ulcers may take years and, as with any wound that heals by scar formation, result in tissue that is only 40%-80% of the tensile strength of undamaged tissue. Development of a pressure ulcer has been implicated in increased mortality, although the causal relationship has not been established. [bib_ref] Issues and dilemmas in prevention and treatment of pressure ulcers: a review, Thomas [/bib_ref] Potential interventions for prevention of pressure ulcers include the repositioning, nutrition, moisturizing dry skin or by preventing damp skin by targeting fecal or urinary incontinence and the use of special support surfaces. Urinary and fecal incontinence place the skin at risk not only due to excessive moisture, but also because of the chemical properties of urine and feces. [bib_ref] Preventing and managing perineal dermatitis: a shared goal for wound and continence..., Gray [/bib_ref] [bib_ref] Skin care protocols for pressure ulcers and incontinence in long term care:..., Thompson [/bib_ref] Specialized support surfaces redistribute pressure on the skin and subcutaneous tissue. Unrelieved pressure can impair circulation and lead to ulcer formation. Pressure redistributing surfaces can be static or dynamic. Dynamic support surfaces mechanically vary the pressure under the patient thus reducing the length of time a particular skin area is under pressure theoretically improving circulation. Example of dynamic support surfaces include alternating pressure mattresses, low air loss mattress replacement systems, and air fluidized mattresses. A dynamic surface is recommended when the patient cannot reposition themselves, be adequately repositioned without weight bearing on a pressure ulcer or if the patient fully compresses the static surface. [bib_ref] Best practice recommendations for the prevention and treatment of pressure ulcers: update, Keast [/bib_ref] Examples of static support surfaces are foam mattresses or static flotation (air cells, gel, fluid overlays that are placed on top of the mattress). [bib_ref] Best practice recommendations for the prevention and treatment of pressure ulcers: update, Keast [/bib_ref] Foam overlays are no longer used for pressure redistribution. They do not function well and may violate fire codes.
Repositioning or turning patients while in bed is one of the most important ways to prevent pressure ulcers although passive positioning alone has not been shown to decrease the incidence of ulcer formation. [bib_ref] Issues and dilemmas in prevention and treatment of pressure ulcers: a review, Thomas [/bib_ref] The accepted standard of care is to turn patients at least every two hours. Minimizing pressure in the skin will help maintain circulation to areas that are at risk for breakdown. It is important to keep pressure off areas such as the heels that are at risk for breakdown. This can be accomplished both by turning patients from side to side while in bed and floating the heels above the mattress by placing pillows, etc under the legs.
Nutrition is thought to be important in ulcer prevention although there is not a strong body of supporting evidence. A study by Bourdel-Marchasson and colleagues studied 672 critically ill inpatients older than 65 comparing a standard diet to a standard diet plus two oral nutritional supplements per day. [bib_ref] A multi-center trial of the effects of oral nutritional supplementation in critically..., Bourdel-Marchasson [/bib_ref] Patients receiving a standard diet had a relative risk of 1.57 when compared to a group receiving standard diet plus two nutritional supplements. Nutritional evaluation includes serum albumin and pre-albumin levels, although decreased levels may reflect production of inflammatory cytokines not actual nutritional status. [bib_ref] Issues and dilemmas in prevention and treatment of pressure ulcers: a review, Thomas [/bib_ref] Proper skin moisture is also important in preventing skin ulcers. Dry sacral skin is a known risk factor for developing pressure ulcers. Hence, moisturizing dry skin may help prevent pressure ulcers. Massaging areas at risk over bony prominences while applying moisturizing lotions or cream may increase the risk of breakdown.
Skin that is frequently too wet by contact with urine or perspiration is at greater risk for breakdown. Improvement in continence care with interventions such as toileting patients every two hours, use of skin protectant barrier creams, etc., help maintain skin health. [bib_ref] Skin care protocols for pressure ulcers and incontinence in long term care:..., Thompson [/bib_ref]
## Vascular ulcers
Vascular ulcers result from deficits in either quality of arterial flow or derangement of the venous system. The prevalence of Area at risk is sometimes identified as a "heralding lesion" it indicates potential for increased damage if not addressed immediately. [bib_ref] National Pressure Ulcer Advisory Panel. National Pressure Ulcer Advisory Panel's updated pressure..., Black [/bib_ref] Deep tissue injury
- New category added to the pressure ulcer staging system called deep tissue injury (DTI).
- Nonblanchable purple or maroon discoloration of intact skin that may indicate damage to the underlying tissue.
- DTI can heal or progress to a deep ulcer in a short period of time even with optimal and aggressive treatment.
- Evaluation initially may be confusing if the hyperpigmented or bruised area is a result of DTI. One should err on the side of caution and identify the lesion as a suspected DTI.
[formula] 5-7 - - - STAGE 1 SUSPECTED DEEP TISSUE INJURY [/formula]
## Stage ii
- Ulcers involve the epidermis and dermis only.
(Partial thickness)
- Ulcer presentation may be as a serum-filled blister or a shallow, superficial ulcer with a pink or red wound base.
- Lesions may be painful.
## Stage iii
- Ulcers are full thickness; however tissue damage does not expose tendon, bone or joint capsule.
- Anatomical location and the depth or composition of the underlying tissue determine whether the ulcer is relatively shallow or deep.
Areas that have no subcutaneous tissue may be relatively shallow, but areas with a thick cushion of adipose tissue may be deep but bone is not exposed or directly palpable.
- Undermining or tunneling may be seen with these lesions.
- Undermining is tissue destruction extending under the skin along the perimeter of the wound with a relatively wide opening.
- Tunneling, on the other hand, has a relatively small opening, but the tissue loss may extend deeply into the adjacent tissue.
## 5-6
## Stage 3
Stage IV
- Pressure ulcer may extend into muscle, and in some cases, erode bone, tendons, ligaments and other supporting structures.
- Ulcer may have slough or eschar in portions of the wound bed and undermining and/or tunneling is common.
- Anatomical location dictates the depth of the wound, but the hallmark is the exposure of deeper supporting structures. [bib_ref] National Pressure Ulcer Advisory Panel. National Pressure Ulcer Advisory Panel's updated pressure..., Black [/bib_ref] Unstageable - Ulcers covered with slough, eschar or necrotic debris to the extent that the wound base is not visualized are classified as unstageable.
- Slough is soft nonviable tissue that may be yellow, gray, tan or green. Eschar is desiccated necrotic tissue and may be black, gray or brown. [bib_ref] National Pressure Ulcer Advisory Panel. National Pressure Ulcer Advisory Panel's updated pressure..., Black [/bib_ref] - Presence of slough or eschar indicates that the ulcer is at least Stage III because these tissues arise from subcutaneous fat or devitalized muscle. wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com Dovepress peripheral arterial and peripheral venous disease is increased in the geriatric population. Evaluation must include both systems because these problems are not mutually exclusive.
## Arterial ulcers
Arterial ulcers are a result of restriction of flow or occlusion of an artery. They have a discrete "punched out" appearance, a pale wound bed and minimal exudate. Gangrene or necrotic tissue is common. Due to the ischemia underlying the development of these ulcers, they are often very painful and difficult to treat or heal. [bib_ref] Leg ulcers, Paquette [/bib_ref] The most frequent locations for these ulcers are the tips of or between the toes, over the phalangeal heads, or over the lateral malleolus. Even a bump or scrape can precipitate an ulceration. [bib_ref] Management of vascular leg ulcers, Wipke-Tevis [/bib_ref] If an arterial or ischemic ulcer is suspected, then the site should be evaluated for adequate blood flow. For leg and foot ulcers if there is a palpable dorsalis pedis pulse, the ankle systolic pressure is at least 80 mm Hg or higher and the brachial systolic pressure at least 100 mm Hg. These values are usually indicative of enough pressure to heal a wound. If a pulse is nonpalpable then evaluation of blood flow by Doppler ultrasound is important. The Ankle Brachial Index (ABI) is used to determine quality of blood flow and ascertain if a wound is healable. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] The ABI is calculated by dividing the dorsalis pedis systolic pressure by the brachial artery systolic pressure. A value less than 0.5 (moderately severe ischemia) means that it will be difficult for the wound to heal. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] If the ABI is greater than 1.2 (especially in the diabetic or renal failure patient) it may mean that the vessels are heavily calcified, which can produce a falsely elevated ABI. Toe pressures can then be used because the vessels supplying the toes do not have calcium deposits. Toe pressures greater than 50 mm Hg are normal; whereas values of less than 30 are grossly abnormal. Frequently, wounds can heal with toe pressure values between 30-50 mm Hg. [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] The treatment of arterial ulcers is based on optimization or restoration of adequate perfusion and may be achieved by risk factor modification, activity and positioning, medication or surgical treatment. [bib_ref] Management of vascular leg ulcers, Wipke-Tevis [/bib_ref] Risk factor modification includes smoking cessation, controlling diabetes, hypertension, and hyperlipidemia. A daily walking program is encouraged. Leg elevation is relatively contraindicated in patients with arterial ulcers. Patients with arterial ulcers should wear compression bandages or stockings only with careful consideration of the risks and benefits and with close monitoring.
Two drugs are approved by the Federal Drug Administration (FDA) for promoting blood flow in patients with arterial ulcers or claudication: pentoxyphilline (Trental), and cilostazol (Pletal). These medications are phosphodiesterase inhibitors that prevent platelet aggregation. Trental improves erythrocyte and leukocyte flexibility and thins the blood. Pletal also increases vasodilatation. However, its use is contraindicated in patients with congestive heart failure. [bib_ref] Peripheral arterial disease, Aronow [/bib_ref] Surgical treatment is recommended for patients with rest claudication who are at risk for limb loss. [bib_ref] Management of vascular leg ulcers, Wipke-Tevis [/bib_ref] The site of arterial occlusion can partially be determined by the location of pain or fatigue. Occlusion of the aorta or iliac arteries causes symptoms in the buttocks or hips, femoropopliteal disease causes symptoms in the calf. [bib_ref] Peripheal arterial disease: tips on diagnosis and management, Holman [/bib_ref] Bypass grafting, stent placement, or angioplasty are possible options for intervention.
## Venous ulcers
Venous ulcers are caused by venous hypertension resulting in uncontrolled edema. Venous hypertension may result from a deep vein thrombosis (DVT) or postphlebitic syndrome, obesity, congestive heart failure or severe trauma to the leg. The visible changes in the tissue of the leg associated with venous hypertension are: hemosiderin staining, the hyperpigmentation resulting from red blood cells leaking into the tissue and deposition of the heme; stasis dermatitis marked by erythema, edema, scaling, and weeping of serous fluid; lichenification, thickening of the tissue; and skin ulceration. [bib_ref] Leg ulcers, Paquette [/bib_ref] [bib_ref] Patient with chronic venous stasis ulcer, Gunder [/bib_ref] Long-term untreated or under-treated venous hypertension, may result in development of lymphedema, which further complicates treatment.
Venous ulcers are generally shallow with irregular wound margins. The ulcer base tends to be beefy red and granulation is frequent with exudates most often moderate to heavy. If there is an arterial component, the color and amount of exudates may be muted. These wounds may have a strong odor that may be concerning to the patient and must be addressed when providing wound care. Venous ulcers are classically located over the medial malleolus although sometimes they can be near the lateral malleolus. In severe cases they may encircle the entire ankle.Other disease processes such as vasculitis, pyoderma gangrenosum, or neoplasms may be mistaken for venous ulcers. Ulcers that do not respond to standard treatment should be referred for further evaluation, ie, dermatology, vascular or plastic surgery, etc.
The treatment of venous ulcers includes specific recommendations regarding activity, positioning and compression therapy. Standing or sitting with the legs in a dependent position should be discouraged. Patients should shift their weight frequently when standing and should be encouraged to have a daily walking program. Leg elevation above the level of the heart is recommended to reduce edema. Compression therapy is essential to venous ulcer healing and prevention of recurrence. Before compression therapy can be used arterial Dovepress status should be evaluated and deemed to be adequate. ABIs of 0.5 or greater are required to initiate compression therapy. Many types of compression devices are available that provide sustained graduated compression at a therapeutic level. These include elasticized tubular support bandages, elastic wraps, custom fitted compression stockings and wraps with hook and loop closure such as CircAids. [bib_ref] Compression therapy of venous ulcers, Hunter [/bib_ref] The Unna boot is an example of rigid compression therapy that goes on wet but dries to becomes rigid, it works through enhancement of the calf muscle pump and is appropriate for ambulatory patients. [bib_ref] Compression therapy of venous ulcers, Hunter [/bib_ref] There are several multi-layer elastic compression systems available that provide moist wound healing, wound protection, absorption of drainage and continuous therapeutic levels of compression. [bib_ref] Patient with chronic venous stasis ulcer, Gunder [/bib_ref] Careful consideration of requirements for use of these devices as outlined in the manufacturers guidelines is warranted.
## Neuropathic ulcers
Neuropathy is most often associated with diabetes, but may result from other diseases. Neuropathy falls into three categories: sensory, motor, and autonomic. The most common is sensory neuropathy resulting in an insensate foot. Ulcer causality is multifactorial, the presence of sensory neuropathy, peripheral occlusive vascular disease and the presence of bony deformities increase the incidence of foot ulcers. [bib_ref] The treatment of diabetic foot ulcers: Reviewing the literature and a surgical..., Kravitz [/bib_ref] Diabetic foot ulcers may be classified in stages according to the natural history progression: 1) normal foot; 2) high risk foot; 3) ulcerated foot; 4) infected foot; 5) necrotic foot. [bib_ref] A natural history and framework for managing diabetic foot ulcers, Edmonds [/bib_ref] Patients with loss of protective sensation have been shown to have a 15 fold increase in the risk of developing a foot ulcer. [bib_ref] Management of the diabetic foot ulcer in the elderly population, Mulder [/bib_ref] When the protective sensory threshold is lost, patients may continue to experience tissue damaging pressure or trauma. The ulcers are typically located in areas exposed to repetitive pressure and the precursor of callus or blister formation can alert patients and providers alike to an impending ulcer. Callus formation is a normal response to the stress of elevated pressures on an area of the foot. The callus can act like a stone (local pressure) in the shoe. [bib_ref] Best practices for the prevention, diagnosis and treatment of diabetic foot ulcers, Inlow [/bib_ref] Repetitive pressure can lead to formation of an ulcer at the site of the callus. Ulcers can occur undetected under a callus and only declare themselves with an infection that threatens the foot or lower limb. Testing for protective sensory threshold is done with a Semmes-Weinstein 5.07/10 gm monofilament (Semmes-Weinstein Corp., NY, USA). [bib_ref] Best practices for the prevention, diagnosis and treatment of diabetic foot ulcers, Inlow [/bib_ref] Motor neuropathy causes an imbalance in the forces of flexor versus extensor muscles, with the stronger flexor muscles generally overpowering the extensors causing deformities such as hammer toes, claw toes, etc. These deformities change the configuration of the protective pads of the foot leaving the joints in jeopardy for breakdown. As the toes contract the patient walks on the tips of the toes instead of the plantar surface, the phalangeal heads become more prominent and subject to pressures from the shoes. The protective pads over the metatarsal heads are also pulled forward leaving the tissue over them at high risk for development of pressure ulcers.
Autonomic neuropathy causes a decrease in sweating leading to excessively dry skin. This can cause the skin to dry and thicken, often resulting in painful fissures that are a potential portal for infection. [bib_ref] Acute Charcot's arthropathy: A difficult diagnosis, Johnsen [/bib_ref] Loss of autonomic regulation allows dilatation of the blood vessels of the foot, demineralization, and softening of the bones leading to Charcot foot deformities. [bib_ref] Acute Charcot's arthropathy: A difficult diagnosis, Johnsen [/bib_ref] Prevention of diabetic neuropathic foot ulcers is paramount; 60% of nontraumatic lower-limb amputations are diabetes-related.People with diabetes should wear comfortable well fitting shoes and never walk barefoot. Glucose control needs to be optimized to help prevent neuropathy and aid in healing. Calluses or corns should be treated by a professional. Patients providing self-care must follow their professional's guidelines and instructions carefully. Off-loading pressure on the ulcer is necessary using devices such as wheelchairs, crutches, walkers, total contact casts, total contact sandals, short leg walkers and wedged healing shoes.If adequate blood flow is present, periwound hyperkeratosis needs to be debrided with the wound margin being "saucerized" (debrided so wound margins slope like a shallow bowl, thereby reducing pressure and optimizing wound healing). Surgical correction of a specific foot deformity may be required. [bib_ref] Management of the diabetic foot ulcer in the elderly population, Mulder [/bib_ref] Other modalities are being used for treatment of nonhealing diabetic foot ulcers including negative pressure wound therapy, recombinant human growth factors and skin substitutes. The US FDA has issued an ongoing safety review regarding becaplermin (Regranex), a topical recombinant form of human platelet-derived growth factor: "… there may be some evidence for an increased risk of death from cancer in patients who had repeated treatment with Regranex."The risk of a nonhealing diabetic foot ulcer should be weighed against the possible risk of using Regranex on an individual basis. 27
## Stages of healing
There are four major stages of healing of acute wounds. They include coagulation, inflammation, proliferation and maturation. During coagulation platelets start healing by releasing mediators such as growth factors. In inflammation, wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com Dovepress there is vasodilatation, an increase in capillary permeability, complement activation and migration of neutrophils and macrophages to the site. The neutrophils and macrophages destroy bacteria, release proteases, and secrete growth factors. Inflammation is regulated by bacteria and cytokines.
Cell proliferation occurs when fibroblasts, endothelial cells, and keratinocytes promote cell migration, proliferation, angiogenesis and synthesis of extracellular matrix components. Remodeling or maturation occurs over several weeks to months after a new scar is formed and collagen fibers are rearranged into a more orderly configuration modifying the appearance of the scar tissue. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] Unfortunately chronic wounds become stalled in the inflammation stage and do not progress to the proliferation stage. Chronic wounds have an increase in proinflammatory cytokines. They also have an increase in matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent endopeptidases secreted as inactive zymogens. They are capable of degrading most extracellular components and basement membrane proteins at a neutral Ph. Within the skin they are produced by keratinocytes, fibroblasts, neutrophils, macrophages, mast cells and eosinophils. This proinflammatory environment contains many proteins that break down the developing collagen matrix and impede healing. [bib_ref] Pathophysiology of chronic nonhealing wounds, Medina [/bib_ref]
## Major considerations in treating a chronic wound
Bacterial balance, necrotic tissue, and moisture balance comprise the three major considerations in treating a chronic wound. These areas must be addressed in order for healing to occur.
## Bacterial balance
All wounds contain some bacteria. The question is whether the amount of bacteria merely represents colonization of the wound or represents bacterial overgrowth and infection. The latter can lead to impaired wound healing. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] There are many signs and symptoms which suggest bacterial over growth and infection. Either a subclinical or overt infection may prevent wound healing. Signs and symptoms include change in wound exudate that may be clear or serous then become purulent in appearance, an unpleasant odor from the wound can indicate superficial or deep infection. Bacterial stimulation of vascular endothelial growth factor (VEGF) can occur which stimulates new blood vessel formation and hence makes the area more likely to bleed. An increase in cutaneous temperature and increased surrounding erythema suggests an infection. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] [bib_ref] The edge effect: current therapeutic options to advance the wound edge, Woo [/bib_ref] Treatment of bacterial overgrowth and infection is determined by whether the infection is superficial or deep. Superficial bacterial overgrowth can be treated with topical agents. Deep tissue infections such as cellulitis, muscle, and bone (osteomyelitis) infections must be treated with systemic antibiotics and frequent debridement. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] Oral antibiotics may be used for less severe infections with intravenous (IV) antibiotics reserved for more severe infections such as advancing cellulitis or osteomyelitis. Antibiotics are determined by Gram stain and suspicion initially and adjusted based on bacterial identification with antibiotic sensitivity. The microbial flora of a chronic wound changes over time.
In an early wound, Gram-positives such as Staphylococcus aureus and beta-hemolytic streptococci predominate. After four weeks, Gram-negative rods ie, Proteus, Escherichia coli and Klebsiella will colonize the wound. Later on anaerobes come into play and after several months the average wound may be colonized by 4-5 different organisms. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] Superficial bacterial overgrowth can usually be treated with topical agents, sometimes as simple as normal saline, which can keep a chronic wound clean. Wounds that have no chance of healing should also be treated with topical agents with the goal of preventing the nonhealing wound from becoming infected.
Examples of topical agents are chlorhexidine, betadine, acetic acid, hydrogen peroxide, scarlet red dye, and bacitracin. Chlorhexidine and betadine are broad spectrum and have low tissue toxicity. Acetic acid is effective against pseudomonas. Dyes are effective against Gram-positive cocci but not against Gram-negative bacteria. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] These agents should be used for cleansing dirty or infected wounds only and not for clean wounds as they may cause damage to the wound bed. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] Broad-spectrum antimicrobials such as silver products, polyhexamethylene biguanide, Cadoxemer Iodine or polyacrylates are beneficial in decreasing the bioburden of a chronic wound and are also used to treat superficial infections. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] Silver-based agents are also used to treat superficial infection. There are a wide range of silver-based agents. Examples of such agents include: hydrogels, alginates, foams, negative pressure wound therapy foam dressings, and some compression garments. The controversy in the industry is whether the Dovepress total amount of silver or the chemically available amount is more important in bacterial control. Silver dressings should be chosen according to the amount of silver released and their relative moisture absorption and retentive properties. 33
## Dealing with necrotic tissue through debridement
Necrotic tissue impedes the healing of both acute and chronic wounds. It impairs the development of granulation tissue and impairs the migration of keratinocytes that are required for re-epithelialization. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] [bib_ref] Wound debridement: therapeutic options and care consideration, Beitz [/bib_ref] Necrotic tissue can also serve as a medium for bacterial overgrowth. Removing the necrotic tissue reduces the bacterial burden. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] Evaluation of a wound's potential for healing is a prime consideration in treatment choices. Determining whether a wound can heal is accomplished by evaluating whether the wound has an adequate blood supply. Checking for pulses, capillary refill and Doppler pressures are ways to determine if a blood supply is adequate. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] Debridement of an ischemic wound increases tissue loss and the risk of infection and is therefore contraindicated. An example of this is dry gangrene of the toes. Debriding or increasing moisture in ischemic lesions may convert dry gangrene to wet gangrene and hasten a life threatening infection. If a wound has an adequate blood supply, then debridement of necrotic tissue should be considered. Several methods of debridement are available. Sharp or surgical debridement is removal of necrotic tissue with scissors or a scalpel blade and forceps. This can be performed in a clinic setting or at the bedside. If indicated, premedication of the patient with topical, oral or parenteral pain medication reduces patient discomfort and facilitates adequate debridement. Sharp surgical debridement is the most rapid way to remove necrotic tissue and has been shown to be effective in treating diabetics with neuropathic foot ulcers. It has also been shown to be effective in treating difficult to heal venous ulcers when compared to no debridement. [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] Autolytic debridement occurs when the wound is covered with a dressing and the body's own proteolytic enzymes released by macrophages act to liquefy necrotic tissue. Hydrogels, films and hydrocolloids are some of the dressing types that facilitate autolytic debridement [fig_ref] Table 1: examples of dressing types [/fig_ref]. Use of these dressing must be carefully considered in the presence of an incompetent immune system. Wounds must be carefully cleansed prior to application of a new dressing. [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] The third method of debridement is with an enzymatic agent (EA). Collagenase remains the only EA still available in the United States. Dressings with EAs must be changed at least once a day. Wounds should be closely monitored, as the EA can sometimes damage healthy tissue at the margins of the dressing. [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] Mechanical debridement is achieved by use of the classic wet-to-dry dressing and whirlpool or hydrotherapy. Wet-to-dry dressings involve packing a wound with salinemoistened gauze and covered with a dry dressing. The dressing is allowed to dry and is then quickly removed along with adherent tissue. Unfortunately, it is nonselective and can remove vital tissue as well as necrotic tissue, may be very painful and may prolong healing time. This dressing technique is never indicated for use in clean, granulated wounds. Whirlpool therapy uses the action of fast-moving water to remove necrotic tissue. This strategy is rarely used, as it has not been shown to be very effective and may increase lower extremity edema. [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] Maggot therapy can also be used to debride some wounds. Sterilized greenbottle fly maggots are applied to the wound every 2-3 days. Maggots secrete enzymes into the wounds which act like proteases to degrade necrotic tissue. The larvae have the ability to digest bacteria and can help with heavily colonized wounds. Maggot therapy has been shown to decrease wound size. [bib_ref] Preparing the wound bed-debridement, bacterial balance, and moisture balance, Sibbald [/bib_ref] [bib_ref] Debridement: a vital component of wound bed preparation, Kirshen [/bib_ref] [bib_ref] Wound debridement: therapeutic options and care consideration, Beitz [/bib_ref]
## Moisture balance
In addition to bacterial burden and debridement of necrotic tissue, moisture balance must be considered when looking at the bed of a chronic wound. In the remote past it was considered best to have a dry wound to facilitate healing but more recent research has proven that healing is accelerated in a moist wound environment. A wound with too much moisture and drainage can damage surrounding tissue causing maceration. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] The components in the exudate of chronic wounds differ significantly from those of acute wounds. Acute wounds are rich in growth factors that help promote the generation of fibroblasts, epithelial cells and keratinocytes. In contrast, exudate in the chronic wound has higher concentrations of MMPs, proenzymes that tend to degrade the wound matrix that is optimal for wound healing. Hence for chronic wounds it is necessary to remove excessive exudate or moisture high in MMPs. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] The wound environment must maintain proper moisture balance. This can be done by increasing moisture to wounds that are too dry and decreasing the moisture of wounds that are too wet. Some of the newer generation of wound dressings are designed to do this. Research continues to work toward the development of the "ideal dressing" that can wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com Dovepress address the major concerns in wound care. In 1979, Turner described the ideal dressing as possessing the following characteristics:
- Removes excess exudates and toxins - Provides high humidity at the dressing wound interface - Allows for gaseous exchange - Provides thermal insulation - Protects against secondary infection - Remains free from particulate and toxic components - Causes no trauma on removal. [bib_ref] Hospital usage of absorbent dressings, Turner [/bib_ref] These characteristics are still pertinent today. Some of the many types of wound dressings will be discussed below.
## Dovepress
## Treatments or dressings that increase moisture transparent films
Film dressings exist in either adhesive or non-adhesive forms. They are made of either polyurethane or synthetic polymer sheets. They are frequently used to cover IV catheter sites and are sometimes used to help protect or heal partial thickness wounds. They have no ability to absorb excess moisture and cannot be used as a primary dressing for exudating wounds. They are best used for treating superficial, nonexudative wounds or during the late stages of wound healing. [bib_ref] A review of moisture-control dressings in wound care, Brett [/bib_ref] Combined with cross-hatching of eschar (cutting through the thick, dry tissue in a grid pattern) with a scalpel, covering the wound with a transparent film can quicken debridement. 28
## Hydrocolloids
Hydrocolloids have an inner hydrocolloid gelling agent that is made of carboxymethylcellulose (CMC) combined with pectin. This is sandwiched between an inner adhesive layer that rests on the wound surface and an outer coating of foam or a film. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] The hydrocolloid gel absorbs moisture when exposed to the wound exudate and forms a gel. As hydrocolloids absorb more moisture, they become more water permeable. This allows them to transmit moisture and manage wound exudate. Due to their hydrating properties with the mixture of the patient's own endogenous enzymes, hydrogel dressings are useful in autolytic debridement of the wound bed. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] When the dressings are removed they may leave some of the gelling agent behind which can have a foul odor and be confused with infection, therefore it is imperative to cleanse and assess the wound after removal of the dressing. These should be changed every 2-7 days as needed, indicated either by leaking of exudate, loosening or displacement of the dressing. Hydrocolloids are useful in treating venous ulcers, pressure ulcers and (if used judiciously) diabetic foot ulcers. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] They are relatively contraindicated with treating arterial insufficiency, vasculitis, and infection. [bib_ref] Wound debridement: therapeutic options and care consideration, Beitz [/bib_ref]
## Hydrogel dressings
Hydrogel sheet dressings are semi-occlusive and composed of a cross linked network of hydrophilic polymers. [bib_ref] The role of moisture balance in wound healing, Okan [/bib_ref] Semiocclusive dressings enhance autolytic debridement of dry to mildly exudative wounds. They are useful in burn patients because they have a soothing effect, but should not be used in ischemic ulcers. All hydrogel sheets are somewhat transparent and allow monitoring of the wound without removing the dressing. They should be changed every 24 to 72 hours. Wound treatment gels are either isotonic or hypertonic depending on whether the intent is to maintain a moist wound environment or to enhance debridement and are used with a secondary dressing.
## Treatment or dressings that reduce moisture calcium alginate dressings
Alginate dressings are made from kelp and consist of calcium alginate polysaccharides. They work through a sodium-calcium ion exchange between the wound exudate and the dressing. This produces a sodium-alginate gel with moisture retaining or absorbing properties making them useful for treating draining or infected wounds. The calcium ions released by these dressings also aid in the coagulation pathway. This type of dressing may be useful in bloody exudative wounds. Alginates high in mannuronic acid form a soft gel that partially dissolves in solutions containing sodium ions, such as wound exudate. Alginates with high concentrations of guluronic acid, retain their integrity and structure throughout the gelling process. [bib_ref] Understanding wound dressings: Alginate, Fletcher [/bib_ref] When removed from the wound, these dressings frequently have an odor and may give the appearance of an infected wound. It is important to cleanse the wound before assessing for the presence of infection. Alginates are useful in wounds with moderate to heavy exudates. The concomitant use of topical antibiotics or other antimicrobial agents may decrease absorption of fluids. [bib_ref] Therapeutic behavior of a hydrocolloid dressing. Its evolution in the treatment of..., Gallego [/bib_ref]
## Foam
Foam dressings have an absorbent porous hydrocellular polyurethane center which is laminated with a semi-occlusive outer layer. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] Foam dressings are configured with different outer layers that have varying moisture vapor transmission rates. Dressings that have higher moisture vapor transmission rate allow moisture to be transmitted through the dressing and evaporated. This property makes these dressings useful in highly exudative wounds. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref] Foam dressings may be either adherent or nonadherent and come in a variety of shapes and absorbency. They have different degrees of flexibility and density and many can be shaped to fit wounds of any size and depth. The opacity of the dressing prevents inspection of the wound without removal.
wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com
## Dovepress
## Hydrofiber
Hydrofiber dressings are available in sheets or ropes. They are composed of highly absorbent sodium CMC. They are indicated for wounds that have moderate to heavy exudate. Because they are nonadherent, they require a secondary dressing to keep them in place. Hydrofiber dressings are useful in the treatment of draining chronic venous ulcers. Both foam and hydrofiber dressings have wear times of up to one week. [bib_ref] Wound bed preparation: a systemic approach to wound management, Schultz [/bib_ref]
## Composite dressings
Composite dressings are comprised of multiple layers with varying absorbent properties. They are useful in exudate control and as a secondary dressing for foams, alginates, and hydrofibers. They can be used under a compression dressing alone or in combination with other dressings have a wear time of up to one week. [bib_ref] Pathophysiology of chronic nonhealing wounds, Medina [/bib_ref]
## Synthetic skin grafts
Synthetic skin grafts, also called skin substitutes, were developed to avoid the morbidity associated with full thickness skin grafts and to decrease time required for wound healing. [bib_ref] The edge effect: current therapeutic options to advance the wound edge, Woo [/bib_ref] Several products (Alloderm, Integra Neurosciences, Plainsboro, NJ, USA; Integra, Integra Life Sciences, Plainsboro, NJ, USA; Apligraf, Novartis, Basel, Switzerland; Dermagraft, Advanced BioHealing, LaJolla, CA, USA) are on the market and are bioengineered from cells derived from different sources. [bib_ref] The edge effect: current therapeutic options to advance the wound edge, Woo [/bib_ref] Synthetic skin grafts have been shown to increase the likelihood of healing of venous ulcers when compared to conventional dressings. 31
## Negative pressure wound therapy
Negative pressure wound therapy (NPWT) consists of applying a foam or gauze dressing over the surface of the wound. It is secured with a transparent film to form an airtight seal. Continuous or intermittent negative pressure is applied. Wound healing is accelerated by removing excess interstitial fluid, improving blood flow reducing bacterial count, stimulating angiogenesis and increasing tissue perfusion. [bib_ref] The edge effect: current therapeutic options to advance the wound edge, Woo [/bib_ref]
# Conclusion
The aging population presents many challenges to the medical community in the provision of care; among them is wounds care. Although it is a complex subject, basic understanding of the healing process, the most common types of ulcers and some of the dressing options available is necessary in caring for these patients. A multidisciplinary approach maximizes wound care in the elderly by drawing on each team member's expertise. The majority of elderly patients with ulcers heal if treated assertively and appropriately. Healing is not possible in many instances. A holistic approach to wound care is paramount. Symptom management, whether healing or palliation is the goal, will improve overall outcomes for the elderly patient. The reward of providing wound care is in assisting a person to an enhanced health-related quality of life.
## Case studies
To illustrate the decision-making process that is involved in care related to specific types of wounds, consider the following case studies:
## Case study dm: venous stasis ulcers
Mr. DM was a morbidly obese, 66-year-old man when admitted to the hospital with left lower extremity cellulitis and venous ulcers. Pertinent diagnoses: Type II diabetes mellitus, vitamin D deficiency, sleep apnea, history of congestive heart failure, hypertension and hyperlipidemia. Of note, his glycosylated hemoglobin (HbA1c) was 6.9, albumin was 3.0 (normal 3.5-5.2) and his white blood cell (WBC) count 11,600. Weight was 274.2 pounds (124.4 Kg), height 73 inches (160.6 cm), with a body mass index (BMI) of 36.25.
He was given furosemide 40 mg IV on day one, 40 mg IV bid day two, and another 40 mg IV on day three. He was also started on sulfamethoxazole 800/trimethoprim 180 DS bid. Profore 4-layer compression dressings were applied to bilateral lower extremities for 4+ interstitial edema. Followup care in the wound clinic after discharge from the hospital involved debridement of crusting lesions and Unna boot wrap with compression. Within two weeks, the cellulitis had resolved and the edema was reduced.
Compression stockings (knee high 20-30 mm Hg) were obtained and donned. The following week, his cellulitis had exacerbated. Keflex and clindamycin were given with complete resolution of the infection. He experienced continued edema with use of compression stockings so compression wraps were again initiated and he was measured for CircAids. When the CircAids were received, he was converted to these devices and did well. Edema was controlled and he has had no recurrence of ulcers [fig_ref] Figure 2: Mr [/fig_ref]. Initially, all lesions were cleansed with normal saline. Silver alginate was applied to wound bases of Stage III and IV ulcers, covered with composite absorbent dressings (ABDs) and secured with hypoallergenic tape. The silver alginate was intended to decrease bacterial load and absorb exudate and the composite dressings for exudate control. Nonadhering and composite dressings were applied to left foot and secured with roller gauze. The nonadhering dressing was to prevent the dressing from sticking to the wounds and the composite dressings for exudate control. A hydrocolloid dressing was applied to the right thigh ulcer to provide moist wound healing and a bacterial barrier. Pressure-relieving boots were ordered to enhance healing and prevent further damage to feet.
## Case study al: pressure ulcers
An aggressive plan consisting of a turning and positioning schedule, wound care nutritional support, physical therapy for positioning assistance and occupational therapy for contracture mobilization was instituted. He was placed on a low air loss mattress for pressure redistribution. He had a Foley catheter to gravity drainage for urinary retention due to neurogenic bladder. Care was complicated by fecal incontinence.
Changes were made in the dressing procedures: Collagenase (Santyl) was applied daily to debride left hip ulcers and a composite dressing cover that was secured with hypoallergenic tape. A hydrocolloid dressing over silver alginate was applied to the left hip and coccyx lesions to prevent fecal contamination of wounds, control exudate and provide for moist wound healing. Double layer foam dressings were applied to the bilateral left ankle lesions which consisted of an intact dressing to cover the ulcer for exudate control and to protect the periwound skin, and second layer with hole in center to aid in pressure redistribution. These dressings were secured with roller gauze.
Mr. AL developed contact dermatitis from the foam dressings. Triamcinolone 0.1% and hydrophilic ointment were ordered and applied every day. The ulcers were healed, no dressings required, use of pressure reducing boots for left foot was continued. His rash resolved.
## Case study wc: diabetic foot ulcers
Mr WC was a 64-year old man status post cerebral vascular accidents × 5 with residual right hemiparesis and myocardial infarction × 2. Other pertinent diagnoses include: Type II diabetes.
He developed bilateral posterior heel pressure ulcers and was initially seen in the Podiatry clinic where debridement was performed. Care was coordinated between the Wound Care Clinic, Podiatry and a Home Health Agency Registered Nurse.
The left heel ulcer (Stage III) was 50% yellow slough and 50% granulation tissue. The lesion was cleansed with normal saline, a silver hydrogel was applied and a foam dressing with a moisture barrier as a secondary dressing and secured with roller gauze. The right heel ulcer was larger and probed to bone (Stage IV). The wound base was 50% yellow slough and 50% granulation tissue. There was a border of hyperkeratotic tissue along the plantar surface. The lesion was cleansed with a solution of 5cc betadine solution in 500 cc saline and rinsed with normal saline (NS). Kovia ointment (an enzymatic debriding agent) was applied to slough, covered with a saline-moistened gauze and then a dry sterile dressing. Heel protective boots were obtained and donned bilaterally. The patient was provided with supplies for continued treatment by his home health nurse. wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com
## Dovepress
Mr WC had not been wearing the protective devices at night and the ulcers worsened. The right heel ulcer measured 6.5 cm × 5.5 cm with large amount yellow slough in center of the wound. Conservative sharp debridement was completed to decrease necrotic tissue and reduce bioburden. The left heel ulcer was clean, the wound base, a combination of slough and granulation tissue, measured 1.2 cm × 1.7 cm. The lesions were irrigated with NS and the initial dressing options were continued. At this time it was decided to begin negative pressure wound therapy to the right heel ulcer.
A VAC Freedom (Kinetic Concepts, Inc.) was applied with the unit set at 125 mmHg continuous negative pressure. In two weeks, the right heel ulcer measured 6.0 cm × 5.0 cm, the wound base was 80% granulation and 20% slough. The left heel ulcer measured 1.0 cm in diameter. Two weeks later, the right heel ulcer measured 5.5 cm × 4.5 cm × 1.0 cm, did not probe to bone and the wound base was fully granulated.
The VAC dressing was discontinued and a foam dressing was applied with a light compression wrap to secure it. The foam dressing provided absorption and cushioning of the wound, compression controlled edema.
With the improvement in the wound, the decision was made to apply Dermagraft dressings to facilitate wound closure. Dermagraft was applied once a week for five weeks with continued attention to the control of exudate and edema. The ulcer on the left heel progressed to complete closure with conservative dressing applications and continued pressure relief. The right heel also progressed to complete closure with application of Dermagraft combined with other conservative dressing techniques to control for infection through use of topical antimicrobials (silver alginates, Iodosorb gel), exudate management (foam and composite dressings) and edema reduction with light compression wraps. Knee high compression stockings (20-30 mmHg) were used after healing was complete [fig_ref] Figure 2: Mr [/fig_ref]. Clinical Interventions in Aging is an international, peer-reviewed journal focusing on evidence-based reports on the value or lack thereof of treatments intended to prevent or delay the onset of maladaptive correlates of aging in human beings. This journal is indexed on PubMed Central, MedLine, the American Chemical Society's 'Chemical Abstracts Service' (CAS), Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
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wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com
## Dovepress
## Dovepress
# Disclosure
The authors report no conflicts of interest in this work.
[fig] Figure 2: Mr. AL was a 67-year-old man admitted to the Nursing Home Care Unit because of adult failure to thrive, malnutrition, depression, and multiple pressure ulcers. He was s/p CVA with left hemi paresis and contractures of both upper and lower extremities. His weight was 105 pounds (47.6 Kg),Gist et al Dovepress submit your manuscript | www.dovepress.com Dovepress Anterior left lower leg, venous hypertension ulcer. [/fig]
[fig] Figure 3: Posterior left lower leg, venous hypertension ulcer. [/fig]
[fig] Figure 5, Figure 6: Posterior right thigh, Stage III. wound care in the geriatric client Dovepress submit your manuscript | www.dovepress.com Dovepress Coccyx, Stage Iv. [/fig]
[fig] Figure 7, Figure 8: Left hip, Stage Iv; unstageable eschar covered. Gist et al Dovepress submit your manuscript | www.dovepress.com Dovepress Lateral left ankle, Stage Iv; lateral mid-foot, unstageable. [/fig]
[fig] Figure 9: Full body view, multiple ulcers. [/fig]
[fig] Figure 10: right posterior heel, Stage Iv, with slough and eschar. Gist et al Dovepress submit your manuscript | www.dovepress.com Dovepress Figure 11 right posterior heel with granulation tissue. [/fig]
[fig] Figure 12: right posterior heel almost healed. Clinical Interventions in Aging 2009:4 Clinical Interventions in Aging Publish your work in this journal Submit your manuscript here: http://www.dovepress.com/clinical-interventions-in-aging-journal [/fig]
[table] Table 1: examples of dressing types [/table]
[table] Table 2: Mr. AL's pressure ulcer submit your manuscript | www.dovepress.comFigure 4 Left foot, medical hallux, Stage III; Medial first metatarsal head, Stage II; Medial mid-foot, Stage II; Medial malleolus, Stage IV. [/table]
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Novel lead structures with both Plasmodium falciparum gametocytocidal and asexual blood stage activity identified from high throughput compound screening
Background: Blocking malaria transmission is an important step in eradicating malaria. In the field, transmission requires the production of sexual stage Plasmodium parasites, called gametocytes, which are not effectively killed by the commonly used anti-malarials allowing individuals to remain infectious after clearance of asexual parasites.Methods:To identify new gametocytocidal compounds, a library of 45,056 compounds with diverse structures was screened using a high throughput gametocyte viability assay. The characteristics of active hits were further evaluated against asexual stage parasites in a growth inhibition assay. Their cytotoxicity were tested against mammalian cells in a cytotoxicity assay. The chemical scaffold similarity of active hits were studied using scaffold cluster analysis.Results:A set of 23 compounds were identified and further confirmed for their activity against gametocytes. All the 23 confirmed compounds possess dual-activities against both gametocytes responsible for human to mosquito transmission and asexual parasites that cause the clinical symptoms. Three of these compounds were fourfold more active against gametocytes than asexual parasites. Further cheminformatic analysis revealed three sets of novel scaffolds, including highly selective 4-1H-pyrazol-5-yl piperidine analogs.Conclusions:This study revealed important new structural scaffolds that can be used as starting points for dual activity anti-malarial drug development.
# Background
Malaria remains a major health problem in underdeveloped countries. In the past decade, mortality rates have dropped significantly owing to the combined efforts of insecticidal bed nets and artemisinin-based combination therapy (ACT) [bib_ref] Malaria medicines: a glass half full?, Wells [/bib_ref] [bib_ref] Control of malaria, Kar [/bib_ref] , but 214 million estimated malaria cases still occurred worldwide in 2015. The success of ACT has renewed hopes and provided a unique opportunity for researchers to consider new approaches to eliminate malaria [bib_ref] Targeting Plasmodium PI(4)K to eliminate malaria, Mcnamara [/bib_ref] [bib_ref] The open access malaria box: a drug discovery catalyst for neglected diseases, Spangenberg [/bib_ref]. Due to the complex life cycle of parasites, currently no single strategy effectively treats human disease and controls parasite transmission. Malaria vaccine development also continues to be a challenge. RTS, S, the only malaria vaccine approved for use outside of trials, has low efficacy (26-50%) in young children and is being evaluated by the World Health Organization (WHO) [bib_ref] Efficacy and safety of RTS, S/AS01 malaria vaccine with or without a..., Rts [/bib_ref] , who recently announced a pilot roll-out of the vaccine in three African countries.
One of the major hurdles to malaria elimination is the lack of effective agents to prevent and control malaria transmission from mosquito to human. The malaria life cycle requires a mosquito vector ingest sexual stage parasites, called gametocytes, during a human blood meal. In the human host, Plasmodium falciparum gametocytes develop through five stages (I-V) over 10-12 days after RBC invasion by a merozoite committed to sexual differentiation. The mature stage V gametocytes then circulate in the peripheral blood for 4-6 days. Once taken up in a blood meal by a mosquito, male and female stage V gametocytes are stimulated to undergo fertilization and form oocysts on the basal surface of the midgut. The infectious form of the parasite, sporozoites, are formed in the oocysts and after maturation they are released and migrate to the salary glands. During a subsequent blood meal the sporozoites are transmitted to humans with the saliva. Most of the current drug development efforts have been devoted to controlling the asexual parasites that are responsible for the disease symptoms in patients. Currently, the only drugs that are active against gametocytes and can block malaria transmission are 8-aminoquinolines such as primaquine. However, 8-aminoquinolines can cause haemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a highly prevalent genetic condition in malaria-endemic regions [bib_ref] G6PD deficiency: global distribution, genetic variants and primaquine therapy, Howes [/bib_ref]. To date, only a few drug candidates in preclinical or clinical stages have the potential to block malaria transmissions [bib_ref] Malaria medicines: a glass half full?, Wells [/bib_ref] [bib_ref] Antimalarial compounds in Phase II clinical development, Held [/bib_ref]. This deficit is partly due to difficulty in producing P. falciparum gametocytes in culture, a process that takes at least 12-14 days with very limited yield [bib_ref] Malaria, sexual development and transmission: retrospect and prospect, Sinden [/bib_ref]. This hurdle limits the capacity of malaria gametocytes for compound screening even with the recent development of several high throughput assays [bib_ref] High-throughput assay and discovery of small molecules that interrupt malaria transmission, Plouffe [/bib_ref] [bib_ref] A combination of new screening assays for prioritization of transmission-blocking antimalarials reveals..., Bolscher [/bib_ref] [bib_ref] A quantitative high throughput assay for identifying gametocytocidal compounds, Tanaka [/bib_ref] [bib_ref] A simple and predictive phenotypic high content Imaging assay for Plasmodium falciparum..., Lucantoni [/bib_ref] [bib_ref] Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking..., Delves [/bib_ref]. Consequently, only limited compound collections have been screened, including two screens of FDA approved drugs collections [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref] [bib_ref] A male and female gametocyte functional viability assay to identify biologically relevant..., Ruecker [/bib_ref] , several screens of MMV Malaria Box library [bib_ref] Open source drug discovery with the malaria box compound collection for neglected..., Van Voorhis [/bib_ref] , and additionally, three relatively large scale screens of ~10,000 molecules [bib_ref] High-throughput assay and discovery of small molecules that interrupt malaria transmission, Plouffe [/bib_ref] [bib_ref] Open source drug discovery with the malaria box compound collection for neglected..., Van Voorhis [/bib_ref] [bib_ref] A new set of chemical starting points with Plasmodium falciparum transmission-blocking potential..., Almela [/bib_ref]. These initial screens are a good start, but additional novel lead compounds with dual-activities against both gametocytes and asexual parasites are highly needed.
The emergence of anti-malarial resistance is of substantial concern to the malaria community [bib_ref] Antimalarial drug resistance: literature review and activities and findings of the ICEMR..., Cui [/bib_ref]. Recently, artemisinin resistance in P. falciparum has spread in Greater Mekong subregion [bib_ref] The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong Subregion: a..., Imwong [/bib_ref]. Current and future drug screens using new chemical entities with novel modes of action, instead of analogs of the previous anti-malarials, will have a better opportunity to address the drug-resistance [bib_ref] Diversity-oriented synthesis yields novel multistage antimalarial inhibitors, Kato [/bib_ref] [bib_ref] A novel multiple-stage antimalarial agent that inhibits protein synthesis, Baragana [/bib_ref]. In the previous studies, a 1536-well high throughput gametocyte viability assay was developed [bib_ref] A quantitative high throughput assay for identifying gametocytocidal compounds, Tanaka [/bib_ref] [bib_ref] A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue, Tanaka [/bib_ref] and used for screening of several known compound libraries, including 1280 compounds in the LOPAC library [bib_ref] A quantitative high throughput assay for identifying gametocytocidal compounds, Tanaka [/bib_ref] , 4265 approved drugs and 400 from the MMV Malaria Box library [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref]. Here, this study reports the results of high throughput screening of 45,056 compounds with diverse structures with the gametocyte viability assay. The results revealed 3 groups of novel structures that actively suppress both gametocytes and asexual parasites.
# Methods
## Cell culture
Asexual parasites of P. falciparum strain NF54 were cultured as described previously [bib_ref] Human malaria parasites in continuous culture, Trager [/bib_ref]. Briefly, parasites were maintained in RPMI 1640 medium containing 10% positive human serum + erythrocytes (5% haematocrit), 2.5% sodium bicarbonate and 11 µg/mL gentamicin at 37 °C with 5% CO 2 , 5% O 2 and 90% N 2 . Gametocyte cultures were set up at 0.1% parasitaemia and on days 9-10 treated with 50 mM N-acetylglucosamine (NAG) to block further asexual growth. Stage III-V gametocytes were isolated by Percoll density gradient centrifugation on day 12 and returned to culture for 24 h before being used in the assay [bib_ref] A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue, Tanaka [/bib_ref]. At the time of the assay over 73% of the gametocytes were stage IV or V (Additional file 1). HepG2 cells (ATCC, cat. no. 77400) were cultured in 175-cm 2 tissue culture flasks with 30 ml growth medium at 37 °C in a 5% CO 2 and 5% O 2 humidified atmosphere. Growth medium was made with Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS). Growth medium was replaced every other day and cells were passaged at 75% confluence.
## Compound library and gametocyte assay screen
Compounds from the Sytravon library (a retired Pharma screening collection that contains a diversity of novel small molecules, with an emphasis on medicinal chemistry-tractable scaffolds) were obtained as powder samples and dissolved in DMSO as 400 and 80 µM stock solutions. Compound screening experiments were performed as previously described [bib_ref] A quantitative high throughput assay for identifying gametocytocidal compounds, Tanaka [/bib_ref] [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref]. In the primary screen, two concentrations for each compound were tested. No technical and biological replicates were involved. In the follow-up confirmation studies, three biological replicates were tested for each compound in both parasite assays. The positive control was 46 µM of Torin 2 and negative control was DMSO. Briefly, 2.5 μL/ well incomplete medium was dispensed into each well of 1536-well plates using the Multidrop Combi followed by 23 nL compound transferring using the NX-TR Pintool (WAKO Scientific Solutions, San Diego, CA). Then, 2.5 μL/well of gametocytes was dispensed with a seeding density of 20,000 cells/well using the Multidrop Combi. The assay plates were incubated for 72 h at 37 °C with 5% CO 2 . After addition of 5 μL/well of 2X AlamarBlue dye (Life Technologies, cat. no. DAL1100), the plates were incubated for 24 h at 37 °C with 5% CO 2 and then were read in a fluorescence detection mode (Ex = 525 nm, Em = 598 nm) on a ViewLux plate reader (PerkinElmer).
## Asexual stage parasites drug activity assay
Drug activity on asexual stage parasites was tested using a SYBR Green assay as described previously [bib_ref] A class of tricyclic compounds blocking malaria parasite oocyst development and transmission, Eastman [/bib_ref]. Briefly, 2.5 μL/well complete culture medium was dispensed into each well of 1536-well plates using the Multidrop Combi followed by 23 nL compound transferring using the NX-TR Pintool. Parasites were diluted to 0.5% parasitaemia in complete culture medium with 2% haematocrit and drugs diluted in DMSO (≤0.5%). The pre-diluted parasites were dispensed into a 1536-well plate (2.5 μL/ well). After 72 h incubation under the standard culture conditions, 5 μL/well of lysis buffer containing SYBR Green I was added to the parasite culture and incubated for 30 min at room temperature. The fluorescence of each well was measured in a fluorescence detection mode (Ex = 490 nm, Em = 520 nm) using a ViewLux plate reader (PerkinElmer).
## Human cell line cytotoxicity assay
Drug activity on HepG2 was tested using an AlamarBlue assay as previously described [bib_ref] Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on..., Rudzok [/bib_ref]. Briefly, 5 μL/well of HepG2 cells were dispensed with a seeding density of 1000 cells/well using the Multidrop Combi. 23 nL compound was transferred using the NX-TR Pintool. After 72 h incubation under the standard culture condition, 5 μL/well of 2X AlamarBlue dye was added to the cells, the plates were incubated for 2 h at 37 °C with 5% CO 2 and then were read in a fluorescence detection mode (Ex = 525 nm, Em = 598 nm) on a ViewLux plate reader.
# Data analysis
The primary screen data was analysed using customized software developed internally [bib_ref] A grid algorithm for high throughput fitting of dose-response curve data, Wang [/bib_ref]. IC 50 values were calculated using the Prism software (Graphpad Software, Inc. San Diego, CA). The Z' factors of the five HTS screens were 0.46, 0.38, 0.53, 0.55, and 0.62. The Z' factors of asexual stage parasites and cytotoxicity assays were >0.5. The scaffold cluster analysis was performed based on the chemical scaffold similarity of 32 hits. Hit molecules with similar core structures were grouped into a cluster. Known anti-malarial scaffolds were confirmed in both the asexual and the gametocyte assays.
# Results
## High throughput screening with a gametocyte viability assay identified 32 primary hits
A total of 45,056 compounds were screened against enriched stage III-V gametocytes using the previously developed high throughput viability assay [bib_ref] A quantitative high throughput assay for identifying gametocytocidal compounds, Tanaka [/bib_ref] [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref] [bib_ref] A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue, Tanaka [/bib_ref] [fig_ref] Figure 1: Flowchart of malaria gametocyte viability screens and compound confirmation in gametocyte, asexual... [/fig_ref]. Due to the intrinsic difficulty in producing a large amount of gametocytes, the primary screen was split into five campaigns. To identify compounds with activities in the low micromolar range, each compound at 400 µM (final concentration was 1.84 µM) and 80 µM (final concentration was 0.368 µM) were added to the gametocytes in singlet in the primary screen. These concentrations were selected because the available stock concentrations in the compound library. A set of 128 primary hits that had >50% gametocytocidal activity at either compound concentration were selected for validation in the same gametocyte assays using an 11 point concentration titration ranging from 0.001 to 46 µM. The activity of 32 of these compounds was confirmed and 23 of the 32 had an IC 50 less than 10 µM with >75% maximum activity [fig_ref] Table 1: Cluster and activity of 32 compounds against Plasmodium falciparum NF54 gametocytes, asexual... [/fig_ref] ; [fig_ref] Figure 2: Structures and activities of 10 selected anti-gametocyte compounds [/fig_ref].
## Activities of confirmed compounds against asexual parasites
In order to identify the compounds active against both gametocytes and asexual parasites, the activity of the 32 confirmed gametocytocidal compounds was determined using a parasite growth inhibition assay [bib_ref] Thousands of chemical starting points for antimalarial lead identification, Gamo [/bib_ref]. Among the compounds tested, 27 of them inhibited the growth of asexual parasites with IC 50 s less than 10 µM and maximum responses >75% [fig_ref] Table 1: Cluster and activity of 32 compounds against Plasmodium falciparum NF54 gametocytes, asexual... [/fig_ref] ; [fig_ref] Figure 2: Structures and activities of 10 selected anti-gametocyte compounds [/fig_ref]. The remaining five compounds showed IC 50 s higher than 10 µM [fig_ref] Table 1: Cluster and activity of 32 compounds against Plasmodium falciparum NF54 gametocytes, asexual... [/fig_ref]. Three compounds (NCGC00134126, NCGC00134124, and NCGC00110901) showed slightly lower IC 50 s in the gametocyte viability assay than that in the asexual parasite growth assay (ratio of IC 50 s = ~fourfold) and 9 compounds (NCGC00134795, NCGC00100599, NCGC00101506, NCGC00141020, NCGC00100597, NCGC00127015, NCGC00126987, NCGC00104044, and NCGC00140326) showed higher IC 50 s in the gametocyte viability assay than that in the asexual parasite growth assay (ratio of IC 50 s from 3.21 to 17.5-fold). The other 20 compounds showed similar activity (ratio of IC 50 s within threefold). Among these confirmed hits, 16 compounds decreased gametocyte viability to <10% in the preparations of late stage gametocytes, including the 3 molecules with an IC 50 gametocyte/asexual ratio of ~4.
## Cytotoxicity of confirmed compounds in hepg2 cells
To eliminate the general cytotoxic compounds from the confirmed compounds, the cytotoxic effect of these compounds was determined in mammalian HepG2 cells with an AlamarBlue assay using an 11-concentration titration from 0.001 to 46 µM. Of the 32 compounds tested 28, including the 3 compounds that preferentially targeted gametocytes, were not cytotoxic at the highest compound concentration of 46 µM in the HepG2 cells. The remaining four compounds showed weak cytotoxicity with respect to HepG2 cells: NCGC00127017 (IC 50 = 25.32 ± 5.41 µM, maximum response at 87.6%), NCGC00127015 (38.39 ± 0.48 µM, maximum response at 87.2%), NCGC00126987 (38.6 ± 0.42 µM, maximum response at 86.1%), and NCGC00126892 (34.37 ± 5.36 µM, maximum response at 93.0%).
## Three novel structural clusters identified from confirmed active compounds
The chemical structures of the confirmed compounds were then analysed based on their chemical similarity and found 10 diverse clusters [fig_ref] Table 1: Cluster and activity of 32 compounds against Plasmodium falciparum NF54 gametocytes, asexual... [/fig_ref]. Several structural clusters are known anti-malarial scaffolds including 2,4-diaminopyrimidines (for example, NCGC00134128, pyrimethamine-like), 4H-chromen-4-ones (for example, NCGC00100599, MMV665820-like) [bib_ref] The open access malaria box: a drug discovery catalyst for neglected diseases, Spangenberg [/bib_ref] , and 8-quinolinols (for example, MMV000788-like). 4H-chromen-4ones are more active against sexual parasites than against asexual parasites. Notably, three novel clusters containing 3-amino-imidazo[1,2-a]pyridines (for example, NCGC00104490), 3H-imidazo[4,5-b]pyridines (NCGC00134795), and 4-1H-pyrazol-5-yl piperidines (for example, NCGC00127017) were active against both asexual and sexual parasites. To the best of our knowledge, these new scaffolds have not been reported as anti-malarial agents before.
## Human kinase profiling of the two most potent hits
To avoid compounds that also inhibit human kinases, as has been a problem for some previous anti-malarial drug candidates [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref] [bib_ref] Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous..., Hanson [/bib_ref] , the binding affinities to 468 human kinases were tested for the two most potent compounds identified in this screen. NCGC00100599 is the most potent compound against asexual parasites with an IC 50 of 0.339 µM and a gametocytocidal IC 50 of 4.18 µM. The other compound NCGC00114940 was similarly effective against gametocytes (IC 50 = 0.620 µM) and asexual parasites (IC 50 of 1.16 µM). At 10 µM, NCGC00100599 had relatively low affinity inhibitory activity against 13 out of 468 human kinases [fig_ref] Figure 2: Structures and activities of 10 selected anti-gametocyte compounds [/fig_ref] , whereas NCGC00114940 did not affect the activity of any of the 468 human kinases .
# Discussion
Ideally, the next generation of anti-malarial drug combinations will treat clinical symptoms and eliminate malaria transmission [bib_ref] Designing the next generation of medicines for malaria control and eradication, Burrows [/bib_ref]. This target profile will require new Primary Screen in gametocytes stage III-V small molecules with efficacy against gametocytes with dual activity against asexual blood stages. In this work 45,056 diverse compounds were screened for gametocytocidal activity. In the hit confirmation experiments, the activities of primary screening hits were determined in both gametocyte assay and asexual parasite assay. Three compounds were identified that were slightly more active against gametocytes than asexual parasites and an additional twenty compounds had dual-efficacy against both gametocytes and asexual parasites with similar level of potencies. The consistent strong gametocytocidal activity against preparations of late stage gametocytes containing 30-60% stage V gametocytes in the experiments strongly suggests the efficacy against the mature stages directly responsible for transmission. To precisely define the relative activity against male and female gametocytes, additional analysis is needed [bib_ref] A male and female gametocyte functional viability assay to identify biologically relevant..., Ruecker [/bib_ref].
The structural analysis of the 23 confirmed compounds found they could be divided into 10 scaffold clusters, three of which were not previously associated with anti-malarial compounds or to any known anti-malarial scaffolds. Within these three new scaffolds, 3-aminoimidazo[1,2-a]pyridines were reported as PGHS-2 inhibitors with analgesic and anti-inflammatory activities [bib_ref] Discovery of novel analgesic and anti-inflammatory 3-arylamineimidazo[1,2-a]pyridine symbiotic prototypes, Lacerda [/bib_ref]. 3H-imidazo pyridines were inhibitors of luciferases used as reporter enzymes [bib_ref] Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays, Ho [/bib_ref]. Interestingly, 4-1H-pyrazol-5-yl piperidines have been classified as 'dark chemical matter'(DCM) recently [bib_ref] Dark chemical matter as a promising starting point for drug lead discovery, Wassermann [/bib_ref]. The term DCM is used to refer to a collection of small molecules which have not shown any biological activity although these molecules have been extensively tested in a variety of high throughput assays. Hit molecules coming from DCM may provide high selectivity and clean safety profiles with minimum off-target toxicity [bib_ref] Virtual screening targeting the urokinase receptor, biochemical and cell-based studies, synthesis, pharmacokinetic..., Wang [/bib_ref]. Due to the intrinsic Drug safety is critical for development of the next generation malaria agents [bib_ref] The open access malaria box: a drug discovery catalyst for neglected diseases, Spangenberg [/bib_ref]. The ideal compounds for development should have selectivity against targets in Human kinase profiling of NCGC00114940 and NCGC00100599. 10 µM NCGC00114940 (a) and 10 µM NCGC00100599 (b) were tested against 468 human kinases for binding activities, respectively. NCGC00114940 did not interact with any tested human kinases. NCGC00100599 may interact with 16 human kinases. The top three targets are ABL1 (Q252H)-phosphorylated (9.9% of control), PIK3CD (10% of control), and CDK4-cyclin D3 (15% of control) Plasmodium falciparum parasites without significant cytotoxicity to mammalian cells. Inhibition of a few of human kinases, such as PI4KB [bib_ref] Targeting Plasmodium PI(4)K to eliminate malaria, Mcnamara [/bib_ref] , mTOR [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref] , and PI3K [bib_ref] High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations, Mott [/bib_ref] by several previous anti-malarial compounds has been reported [bib_ref] Chemical signatures and new drug targets for gametocytocidal drug development, Sun [/bib_ref] and will need to be avoided for the new generation of anti-malarial drugs. In the lead optimization studies, these activities against human kinases need to be removed, while the anti-malarial activity remains. The most potent gametocytocidal compound NCGC00100599 found in this study did not bind to any of 468 human kinases tested at 10 µM compound concentration. These 468 human kinases include AGC, CAMK, CMGC, CK1, STE, TK, TKL, lipid and atypical kinase families, plus important mutant forms [bib_ref] A quantitative analysis of kinase inhibitor selectivity, Karaman [/bib_ref] [bib_ref] Extending kinome coverage by analysis of kinase inhibitor broad profiling data, Jacoby [/bib_ref]. The kinase profiling results suggest that the lead compound has no relative impact on human kinase targets.
# Conclusions
In summary, a set of novel compounds with dual-efficacy against both gametocytes and asexual parasites were identified from a high throughput screening of 45,056 compounds in the gametocyte viability assay. The structural cluster analysis identified three novel classes of structures in the confirmed compounds. The 4-1H-pyrazol-5-yl piperidine analogs were highly selective against malaria parasites without activities in the 468 human kinases tested. These confirmed compounds provide potential starting points for future anti-malarial drug development.
[fig] Figure 1: Flowchart of malaria gametocyte viability screens and compound confirmation in gametocyte, asexual parasites and mammalian cell assays. a The primary screens of the Sytravon libraries were carried out in malaria P. falciparum gametocyte viability assays. A group of 128 hits from the malaria gametocyte screen were selected for confirmation in the same assays. Further, 32 hits were picked for inhibition of asexual parasites growth and tested for cytotoxicity in the mammalian HepG2 cell line. b Venn-diagram of confirmed hits with activities against P. falciparum gametocytes, P. falciparum asexual parasites, and the mammalian HepG2 cells. NCGC00104528 was clustered as active against both gametocytes and asexual parasites in Venn diagram (IC 50 of 8.91 µM in gametocyte viability assay and IC 50 of 13.9 µM in asexual parasite growth assays) [/fig]
[fig] Figure 2: Structures and activities of 10 selected anti-gametocyte compounds. Structures and concentration-response curves of selected lead compounds (NCGC00114940, NCGC00134134, NCGC00134128, NCGC00134154, NCGC00134126, NCGC00134132, NCGC00104490, NCGC00134130, NCGC00134124, and NCGC00106780) determined in the gametocyte viability assay (green), asexual parasite growth assay (blue) and HepG2 cytotoxicity assay (red). Cluster names are included. Data are presented as mean ± SD with n = 3 independent experiments DCM characteristics of 4-1H-pyrazol-5-yl piperidine analogs, they could serve as a valuable starting point for the medicinal chemistry campaign for drug development. [/fig]
[table] Table 1: Cluster and activity of 32 compounds against Plasmodium falciparum NF54 gametocytes, asexual parasites and cytotoxicity of these compounds against HepG2 [/table]
[table] Table 2: Kinome scan profile of NCGC00100599NCGC00100599 was profiled at 10 μM against a diverse panel of 468 kinases by DiscoverX [/table]
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Current approach in the diagnosis and management of posterior uveitis
Posterior uveitic entities are varied entities that are infective or non-infective in etiology. They can aff ect the adjacent structures such as the retina, vitreous, optic nerve head and retinal blood vessels. Thorough clinical evaluation gives a clue to the diagnosis while ancillary investigations and laboratory tests assist in confi rming the diagnosis. Newer evolving techniques in the investigations and management have increased the diagnostic yield. In case of diagnostic dilemma, intraocular fl uid evaluation for polymerase chain testing for the genome and antibody testing against the causative agent provide greater diagnostic ability.
A thorough diagnostic work-up directed by the history of presenting complaints, patient's symptoms and signs, and clinical examination is mandatory. Ancillary investigations such as fundus fl uorescein angiography (FFA), indocyanine green angiography (ICG), ultrasonography (USG), optical coherence tomography (OCT), and selective laboratory investigations help in confi rming the diagnosis. It is of paramount importance to identify the possible etiology as posterior uveitis can be infective or non-infective. Newer diagnostic modalities have led to early recognition of the condition and its possible etiology, which has a positive infl uence on the management of the disease. This article gives an overview of the current approach in the diagnosis and management of common posterior uveitic entities found in India.
## Clinical approach to a patient of posterior uveitis
History: Thorough history is a critically important step in evaluating any patient with uveitis. It allows the clinician to gain critical evidence regarding general medical history, travel history, social history, associated medications and family history. These oft en provide critically useful information.
Critical questions for diagnosis: Answers to the following set of questions paves the way for an accurate diagnosis and management of posterior uveitic entities. It is also helpful in identifying the possible etiology enabling appropriate treatment.
1. Is it posterior uveitis only or is it part of a panuveitis? 2. Is it choroiditis, retinitis, or retinochoroiditis? 3. Is there associated involvement of the optic nerve head and/ or the retinal vessels? 4. Does the clinical feature fi t into any known infective or noninfective entity? 5. Is there associated anterior segment infl ammation, vitritis, or complications? 6. Is it associated with other systemic features? 7. Is it recurrent? If so, how has it responded to previous therapy? 8. Is it associated with an immunocompromised state? 9. Is it a masquerade syndrome?
Posterior uveitic diseases can be classifi ed based on
## Investigations in the diagnosis of posterior uveitis
Ancillary investigations: A provisional clinical diagnosis can be reached in most cases of posterior uveitis. Ancillary investigations assist in not only confi rming the clinical diagnosis but also in cases of diagnostic dilemma.
Color fundus/FFA: Serial documentation of lesions with color fundus photographs can assist in the follow-up of the disease with treatment. FFA may be useful in confi rming the activity of a choroiditis/retinitis denoted by a characteristic early hypofl uorescence and late hyperfl uorescence in case of active choroiditis. It can be used to detect disease sequelae such as neovascularization, capillary non-perfusion areas, and vascular staining in cases of retinal vasculitis. It can reveal a typical fl ower petal patt ern in cystoid macular edema (CME) or as pooling of dye in late phase in VKH. FFA is most useful to detect the presence, type, and activity of choroidal neovascularization (CNV), which is a vision-threatening complication associated with many posterior uveitic entities.
ICG: ICG is more helpful in case of deeper choroidal lesions, CNV, and in the presence of hemorrhages. It is especially useful in detection, identifi cation, and follow-up of entities with deeper choroidal lesions such as White dot syndromes, where FFA may not be completely confi rmatory.
OCT: OCT is a non-contact imaging tool helpful in detecting and monitoring macular pathologies such as CME, epiretinal membrane, CNV membrane (CNVM), and macular hole. The conventional time domain OCT has a resolution of 10 microns while the newer spectral domain OCT (SD-OCT) has increased the resolution to 5 microns and gives a three-dimensional view enhancing its diagnostic potential.
## Ultrasound b scan (usg):
It is a very useful tool, especially when the media is hazy and in cases with cataract or severe vitritis or vitreous hemorrhage. There is an indication for USG-B scan even when there is a relatively clear media in many posterior and panuveitic conditions. It helps diff erentiate rhegmatogenous and exudative retinal detachment based on the shift ing of fl uid, which is more characteristic of an exudative retinal detachment. An increased choroidal thickening can be a signifi cant fi nding. It may be seen in Vogt Koyanagi Harada's disease (VKH) with signifi cant posterior uveitic manifestations and in posterior scleritis. Normal choroidal thickness is around 1.1 mm. Presence of 'T' sign and/or Tenon's space widening noted in USG-B scan is pathognomonic of posterior sclerits. It is also useful to diagnose intraocular tumors masquerading as uveitis and elevated mass-like lesions such as TB subretinal abscess.
## Laboratory investigations
Tailored lab investigation relevant to the clinical entity in question is the right approach in identifying the etiology of a posterior uveitic entity. Laboratory tests are more useful in infective than in non-infective conditions. Specifi c tests for each entity have been described later. In cases of diagnostic dilemma, intraocular fl uid evaluation for polymerase chain reaction (PCR) and antibody titers helps clinch the diagnosis.
## Systemic examination
It is extremely important that the patient be evaluated thoroughly by an internist to rule out possible associated causes of his/her uveitis and also to evaluate the laboratory test fi ndings, as most of the uveitic entities can have systemic associations. The treatment of the patient is incomplete without simultaneous
## Treatment
Local and systemic steroids along with immunosuppressives in select cases are the mainstay of treatment of non-infective conditions. Infective conditions need to be treated primarily with the specifi c anti-infective agents along with anti-infl ammatory therapy in the form of low-dose steroids. In case of infective uveitis, systemic steroids need to be initiated at least 48-72 h aft er start of specifi c anti-infective therapy and then stopped at least 1 week prior to stoppage of specifi c treatment.
## Infective posterior uveitis
Infective posterior uveitis is a clinical diagnosis based on characteristic fundus picture and relevant positive history. Laboratory investigations are predominantly based on antibody testing against the specifi c antigen and PCR testing for the particular genome. Other tests to detect associated systemic condition may be required to clinch the diagnosis. It is important to treat the underlying systemic disease along with ophthalmic treatment. Basic management approach is given in [fig_ref] Table 2: Basic management approach -guidelines [/fig_ref].
A comprehensive overview of the characteristic clinical appearance of common infective posterior uveitic entities and the current management approach is briefl y described below.
## Ocular toxoplasmosis
Clinical diagnosis: Ocular toxoplasmosis is the most common infective cause of posterior uveitis in immunocompetent patients. Most cases of toxoplasmosis in the immunocompetent host are subclinical or benign. Prevalence is higher in tropical countries than in arid or cold areas.A positive history of contact with pets such as dogs and cats,ingestion of raw, undercooked meat, or contaminated municipal water [bib_ref] Outbreak of ocular toxoplasmosis in Coimbatore, India, Palanisamy [/bib_ref] can identify the source. Transplacental transmission of T. gondii is the only form of human-to-human transmission.
Toxoplasmic retinochoroiditis is unilateral in 72-83% of the cases.Ocular toxoplasmosis occurs from the activation of cysts deposited in or near the retina. Focal necrotizing retinitis is the characteristic lesion. Peripheral retinochoroidal scars are the most common ocular fi nding, occurring in 82% of the patients.
However, toxoplasma has a strong predilection for the posterior pole, particularly the macular region,this location occurring in more than 50% of the cases.Congenital toxoplasmosis Presence of an asymptomatic punched-out macular cicatricial lesion with a central necrotic zone involving the retina, choroid, and vitreous is diagnostic [ [fig_ref] Figure 1: Fundus picture showing a typical punched-out macular scar of a healed congenital... [/fig_ref] ]. Ocular infection may be the only manifestation of congenital toxoplasmosis. Ten percent of the patients have ocular lesions without clear evidence of disease in other organs.Recurrent lesions frequently develop at the borders of the old toxoplasma scars, so-called satellite lesions, and are called reactivation of congenital toxoplasmosis.
## Acquired toxoplasmosis
Patients with ocular toxoplasmosis with macular involvement usually present with diminished vision and/or floaters. "Headlight in the fog" appearance of a focal necrotizing retinochoroiditis with overlying vitritis is the characteristic [ [fig_ref] Figure 2: Fundus picture showing a typical "headlight in the fog appearance" in a... [/fig_ref] ]. Classically, the initial lesion starts in the superfi cial retina, gradually involving the full-thickness retina, adjacent choroid, vitreous, and even sclera. A yellowish white or grey exudative lesion is seen with ill-defi ned borders because of the surrounding area of retinal edema. The size of the lesion varies from a fraction of the disc to about two quadrants of the retina. Adjacent choroiditis, hemorrhage, and vitreitis may be seen. It is relatively asymptomatic in peripheral lesions,seen in 70-90%, and patients may present with only fl oaters.
Vascular involvement may be noted close to the active lesion or in the distant retina and can present as a diff use or segmental vasculitis. This is produced by the antigen-antibody complex deposition and/or localized mononuclear cell infi ltrates in the vessel wall. Although phlebitis is common, arterial involvement is also seen. Kyrieleis arterialitis (exudates or periarterial plaques) can also be seen.The healed scar has well-defined borders around the central retinochoroidal atrophy. Uncommon presentations include associated serous macular detachment, [bib_ref] Toxoplasmic retinochoroiditis presenting as serous detachment of the macula, Kraushar [/bib_ref] retinal vasculitis, neuroretinitis [bib_ref] Optic nerve changes in ocular toxoplasmosis, Eckert [/bib_ref] with papillitis, disc hemorrhages with venous engorgement, and macular star. Anterior uveitis is a complication of the retinochoroiditis and the presence of the parasite in the anterior segment has been demonstrated in immunocompromised patients.In immunocompromised individuals, as in acquired immune defi ciency syndrome (AIDS), punctate outer retinal toxoplasmosis or necrotizing retinitis lesions mimicking viral retinitis may be seen.
Secondary glaucoma is the most common complication. Others include cataract, vitreous hemorrhage, retinal detachment, CNVM, CME, vascular occlusions, and optic atrophy.
Confirmation of diagnosis: Diagnosis is mainly clinical and ancillary investigations like FFA, ICG, [bib_ref] Fluorescein and indocyanine green angiography in ocular toxoplasmosis, Atmaca [/bib_ref] and OCT are complimentary.Although detection of toxoplasma-specifi c antibodies in serum is useful in atypical cases, high titers of positive toxoplasma antibodies in the normal human population may complicate results. Serum antitoxoplasma antibody titers can be determined by several techniques such as Sabin-Feldman dye test (Gold standard), complement fi xation test, hemagglutination test, immunofluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), immunoblott ing, and immunosorbent agglutination assay, but ELISA is the most common serological test employed.
Antibody detection and characterization differentiates recently acquired and chronic infections(immunoglobulin [Ig] M and IgG respectively). Acute systemic toxoplasmosis has traditionally been diagnosed by seroconversion. Anti-Toxoplasma IgG titers present a 4-fold increase that peak 6-8 weeks following infection, then decline over the next 2 years, but remain detectable for life.
Anti-Toxoplasma IgM appears in the first week of the infection and then declines in the next few months. Antibody titers in the serum do not always correlate with ocular disease and hence management has to be based on the clinical diagnosis. Anti-Toxoplasma antibodies may be very low and should be tested in undiluted (1:1) samples if possible. It is to be noted that raised IgG antibody titres, which may be seen in other uveitic conditions, should not form the basis for treatment with anti-Toxoplasma drugs if there is no clinical evidence suggestive of active ocular toxoplasmosis.
PCR is an important tool in the diagnosis of ocular toxoplasmosis, especially in cases of equivocal serology. Various groups have compared PCR and Goldman-Witmer (GW) coeffi cient analysis and have been found to be of equal utility.
In case of diagnostic dilemma, aqueous or vitreous samples may be evaluated for the presence of Toxoplasma DNA sequences, using this technique. Antibodies titers are measured in aqueous humor and serum and GW co-effi cient is calculated.
A combination of PCR testing and GW co-effi cient of antibody titers in aqueous or vitreous [bib_ref] Infectious uveitis in immunocompromised patients and the diagnostic value of polymerase chain..., Westeneng [/bib_ref] [bib_ref] Diagnostic value of specific local antibody production and nucleic acid amplifi cation..., Mahalakshmi [/bib_ref] [bib_ref] Polymerase chain reaction and Goldmann-Witmer coeffi cient analysis are complimentary for the..., Groot-Mij Nes [/bib_ref] has a high degree of specifi city and sensitivity. Analysis of IgG, IgM, and IgA increases the sensitivity of the aqueous humor study [bib_ref] Diagnostic value of specific local antibody production and nucleic acid amplifi cation..., Mahalakshmi [/bib_ref] [bib_ref] Polymerase chain reaction and Goldmann-Witmer coeffi cient analysis are complimentary for the..., Groot-Mij Nes [/bib_ref] [bib_ref] Necrotising retinopathies simulating acute retinal necrosis syndrome, Balansard [/bib_ref] and helps rule out viral etiology, especially in immunocompromised individuals.
Treatment: An ideal combination that destroys tissue cysts and prevents recurrence has not been found [bib_ref] Management of ocular toxoplasmosis, Koo [/bib_ref] [bib_ref] Reactivation of toxoplasma retinochoroiditis under atovaquone therapy in an immunocompetent patient, Baatz [/bib_ref] [bib_ref] Recurrent toxoplasmic retinochoroiditis aft er clindamycin treatment, Benzina [/bib_ref] as current therapies are targeted only on trophozoites. Pyrimethamine(100 mg-1st day, 75 mg-2 nd day, 50 mg-3 rd day, followed by 25 mg once daily) and Sulfadiazine (4 g daily-divided 6 th hourly) for 4-6 weeks, is the most eff ective combination that works synergistically. The other drugs used in the treatment of toxoplasmosis are given in [fig_ref] Table 3: Other anti-Toxoplasmic drugs [/fig_ref].
Treatment regimen consisting of a sulfonamide and a non-sulfonamide with systemic steroids and folic acid supplements is preferred. In patients with sulfa allergy, clindamycin and azithromycin are suitable alternatives. Topical steroids and cycloplegics are used to treat associated anterior uveitis. Immunocompromised individuals require long-term prophylaxis till improvement in immune status of the individual even aft er resolution of lesions.It is important to rule out associated central nervous system involvement in patients with AIDS.
Modifi cations in the treatment regimen in various special situations such as pregnancy and in neonates is given in [fig_ref] Table 4: Anti-Toxoplasma therapy in special situations [/fig_ref].
Toxoplasmic CNVM has been treated effectively with verteporfi n photodynamic therapy. [bib_ref] Verteporfi n photodynamic therapy of choroidal neovascularization secondary to ocular, Wirthlin [/bib_ref] [bib_ref] Toxoplasmosis retinochoroiditis after photodynamic therapy and intravitreal triamcinolone for a supposed choroidal..., Nóbrega [/bib_ref] Pars plana vitrectomy is performed to treat persistent vitreous opacities or vitreoretinal traction. A newer fl uoroquinolone, trovafl oxacin, has potent anti-Toxoplasma activity and appears promising. Sobrin et al. [bib_ref] Intravitreal clindamycin for toxoplasmic retinochoroiditis, Sobrin [/bib_ref] reported favorable response in six patients of toxoplasmic retinochoroiditis with intravitreal clindamycin with/without pars plana vitrectomy.
## Ocular toxocariasis clinical diagnosis:
Toxocariasis is an infection caused by the accidental ingestion of larvae of the dog roundworm Toxocara canis or the cat roundworm Toxocara cati. Children who have pica and are in close contact with puppies are particularly vulnerable. Ocular toxocariasis is diagnosed based on a positive history of contact with pets and suggestive ocular fi ndings. Risk of human infection is higher in children with pica who may ingest contaminated soil or meat. Infections in humans, an end host, result in a focal granulomatous reaction in many organs, including the eye.
Various clinical manifestations according to the decreasing preference of the parasite are: 1. Granuloma in the peripheral retina and vitreous 2. Posterior pole granuloma 3. Chronic endophthalmitis 4. Optic nerve involvement 5. Anterior segment involvement Presence of a posterior pole or a peripheral granuloma [ [fig_ref] Figure 3: Fundus [/fig_ref] ] with tractional retinal detachment or a chronic endophthalmitislike picture [bib_ref] Prevalence, clinical features, and causes of vision loss among patients with ocular..., Stewart [/bib_ref] impairing visualisation is typical. Early lesions may be poorly visualized because of intense vitreous haze. Longstanding masses may have substantial secondary atrophy and hyperplasia of the retinal pigment epithelium (RPE). Whitish/grayish-white granulomas of various sizes may rarely be seen in juxta papillary and subfoveal locations. Inferiorly located lesions can sometimes be mistaken for parsplanitis. Dead larvae are sometimes seen as a dark grey area within the whitish mass at the posterior pole. CNVM and a subretinal toxocara granuloma have to be ruled out. [bib_ref] Optical coherence tomographic and angiographic fi ndings of a case with subretinal..., Higashide [/bib_ref] Confi rmation of diagnosis: Diagnosis is mainly clinical, although ELISA with Toxocara excretory-secretory antigen (TES-Ag) has been shown to be highly specifi c for toxocara
infection. An increase of anti-TES-Ag IgE level indicates acute toxocara infection or progressive infl ammation. An increase in the IgG level confi rms a past or present infection with minimum infl ammation. Toxocara GW co-effi cient analysis from aqueous and serum can be of value when diagnosing patients with posterior focal lesions or vitritis of unknown etiology. [bib_ref] Canninga-van Dij k MR. Diagnosis of ocular toxocariasis by establishing intraocular antibody..., De Visser [/bib_ref] USG/ computerized tomography (CT) fi ndings have additional value.
## Diff erential diagnosis:
- Retinoblastoma may mimic toxocariasis with calcifi cation - Rule out endophthalmitis, severe pars planitis, juvenile idiopathic arthritis-associated uveitis, Coats' disease, primary hypertrophic proliferative vitreopathy, familial exudative vitreoretinopathy, and late stages of retinopathy of prematurity.
## Treatment:
No large case control trial as yet has compared anti-helminthic therapy for ocular toxocariasis against observation alone. Systemic steroids is the mainstay and it is important to monitor its side effects, especially growth retardation in children. Surgical treatment such as pars plana vitrectomy, cryopexy, and laser photocoagulation has been used to treat complications. Vitrectomy may be benefi cial for patients with endophthalmitis, unrelieved vitreoretinal traction with retinal detachment, [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] and also as an optical indication.
## Tubercular posterior uveitis
Ocular TB can be primary, where the eye is the initial site of entry or secondary, where organisms spread to the eye hematogenously; this type includes tuberculous uveitis. Hypersensitivity reaction to tuberculous protein can also cause retinal vasculitis. A study from our center noted that only 1.39% patients with systemic TB had tubercular uveitis.
Clinical diagnosis: The most common presentation of tuberculous uveitis is of disseminated choroiditis. [bib_ref] Ocular tuberculosis, Thompson [/bib_ref] Choroidal tubercles may be one of the earliest signs of disseminated disease. The lesions may vary from few numbers to several hundred. The lesions range from 0.5 to 3.0 mm in diameter and may vary in size and elevation within the same eye. They are deep in the choroid, appear yellow, white, or gray, and are fairly well circumscribed. In the vast majority of cases, the lesions present in the posterior pole.
The next most common presentation is a single tubercle, also termed focal choroiditis, [bib_ref] Tuberculoma of the choroid, Cangemi [/bib_ref] which can occur at the posterior pole. A single choroidal mass is the characteristic feature on presentation, although multiple choroidal tubercles can be seen in cases of miliary tubercles and in immunosuppressed individuals. A large tubercle may measure up to 4.0 mm in diameter; however, choroidal masses up to 14 mm in diameter have been reported . The mass is typically elevated and may be accompanied by an overlying serous retinal detachment [bib_ref] Primary choroidal tuberculoma, Bernstein [/bib_ref] [fig_ref] Figure 4: Fundus picture showing a tuberculous subretinal abscess Sudharshan, et al [/fig_ref] ]. Subretinal abscess is formed progressively from a choroidal tubercle, which can be single or multiple. Diagnosis is based on clinical features, suggestive of systemic fi ndings and supportive investigations.
Solitary tubercular choroidal granuloma [bib_ref] Choroidal granuloma revealing tuberculosis. A case report, Karim [/bib_ref] [bib_ref] Solitary choroidal tuberculoma in an immunocompetent patient, Levecq [/bib_ref] can be misdiagnosed as choroidal melanoma due to multiplicity of clinical fi ndings, which also causes diagnostic delay. Other presentations include serpiginous-like choroiditis, [bib_ref] Presumed tubercular serpiginouslike choroiditis: clinical presentation and management, Gupta [/bib_ref] retinal vasculitis, [bib_ref] PCRpositive tubercular retinal vasculitis: Clinical characteristics and management, Gupta [/bib_ref] intermediate uveitis, panuveitis, and neuroretinitis.
## Confirmation of diagnosis:
Angiography confirms activity in choroiditis [bib_ref] Fundus fl uorescein angiography of choroidal tubercles: case reports and review of..., Mehta [/bib_ref] and reveals a classical ring of fire appearance in the subretinal abscess/choroidal granuloma. Retinal vasculitis can present with neovascularization and capillary non-perfusion areas due to inflammatory vessel obstruction, which may need prophylactic laser panretinal photocoagulation. OCT scans through areas of suspected granuloma can be helpful in differentiating choroidal granulomas from other non-infl ammatory conditions, such as central serous chorioretinopathy (CSR), exudative age-related macular degeneration, and choroidal tumors mimicking choroidal granuloma. In TB granuloma, OCT [bib_ref] Optical coherence tomography in choroidal tuberculosis, Salman [/bib_ref] reveals an area of localized adhesion between the choriocapillaris-RPE layer and overlying the neurosensory retina ("contact" sign), possibly due to infl ammatory adhesions overlying the granuloma that cause the neurosensory retina to stick to the RPE at that point. Infl ammatory cells appear as increased refl ectivity in the deeper retinal layers over the granuloma. These features, unique in TB and other infl ammatory conditions, are unusual in noninfl ammatory lesions. USG-B scan helps rule out tumors in large mass like subretinal abscesses.
Associated systemic latent/manifest TB confi rms diagnosis. Mandatory investigations include hemogram, erythrocyte sedimentation rate (ESR), Mantoux test, and radiological imaging such as chest X-ray/CT scan.
It is important to note that tuberculin test can be false positive in patients hailing from endemic countries like India and is also aff ected by previous BCG vaccination.
Newer tests based on gamma interferon assays such as the Quantiferon TB-Gold test are promising. [bib_ref] Utility of Quantiferon TB gold test in a South Indian Patient population..., Babu [/bib_ref] Others include BACTEC MGIT 960 system, MB/BacT system, and the ESP II culture system.
Histopathological and/or microbiological confi rmation of mycobacterium infection, especially by PCR, from intraocular specimens is diagnostic. IS6I I0 primer-based PCR is widely used for detection of the M. tuberculosis complex. However, nested PCR technique employing the MPB64 gene is 10,000 times more sensitive and 100% specifi c and is used in cases of doubt. [bib_ref] Polymerase chain reaction for detection of Mycobacterium tuberculosis in epiretinal membrane in..., Madhavan [/bib_ref] [bib_ref] Application of nested polymerase chain reaction (nPCR) using MPB 64 gene primers..., Therese [/bib_ref] [bib_ref] Detection of mycobacterium tuberculosis by nested polymerase reaction in a case of..., Biswas [/bib_ref] Real-time PCR technology can diff erentiate commensals and contaminants from infecting microbes. Dot-blot hybridization of the PCR product by 32 P-labeled specifi c probes improves sensitivity. [bib_ref] Diagnostic effi cacy of polymerase chain reaction in granulomatous uveitis, Arora [/bib_ref] NOTE: ELISA and PCR testing for TB on serum are not useful, especially in endemic regions like India, and should not form the basis of diagnosis of intraocular TB.
Treatment: It is imperative that anti-tuberculous therapy (ATT) be initiated under care of an internist once tuberculous etiology is confi rmed. Concomitant systemic steroids for 4-6 weeks have a protective eff ect against tissue damage from delayed type of hypersensitivity (DTH). Use of corticosteroids alone should be avoided as it promotes multiplication of bacilli and can lead to panophthalmitis. Guidelines described by Gupta et al. [bib_ref] Intraocular tuberculosis--an update, Gupta [/bib_ref] can form the basis for diagnosis and management of ocular TB. Successful medical management of subretinal tubercular granuloma have also been described, [bib_ref] Successful management of tubercular subretinal granulomas, Gupta [/bib_ref] where authors have concluded that once the diagnosis of presumed or confi rmed TB is established, surgical intervention should be avoided and successful resolution of lesions can be noted with ATT and steroids alone. Surgery is an option mainly for complications. [bib_ref] Macular subretinal neovascularization in choroidal tuberculosis, Chung [/bib_ref] What's new?
- Newer quinolones and rifamycins like rifabutin and macrolides are part of ATT regimes, especially in multidrugresistant TB. - Usefulness of Retinalamine in patients with TB chorioretinitis is being evaluated. [bib_ref] Results of retinalamine use in the treatment of tuberculous chorioretinitis, Aleksandrova [/bib_ref] Viral retinitis A retinitis should evoke a suspicion of a viral infection, which is usually caused by Herpes simplex, Varicella zoster, and CMV. Other rare causes include chikungunya and rubella viruses.
Herpetic eye disease is among the most common causes of infectious uveitis. It may aff ect healthy as well as immunocompromised hosts, although its clinical presentation varies accordingly. Posterior uveitis caused by herpes viruses may appear as part of herpetic disease elsewhere (skin, brain, anterior segment of the eye) or as an isolated fi nding. Most forms of herpetic posterior uveitis are acute and fulminant, oft en resulting in serious complications such as retinal detachment and proliferative vitreoretinopathy.
Presentation of herpetic infections varies based on the immune status of the individual. Associated systemic herpetic manifestations, a past history of chicken pox, or signs and symptoms of an immunocompromised state may be diagnostic clues.
Clinical diagnosis: Acute retinal necrosis (ARN) is the classical presentation of herpetic viruses. Characteristic clinical triad of moderate to severe vitritis, arteritis, and periphlebitis and confl uent peripheral retinal necrosis is diagnostic of ARN, which can present as a panuveitis. Although the posterior pole is not typically aff ected early in the disease process, it can also be involved primarily in necrotizing herpetic retinopathies.
Progressive outer retinal necrosis (PORN): Necrotizing retinitis, confl uent areas of outer retinal whitening with minimal vitritis involving the posterior pole and sparing of retinal vessels at the early stage, the typical "cracked mud appearance" [fig_ref] Figure 5: Fundus picture showing the classical "cracked mud appearance" in progressive outer retinal... [/fig_ref] ] is virtually diagnostic. [bib_ref] Rapidly progressive outer retinal necrosis in the acquired immunodefi ciency syndrome, Forster [/bib_ref] Although ARN and PORN are considered to be two diff erent entities, they are considered essentially as diff erent manifestations of the same disease and the varied presentations occurring due to diff erences in the immune status of the host. PORN is frequently bilateral, occurring exclusively in immunocompromised state, such as in patients with HIV infection, [bib_ref] Rapidly progressive outer retinal necrosis in the acquired immunodefi ciency syndrome, Forster [/bib_ref] and is associated with rapid development of rhegmatogenous retinal detachment or optic atrophy.
Viral posterior uveitis can also rarely present as patchy, single, or multiple retinitis patches, which includes acute varicella retinitis/choroiditis seen in children as well as chronic choroiditis or non-necrotizing retinal vasculitis in adults.
Chikungunya retinitis [bib_ref] Ocular manifestations associated with chikungunya, Mahendradas [/bib_ref] [bib_ref] Bilateral retinitis following chikun-gunya fever, Murthy [/bib_ref] [bib_ref] A case of bilateral presumed chikungunya neuroretinitis, Mahesh [/bib_ref] mimics herpetic or CMV retinitis and is diagnosed based on a history of ailment with chikungunya disease and detection of specifi c antibodies to the virus from serum or intraocular fl uid. It is oft en self-limiting and, although not specifi c, acyclovir or intravitreal ganciclovir have been found to be benefi cial.
CMV retinitis, usually seen in immunocompromised states such as in AIDS or post organ transplant patients on immunosuppressives, has a characteristic granular or pizza pie appearance [ [fig_ref] Figure 6: Fundus picture showing a typical "pizza pie appearance" in a patient with... [/fig_ref] ] and a typical brushfi re patt ern of spread along the blood vessels.
Although rubella virus, which is part of the 'TORCH' complex, can also cause ocular lesions, rubella retinopathy can be clearly diff erentiated from retinitis due to herpetic viruses as it has a characteristic salt-pepper appearance and can mimic retinitis pigmentosa.
Treatment: Long-term systemic antiviral drugs such as acyclovir or valaciclovir are the treatment of choice in case of herpetic retinitis. Aciclovir is very eff ective against HSV and VZV. Treatment with acyclovir reduces infection of the fellow eye from 70 to 13% in the fi rst year.The dosage is 15 mg/kg body weight in three doses for 7-21 days. Then, 2-4 g daily is recommended for a further 4-6 weeks.Usually, intravenous acyclovir 500 mg 8 th hourly for 1-2 weeks followed by oral acyclovir 800 mg 5-times daily is recommended with additional low-dose systemic steroids. Alternatively, oral valaciclovir, which has a bett er bioavailability, can be given as 1 g three times daily. Antivirals may be continued for almost 3-6 months in some cases. Renal function needs to be monitored if antivirals are administered on a long-term basis, especially in extremes of age. Prophylactic laser barrage adjacent to retinitis lesions is known to prevent retinal detachment, although there is no randomized study that validates the use of laser barrage for preventing a subsequent retinal detachment. Early vitrectomy as an option has been advocated but also needs to be tested in a randomized fashion. In resistant cases, alternatives include a combination of systemic and intraocular antiviral therapy with foscarnet and ganciclovir. [bib_ref] Intravitreal antivirals in the management of patients with acquired immunodeficiency syndrome with..., Scott [/bib_ref] Intravenous ganciclovir-5-7 mg/kg/day in two divided doses for 2 week-induction dose followed by once daily-maintenance dose till complete resolution of lesions and improvement of immune status is the treatment of choice in CMV retinitis. Intravitreal injections of either ganciclovir or foscarnet may be considered, especially in initial cases where the macula is threatened. It can be a way to get the greatest concentration of drug to the aff ected area immediately.
Alternatively, oral valganciclovir-900 mg BD as induction and 900 mg OD as maintenance dose has an additional advantage of being a non-parenteral mode of treatment, avoiding complications related to indwelling catheters, especially in immunocompromised individuals. Appropriate management of [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref]. Pyrimethamine (100 mg-1 st day, 75 mg-2 nd day, 50 mg-3 rd day, followed by 25 mg once daily) + Sulfadiazine (4 g daily-divided 6 th hourly) for 4-6 weeks 2. Clindamycin [bib_ref] Recurrent toxoplasmic retinochoroiditis aft er clindamycin treatment, Benzina [/bib_ref] (300 mg 6th hourly-maximum dose:1.8 gm/day) for 6 weeks 3. Trimethoprim+sulphamethoxazol-DS tab-160 mg/800 mg-one tab twice daily for 6 weeks 4. Spiramycin-2 g/day in two divided doses 5. Azithromycin-loading dose 1 G-1 st day, followed by 500 mg once daily for 3 weeks 6. Atovaquone [bib_ref] Reactivation of toxoplasma retinochoroiditis under atovaquone therapy in an immunocompetent patient, Baatz [/bib_ref] -750 mg every 6 h for 4-6 weeks NOTE: Necrotizing retinopathy due to toxoplasma closely mimics herpetic retinitis, especially in immunocompromised patients.
CMV retinitis can also involve the posterior pole initially and, in early stages, can mimic cott on wool spots of HIV retinopathy.
## Ocular syphilis
Ocular syphilis can mimic any of the uveitic entities, and has to be ruled out especially in any case of infective uveitis. It is the most common intraocular bacterial infection and is re-emerging in varied forms, especially with the advent of AIDS. [bib_ref] Ocular syphilis: the return of an old acquaintance, Durnian [/bib_ref] About 1-2% of HIV-positive patients are found to have ocular syphilis.
Clinical diagnosis: Syphilis is called the great imitator as it can present as chorioretinitis, neuroretinitis, salt-pepper retinopathy, optic neuritis, papilloedema, and optic perineuritis. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] Syphilis can present with placoid choroidal lesions and can mimic APMPPE. An unusual manifestation of syphilis is acute necrotizing retinopathy, which mimics ARN. Syphilitic uveitis can be the fi rst presentation of the systemic disease [bib_ref] Ocular uveitis as the initial presentation of syphilis, Hong [/bib_ref] in both immunocompetent and immunocompromised individuals. In HIV-positive patients, ocular syphilis is more closely associated with neurological abnormalities.
Confi rmation of diagnosis: Laboratory investigations such as venereal disease research laboratory test, rapid plasma reagin test, and treponema pallidum hemagglutination tests are used. Confi rmatory tests include fl uorescent treponema antibody agglutination (FTA-ABs) test and dark ground microscopy. A specifi c treponemal test such as FTA-ABs can be used as an initial test for a syphilis screen as it is more reliable and has low false positivity. Patients oft en show abnormal cerebrospinal fl uid fi ndings. Diagnosis is very challenging as up to 38% of HIVpositive patients can be seronegative despite active syphilitic disease.Treatment: Treatment of ocular syphilis is similar to that of neurosyphilis. Long-acting penicillin is the treatment of choice.
Neuroretinitis: Involvement of the optic nerve head along with retinitis and macular star constitutes the typical appearance of a neuroretinitis [ [fig_ref] Figure 7: Fundus picture showing disc edema and macular exudates characteristic of a neuroretinitis [/fig_ref] ]. It can be caused due to toxoplasma, TB, syphilis, and other non-infective conditions such as collagen vascular diseases, and is treated accordingly.
Cat-scratch disease (CSD) caused due to Bartonella is an important cause of neuroretinitis. It can also present with acute macular neuronopathy. [bib_ref] Bartonella henselae infection presenting as a unilateral acute maculopathy. Bartonella henselae infection..., Wimmersberger [/bib_ref] Doxycycline-100 mg is the treatment of choice. Multifocal chorioretinal lesions associated with B. hensalae can be atypical ophthalmic manifestations of CSD, which may occur in immunosuppressed patients. [bib_ref] Atypical Bartonella hensalae chorioretinitis in an immunocompromised patient, Patel [/bib_ref] Recognition and appropriate treatment of underlying disease gives bett er visual outcomes.
## Dusn
DUSN, a rare entity caused by a worm in the eye, is usually unilateral. Clinical visualization of subretinal nematode makes the diagnosis obvious. Live worm, if detected, can be destroyed by direct laser application. Worm and its by-products can cause severe infl ammation aff ecting the optic nerve head and retina and needs to be treated with high-dose systemic steroids. If a worm is not found clinically, appearance of sub-RPE serpiginous tract in the inferotemporal retina, peripheral RPE hypopigmentation, good clinical response to anti-helminthics, and abnormal ERG supports the diagnosis. [bib_ref] Diff use unilateral subacute neuroretinitis, Anshu [/bib_ref] Treatment with albendazole is benefi cial.
## Intraocular cysticercosis
Cysticercus cellulose cyst, found inside the eye, can cause severe inflammatory reaction. [bib_ref] Parij a SC. Cysticercosis of the eye in South India--a case series, Kaliaperumal [/bib_ref] [bib_ref] Fibrinous anterior uveitis due to cysticercus cellulosae, Mahendradas [/bib_ref] When visualization is impaired, USG-B scan is diagnostic. Neurological association needs to be ruled out. Treatment is with steroids and antihelminthic therapy. [bib_ref] Parij a SC. Cysticercosis of the eye in South India--a case series, Kaliaperumal [/bib_ref] Removal of the cyst is necessary in most cases.
## Non-infective posterior uveitis
Common non-infective posterior uveitic entities include the "WDS" and are named according to their clinical appearance and behavior. FFA and ICG are virtually diagnostic in WDS. Laboratory investigations are not routinely necessary for their diagnosis but are essential for monitoring therapy-related side eff ects. A signifi cant percentage of patients with WDS have a prodromal viral-like illness that triggers the onset of the ocular condition and may be self-limiting, although they need to be followed-up closely for complications such as scarring and CNVM. Patients with WDS present with sudden blurring of vision associated with photopsia, floaters, scotomata, and metamorphopsia. An approach to a case of inflammatory posterior uveitis is dipicted in [ [fig_ref] Table 5: Basic management approach to a case of non-infective posterior uveitis Identify the... [/fig_ref] ].
The comparative clinical characteristics and investigative fi ndings of various WDS are given below [Tables [bib_ref] Optic nerve changes in ocular toxoplasmosis, Eckert [/bib_ref] [bib_ref] Fluorescein and indocyanine green angiography in ocular toxoplasmosis, Atmaca [/bib_ref] [bib_ref] Infectious uveitis in immunocompromised patients and the diagnostic value of polymerase chain..., Westeneng [/bib_ref]. [fig_ref] Figure 8: Fundus picture showing placoid lesions of acute posterior multifocal placoid pigment epitheliopathy... [/fig_ref] ]: It is an infl ammatory retinal/choroidal disease characterized by sudden loss of vision caused by the sudden appearance of multiple yellow-white, fl at infl ammatory lesions lying deep within the sensory retina, most notably at the level of the RPE and the choriocapillaries. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] Atrophy and RPE scarringresults in poor visual acuity. New lesions may be observed in the peripheral fundus for up to 3 weeks and tend to be more linear. Additional fi ndings include disc edema, keratic precipitates, retinal vasculitis, neurosensory detachments, and venous occlusions. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] OCT fi ndings in conjunction with FFA and microperimetry are useful. [bib_ref] Correlation between optical coherence tomography and autofl uorescence in acute posterior multifocal..., Souka [/bib_ref] APMPPE has a self-limited clinical course, with spontaneous recovery of vision in most cases. Of those eyes that are aff ected, 90% of them typically achieve a visual acuity of more than 20/25. In rare cases, recurrences may occur within 6 months of the initial episode, thereby giving a less-favorable prognosis. Corticosteroids and cytotoxic drugs are indicated, especially in cases of associated cerebral vasculitis. [bib_ref] White dot syndromes" in childhood, Spital [/bib_ref] CNV is a rare complication.
## Overview of clinical and diagnostic features of wds apmppe [
NOTE: [bib_ref] White dot syndromes" in childhood, Spital [/bib_ref] [bib_ref] Neurological manifestations of acute posterior multifocal placoid pigment epitheliopathy, Sinan [/bib_ref] - APMPPE mimics non-inflammatory conditions like multifocal choriocapillary infarcts due to hypertension, toxemia of pregnancy, and disseminated intravascular coagulation.- Rule out associated cerebral vasculitis. [bib_ref] Neurological manifestations of acute posterior multifocal placoid pigment epitheliopathy, Sinan [/bib_ref] MEWDS: MEWDS is a rare disorder of unknown etiology characterized by the presence of white lesions deep in the outer retina or at the level of the RPE. It can also present with optic disc edema, mild vitritis, panuveitis, diff use choroidal thickening, and a relative aff erent pupillary defect. Newly recognized angiographic features termed dots and spots, which varied in size and location in the fundus, have been reported. Small dots were in the inner retina or at the level of the RPE, and larger spots were more external in the subpigment epithelial area. Presence of white dots around the nerve is rare and fi eld defect persists even aft er lesions disappear. [bib_ref] Fibrinous anterior uveitis due to cysticercus cellulosae, Mahendradas [/bib_ref] A viral prodrome is known in 50% of the patients, and the multifocal nature of the disease, a viral prodrome, is thought by many. Patients also present with acute, painless, unilateral loss of vision.
It can be distinguished from other WDSs by its distinct morphology, associated macular granularity, transient nature, characteristic angiographic appearance, unilaterality, self-limiting course, lack of signifi cant sequelea, absence of associated systemic involvement, rapid recovery, and excellent visual outcome.
MEWDS is a self-limited disease, with almost all patients regaining good visual acuity within 3-9 weeks. The lesions disappear without scarring and photopsias and scotomata gradually resolve. Occasionally, patients with MEWDS may have persistent blind spot enlargement. Although uncommon, recurrences can occur. However, the prognosis is fairly good for these patients. A rare complication of MEWDS is CNV, which may require laser photocoagulation.
## Note:
- Careful evaluation by slit lamp biomicroscopy [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] is a must. - Characteristic fi nding of MEWDS is foveal granularity. [bib_ref] Multiple evanescent white dot syndrome, Gross [/bib_ref] [bib_ref] Atypical presentation of multiple evanescent white dot syndrome involving granular lesions of..., Khurana [/bib_ref] - Multifocal ERG helps diff erentiate MEWDS from other blind spot enlarging conditions. [bib_ref] Multifocal ERG in multiple evanescent white dot syndrome, Feigl [/bib_ref] [bib_ref] Transient multifocal electroretinogram dysfunction in multiple evanescent white dot syndrome, Chen [/bib_ref] Serpiginous choroiditis [bib_ref] Serpiginous choroiditis, Lim [/bib_ref] (GHPC) It is a rare, chronic, progressive, and recurrent bilateral infl ammatory disease involving the RPE, the choriocapillaries, and the choroid. It is characterized acutely by irregular, graywhite or cream-yellow subretinal infi ltrates at the level of the choriocapillaries and the RPE. Based on clinical presentation, it can be classified into (1) peripapillary, (2) macular, and (3) ampiginous types. The clinical course, regardless of the presentation, is progressive, with multiple recurrences leading to potentially signifi cant visual loss.
Patients present with unilateral or bilateral visual loss when the macula is involved and they may also notice photopsias and scotomata. The anterior segment usually appears quiet, although a non-granulomatous anterior uveitis has been described. Graywhite lesions are noted at the level of the RPE. Active lesions are usually found at the border of inactive lesions and appear in an interlocking polygonal patt ern that spreads out the periphery from the optic nerve [ [fig_ref] Figure 9: Fundus picture showing active geographic helicoid peripapillary choroidopathy Sudharshan, et al [/fig_ref] ]. Macular involvement is common. Mild vitreous and anterior chamber infl ammation is observed in one-third of the cases.
Ampigenous choroiditis mimics placoid lesions of APMPPE and coalesced lesions of GHPC. Persistent placoid maculopathy is a resistant form of serpiginous choroidopathy and resembles macular GHPC, but diff ers in its clinical course and eff ect on visual acuity as a majority of the eyes develop CNVM, [bib_ref] Persistent placoid maculopathy: a new clinical entity, Golchet [/bib_ref] [bib_ref] Serpiginous choroidopathy presenting as choroidal neovascularisation, Lee [/bib_ref] resulting in central vision loss. Ten to 12% can have macular involvement alone. [bib_ref] Clinical profi le, treatment, and visual outcome of serpiginous choroiditis, Abrez [/bib_ref] Mild vitreous and anterior chamber infl ammation is observed in one-third of the cases. Histopathology reveals
## Note:
- To rule out tuberculous etiology in patients with serpiginouslike choroiditis. - PCR-proven viral etiology has also been implicated. [bib_ref] Clinical profi le, treatment, and visual outcome of serpiginous choroiditis, Abrez [/bib_ref] - Toxoplasma infection presenting like serpiginous choroiditis has also been reported. [bib_ref] Serpiginous choroiditis-like picture due to ocular toxoplasmosis, Mahendradas [/bib_ref] MFC Bilateral involvement is present in approximately 66-79% of the patients. [bib_ref] Multifocal choroiditis and panuveitis, Dreyer [/bib_ref] Optic disc edema, rarely, peripapillary scarring, and prominent linear chorioretinal streaks may also be present. Patient may also present with CME and CNVM. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] PIC [bib_ref] Correlation between optical coherence tomography and autofl uorescence in acute posterior multifocal..., Souka [/bib_ref] [bib_ref] Punctate inner choroidopathy: a survey analysis of 77 persons, Gerstenblith [/bib_ref] [bib_ref] Progressive subretinal fi brosis and uveitis, Palestine [/bib_ref] PIC is an inflammatory multifocal chorioretinopathy of unknown etiology. It presents with an acute bilateral loss of vision, photopsias, and scotomata. The anterior segment is quiet. The vitreous is clear without infl ammatory cells. The lack of vitreous infl ammation is a hallmark of PIC and the presence of vitritis should suggest a diff erent diagnosis. [bib_ref] Correlation between optical coherence tomography and autofl uorescence in acute posterior multifocal..., Souka [/bib_ref] No treatment is advised for the majority of patients without CNVM or subretinal fi brosis, which usually occurs within the fi rst year. [bib_ref] Punctate inner choroidopathy: a survey analysis of 77 persons, Gerstenblith [/bib_ref] Patients without CNVM have excellent visual outcomes. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] Note:
PIC occurs in myopic women.
## Absence of vitritis-hallmark of pic presence of vitritis-suggests a diff erential diagnosis
## Sfu
Progressive subretinal fi brosis with multifocal lesions of the RPE and choroid can be seen in association with PIC and recurrent MFC. Although steroids may benefi t initially, progressive fi brotic subretinal lesions leads to severe and permanent visual loss. [bib_ref] Progressive subretinal fi brosis and uveitis, Palestine [/bib_ref] Fibrosis is predominantly at areas of previous infl ammatory lesions and a turbid SRF that overlies the lesions is also noted. SFU is also seen in other infl ammatory and non-infl ammatory conditions, [bib_ref] Progressive subretinal fi brosis in fundus fl avimaculatus, Parodi [/bib_ref] such as late stage of serpiginous choroiditis, SLEassociated CSR, and onchocerciasis. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] Infl iximab has been tried with good results. [bib_ref] Successful treatment with infl iximab in a patient with Diff use Subretinal..., Adán [/bib_ref] Uncommon WDS in India Birdshot chorioretinopathy (BCR)-vitiliginous choroiditis:
Research criteria for its diagnosis have been formulated [bib_ref] Research criteria for the diagnosis of birdshot chorioretinopathy: results of an international..., Levinson [/bib_ref] and more than 90% of the patients with BCR are HLA-A29 positive. [bib_ref] HLA typing in birdshot chorioretinopathy, Priem [/bib_ref] [bib_ref] Birdshot chorioretinopathy, Shah [/bib_ref] Associated non-granulomatous uveitis is seen in about 25% cases. The term "birdshot" is given because the patt ern of lesions in the fundus resembles the shotgun scatt er of a birdshot. A recent longitudinal cohort study has described the baseline clinical characteristics and has concluded that lesion pigmentation may be a marker of decreased visual function that is not refl ected in central visual acuity. [bib_ref] Longitudinal cohort study of patients with birdshot chorioretinopathy. I. Baseline clinical characteristics, Monnet [/bib_ref] POHS: POHS is usually seen in endemic areas of Histoplasma capsulatum and rarely in other non-endemic areas. [bib_ref] Standardization of Uveitis Nomenclature (SUN) Working Group: Standardization of uveitis nomenclature for..., Jabs [/bib_ref] [bib_ref] Presumed ocular histoplasmosis syndrome (POHS) in a non-endemic area, Chryssafi S [/bib_ref] It presents asymptomatically or with central scotoma. H. capsulatum has never been isolated from the choroid. CNVM is managed by argon laser photocoagulation for extrafoveal type, while krypton laser photocoagulation is benefi cial for juxtafoveal type. Dabil et al. [bib_ref] Association of the HLA-DR15/HLA-DQ6 haplotype with development of choroidal neovascular lesions in..., Dabil [/bib_ref] reported a signifi cant association between the HLA-DR15/ HLA-DQ6 haplotype and development of CNVM in POHS Acute retinal pigment epithelitis (ARPE-Krill's disease) [bib_ref] Acute retinal pigment epitheliopathy, Krill [/bib_ref] : It usually presents with unilateral blurred vision and metamorphopsia in young adults without signifi cant prodromal fl u-like illness. Round macular lesions, hallmark of the disease, are manifested by transient and subtle RPE alterations. Discrete clusters of small, hyperpigmented, dark gray spots at the RPE level surrounded by a yellowish white halo or area of macular depigmentation can aff ect vision. Gray dots and halo fade with resolution and become clinically undetectable. Vitritis is rare. VEP and ERG are normal, while EOG may be abnormal. FFA rules out CSR [bib_ref] Fibrinous anterior uveitis due to cysticercus cellulosae, Mahendradas [/bib_ref] and no treatment is needed.
## Treatment of non-infective posterior uveitic conditions
Systemic steroids are the mainstay of therapy in non-infective posterior uveitis. In vision-threatening lesions, such as those with optic nerve head or macular/foveal involvement, pulse therapy of intravenous methyl prednisolone (IVMP) [bib_ref] Intravenous pulse methylprednisolone therapy for acute treatment of serpiginous choroiditis, Markomichelakis [/bib_ref] 1g daily for three consecutive days should be administered on an emergency basis only under care of an internist, preferably in place with an intensive care unit backup/set-up. This is followed by oral prednisolone 1-1.5 mg/kg/day, preferably single morning dose, in a tapering dosage schedule.
Once the diagnosis of non-infectious uveitis has been confi rmed over time and infectious causes have been completely ruled out, intravitreal injections of steroid, such as triamcinolone or dexamethasone, are very useful adjuncts in controlling fl areups.
In patients with repeated recurrences, intolerance to systemic steroids or recalcitrant to therapy, immunosuppressives can be added with/without systemic steroids.
Immunosuppressive options include antimetabolites such as azathioprine, methotrexate, and mycophenolate mofetil, T-cell suppressors such as cyclosporine and tacrolimus, and cytotoxic agents including cyclophosphamide and chlorambucil.
We usually prefer azathioprine, [bib_ref] Clinical profi le, treatment, and visual outcome of serpiginous choroiditis, Abrez [/bib_ref] [bib_ref] Combination of azathioprine and corticosteroids in the treatment of serpiginous choroiditis, Vianna [/bib_ref] considering the cost of therapy and as it has lesser side eff ects. It is given in a dose of 1-2 mg/kg/day in three divided doses and tapered monthly. A thorough knowledge of the usefulness and the side eff ects associated with the various immunosuppressives is a must before initiation of immunosuppressives.
The patient has to be explained the risks and benefits associated with the use of immunosuppressives and the need to monitor the side eff ects with the respective blood tests regularly.
The details regarding the usage of immunosuppressives and other details are described in other appropriate sections. Biologicals such as infl iximab for the treatment of refractory non-infective posterior uveitis and severe SFU [bib_ref] Successful treatment with infl iximab in a patient with Diff use Subretinal..., Adán [/bib_ref] and daclizumab and tacrolimus for the treatment of BCR [bib_ref] Biologics in the treatment of uveitis, Imrie [/bib_ref] [bib_ref] Daclizumab for treatment of birdshot chorioretinopathy, Sobrin [/bib_ref] [bib_ref] Long-term follow-up of tacrolimus treatment in immune posterior uveitis, Figueroa [/bib_ref] have been used with favorable response.
Local drug delivery such as intravitreal triamcinolone [bib_ref] Intravitreal triamcinolone for the treatment of refractory macular edema in idiopathic intermediate..., Hogewind [/bib_ref] [bib_ref] Intravitreal triamcinolone acetonide in serpiginous choroiditis, Karacorlu [/bib_ref] for refractory CME is also effective. Inflammatory CNVM has been satisfactorily treated with intravitreal triamcinolone, bevacizumab [bib_ref] Intravitreal bevacizumab for choroidal neovascularisation secondary to punctate inner choroidopathy, Vossmerbaeumer [/bib_ref] [bib_ref] Therapeutic effi cacy of intravitreal bevacizumab on posterior uveitis complicated by neovascularization, Kurup [/bib_ref] [bib_ref] Intravitreal bevacizumab (avastin) injection as primary treatment of infl ammatory choroidal neovascularization, Adán [/bib_ref] anecortave acetate in serpiginous choroiditisassociated CNVM, [bib_ref] Anecortave may help choroidal neovascularization regression in serpiginous choroiditis, Nóbrega [/bib_ref] and sirolimus for MFC-associated CNVM.Fluocinolone acetonide, a local drug delivery implant, has been found to be useful in a large multicentre trial. [bib_ref] Efficacy of low-release-rate fluocinolone acetonide intravitreal implants to treat experimental uveitis, Mruthyunjaya [/bib_ref] [bib_ref] Fluocinolone acetonide intravitreal sustained release device--a new addition to the armamentarium of..., Brumm [/bib_ref] [bib_ref] Treatment of posterior uveitis with a fl uocinolone acetonide implant: three-year clinical..., Callanan [/bib_ref] Role of interferon-alpha [bib_ref] Interferon-alpha as an eff ective treatment for noninfectious posterior uveitis and panuveitis, Plskova [/bib_ref] in severe, sight-threatening refractory uveitis is being considered, although adverse events like IFN-alpha-associated retinopathy may limit its use. [bib_ref] Uveitis masquerade syndromes, Rothova [/bib_ref] [bib_ref] Masquerade syndromes: malignancies mimicking infl ammation in the eye, Tsai [/bib_ref] : Masquerade syndromes are non-uveitic conditions that mimic and present like uveitis. It is important to diff erentiate neoplastic diseases from various other posterior uveitic entities.
## Masquerade syndromes and other diseases
Tumors such as lymphoma and other secondary malignancies can mimic viral retinitis. Lymphoma has to be ruled in non-resolving or atypical presentations of viral retinitis or intermediate uveitis. They are especially seen in patients with HIV infection. In children, one has to be aware of the possibility of leukemias and retinoblastoma, while in adults malignant melanoma and metastases are important diff erential diagnoses. Knowledge of non-uveitic entities, which can mimic posterior uveitis, is essential before initiating therapy.
A choroidal melanoma or metastases can be mistaken for a choroidal abscess. USG-B scan is very valuable to diff erentiate these entities. Choroidal melanoma shows a high to moderate surface reflectivity and variable low to moderate internal refl ectivity on ultrasound. Other typical features include acoustic hollowing and choroidal excavation. It is generally associated with a shallow retinal detachment, although this may also be seen overlying a subretinal abscess.
Choroidal metastases appear as mass with high surface refl ectivity and uniform high to moderate internal refl ectivity. Acoustic hallowing and choroidal excavation are not present. They are usually associated with a large retinal detachment.
Vitreous and retinal biopsy is very useful in diagnostic dilemma. [bib_ref] Ocular uveitis as the initial presentation of syphilis, Hong [/bib_ref] : Infections such as toxoplasmosis, syphilis, and tuberculosis can have atypical ocular manifestations in AIDS. There are also some unique entities that give a clue to the underlying systemic disease. HIV retinopathy, rare tumors such as lymphomas, and infections such as cryptococcal and pneumocystis choroiditis are a few of them.
# Aids-related posterior uveitis
# Conclusions
Posterior uveitic entities have very characteristic clinical features and diagnosis is mainly clinical. It is essential to diff erentiate infective and non-infective conditions as their management is diametrically opposite. Infective posterior uveitic needs to be managed with specifi c anti-infective therapy and steroids. Empirical use of systemic steroids or immunosuppressives in all cases of posterior uveitis should be absolutely avoided. Judicial use of ancillary tests like FFA, ICG, USG, OCT, and electrophysiological tests as complimentary to clinical examination helps establish the right diagnosis. Intraocular fl uid testing for PCR and antibodies in infective conditions is useful in diagnostic dilemma. [bib_ref] Usefulness of aqueous humor analysis for the diagnosis of posterior uveitis, Rothova [/bib_ref] [bib_ref] Diagnostic vitrectomy for the diagnosis and management of posterior uveitis of unknown..., Margolis [/bib_ref] [bib_ref] Vitrectomy for the diagnosis and management of uveitis of unknown cause, Margolis [/bib_ref] It is essential to follow-up all patients with posterior uveitis even aft er the resolution of lesions for complications related to the disease, such as CNVM, hemorrhage, or breaks. Macular or optic disc involvement can cause irreversible visual impairment and hence early diagnosis and appropriate management is important to save vision.
[fig] Figure 1: Fundus picture showing a typical punched-out macular scar of a healed congenital toxoplasmosis [/fig]
[fig] Figure 2: Fundus picture showing a typical "headlight in the fog appearance" in a patient with acquired toxoplasmosis [/fig]
[fig] Figure 4: Fundus picture showing a tuberculous subretinal abscess Sudharshan, et al.: Diagnosis and management of posterior uveitis [/fig]
[fig] Figure 3: Fundus [/fig]
[fig] Figure 7: Fundus picture showing disc edema and macular exudates characteristic of a neuroretinitis [/fig]
[fig] Figure 5: Fundus picture showing the classical "cracked mud appearance" in progressive outer retinal necrosis [/fig]
[fig] Figure 6: Fundus picture showing a typical "pizza pie appearance" in a patient with cytomegalovirus retinitis underlying systemic disease under care of an internist promotes early resolution of ocular lesions. [/fig]
[fig] Figure 9: Fundus picture showing active geographic helicoid peripapillary choroidopathy Sudharshan, et al.: Diagnosis and management of posterior uveitis [/fig]
[fig] Figure 8: Fundus picture showing placoid lesions of acute posterior multifocal placoid pigment epitheliopathy Vol. 58 No. 1 [/fig]
[table] Table 1: SUN working group classifi cation of uveitis and the primary site of infl ammation [/table]
[table] Table 2: Basic management approach -guidelines [/table]
[table] Table 3: Other anti-Toxoplasmic drugs [/table]
[table] Table 4: Anti-Toxoplasma therapy in special situations [/table]
[table] Table 5: Basic management approach to a case of non-infective posterior uveitis Identify the clinical entity based on characteristic clinical feature Rule out infective causes Systemic steroids are the mainstay of therapy Use intravenous methyl prednisolone therapy in vision-threatening lesions Use immunosuppressives, with caution, in recalcitrant cases Monitor side effects of treatment [/table]
[table] Table 6: Comparative characteristics of clinical presentations of white dot syndrome[52] [/table]
[table] Table 7: Comparative characteristics of FFA and ICG features of white dot syndrome[52] [/table]
[table] Table 8: Clue to diagnosis of white dot syndrome Characteristics of lesions Consider [/table]
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HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia
Introduction: Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow-derived mesenchymal stromal cells) are known to be a component of the tumor microenvironment. BMSCs are multipotent stromal cells that can differentiate into a variety of cell types, including osteocytes, chondrocytes, adipocytes, epithelial cells and endothelial cells. Stem cells found in niches or transplanted into injured tissues constantly encounter hypoxic stress. Areas with very low to no oxygen pressure exist in solid tumors. The differentiation capacity of BMSCs under hypoxic conditions remains controversial. Methods: In this study, a hypoxic workstation, set at an oxygen concentration of 0.2% was used to mimic the hypoxic microenvironment of cancer in vivo. Oil red O staining and alkaline phosphatase staining were used to examine the adipogenic or osteogenic differentiation, respectively, of BMSCs. Real-time PCR was performed to explore the expression of adipocyte-or osteocyte-specific genes. An RT2 Profiler™ PCR Array was used to screen a panel of 84 genes associated with human adipogenesis in BMSCs under normal and hypoxic conditions. A dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were applied to analyze promoter activity to evaluate the possible regulatory mechanism of adipocyte-specific gene expression.Results:We found that this extreme hypoxia impaired osteogenic differentiation as indicated by the attenuation of alkaline phosphatase (ALP) activity and the reduced expression of osteogenic markers osteocalcin and osteopontin. Moreover, extreme hypoxia enhanced adipogenic differentiation, as indicated by the accumulation of lipid droplets and the expression of the adipocyte-specific genes leptin, LPL, CFD, PGAR and HIG2. In the extreme hypoxic conditions (0.2% oxygen), the overexpression of CCAAT enhancer-binding proteins (C/EBPs), especially C/EBPδ, and HIF-1A upregulated the promoter activities of adipocyte-specific genes such as leptin, CFD, HIG2, LPL, PGAR. In the present study, peroxisome proliferator-activated receptor-gamma (PPARγ) exerted a negative effect on the differentiation of BMSCs into adipocytes. Conclusions: In view of these findings, extreme hypoxia induced the adipogenic differentiation of BMSCs through HIF-1A and C/EBPs. These findings might provide clues regarding the roles of BMSCs in the cancer microenvironment.
# Introduction
At sea level the oxygen pressure is approximately 160 mmHg, whereas the oxygen pressure of tissues depends on the organ type. The oxygen pressure in normoxic tissue has been estimated to be 2 to 9% (14.4 to 64.8 mmHg) [bib_ref] Prevention of CCAAT/enhancer-binding protein beta DNA binding by hypoxia during adipogenesis, Park [/bib_ref]. This normal tissue oxygen pressure can therefore be considered hypoxic from a molecular standpoint. In some pathological conditions, such as heart disease, stroke, arthritis, wounds and tumors, oxygen deprivation is closely related to disease development. It has long been known that areas with very low or even zero oxygen pressure exist in solid tumors because aggressive tumor cells rapidly surpass the capacity of the nearest blood vessel. Tumor hypoxia appears to be strongly associated with tumor propagation, malignant progression and therapy resistance. Meanwhile, cancer cells have developed remarkable adaptive mechanisms to survive the severe hypoxia, including angiogenesis, autophagy and glycolysis.
Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow-derived mesenchymal stromal cells) are known to be a component of the tumor microenvironment. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including osteocytes, chondrocytes, adipocytes, epithelial cells and endothelial cells. Bone marrow-derived cells are crucial for the generation of a suitable microenvironment in the primary tumor, as well as for the development of metastasis [bib_ref] Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through..., Peinado [/bib_ref] [bib_ref] VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche, Kaplan [/bib_ref] [bib_ref] Bone marrow cells in the 'pre-metastatic niche': within bone and beyond, Kaplan [/bib_ref]. Many factors participate in the regulation of MSC differentiation. Differentiated MSCs also regulate the biological characteristics of cancer cells, and adipose MSCs have the ability to differentiate into mature adipocytes and initiate cytokine signaling within the tumor microenvironment [bib_ref] Sulforaphane inhibits mammary adipogenesis by targeting adipose mesenchymal stem cells, Li [/bib_ref].
Hypoxia is an important microenvironmental factor in the fate of MSCs. The roles of hypoxia in the differentiation of MSCs remain controversial. However, to investigate the roles of MSCs in the tumor microenvironment, the effect of extreme hypoxia on the differentiation of MSCs must be elucidated. In this study, we set the oxygen pressure at 0.2% to study the differentiation of MSCs in this nearly extreme hypoxic environment.
# Methods
## Mesenchymal stem cell isolation and culture
Human BMSCs were obtained from bone marrow aspirates of ribs from patients undergoing thoracic surgery. The isolation and culture of MSCs were performed using methods described previously [bib_ref] Mesenchymal stem cells as a gene therapy carrier for treatment of fibrosarcoma, Xiang [/bib_ref]. Samples were from the Second Xiangya Hospital, Central South University, Hunan, China. The patients were informed about the sample collection and signed informed consent forms. Collections and use of tissue samples were approved by the ethical review committees of Second Xiangya Hospital. BMSCs are a monolayer cultured in low-glucose Dulbecco's modified Eagle's medium (GE Healthcare Hyclone, Logan, Utah, USA), supplemented with 10% fetal bovine serum (Gibco, Life Technology, Shanghai, China), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells are cultured in 37°C in a humidified atmosphere of 5% carbon dioxide and are subcultured using 0.25% (w/v) trypsin-ethylenediamine tetraacetic acid solution. Osteogenic and adipogenic differentiation were performed using the differentiation media (Cyagen Bioscience Inc., Guangzhou, China). For hypoxia induction, BMSCs were incubated in 0.2% oxygen concentration at 37°C temperature, 5% carbon dioxide concentration and 90% humidity in a Hypoxic Workstation (Don Whitley, West Yorkshire, UK). Cells were lysed for extraction of protein and RNA in the workstation to avoid reoxygenation.
## Plasmids
Human CCAAT enhancer-binding protein (C/EBP) delta expression plasmid, which contains the full-length coding region of human C/EBPδ, was purchased from Origene (Rockville, MD, USA). Human hypoxiainducible factor 1 alpha (HIF-1A) expression plasmid was purchased from Addgene (Cambridge, MA, USA). C/ EBPα and C/EBPβ were constructed by inserting fulllength coding regions into PCDNA3.1. The firefly luciferase reporter plasmid pGL3-Basic plasmid was purchased from Promega (Madison, WI, USA). For screening promoter activity, five reporter gene vectors with promoter segments were constructed. The putative promoters, about 1,000 base pair upstream fragments of genes such as leptin, CFD, LPL, HIG2 and PGAR, were amplified by PCR from genomic DNA that was isolated using the Genomic DNA Extraction Kit (Takara, Dalian, China). Primer sequences are presented in [fig_ref] Table 1: PCR primers used for luciferase constructs [/fig_ref]. Cloning of different fragments to pGL3-Basic (Promega), which is a vector carrying the firefly luciferase gene, was performed after the restrictive enzyme cut. Finally, all plasmids were confirmed by sequencing. For the HIF-1A short hairpin RNA construct, oligo (5′-CTGATGACCAGCAACTTGA-3′) was designed to construct the lentivirus gene transfer vectors. The double-stranded short hairpin RNA oligo was cloned into the BLOCK-iT™ lentivirus vector (Invitrogen, Carlsbad, CA, USA). The lentivirus vector was cotransfected with the lentivirus package plasmids into 293FT cells. Forty-eight hours post transfection, the virus containing supernatant was harvested by collecting the medium. For infection of BMSCs, the cells were cultured in six-well plates, and when the culture reached 80% confluence the concentrated lentivirus was added to the culture dishes.
## Inhibitors and chemicals
The peroxisome proliferator activated receptor gamma (PPARγ) inhibitor GW9662 and C/EBP inhibitor betulinic acid were purchased from Sigma (St. Louis, MO, USA).
## Flow cytometry
Flow cytometry was performed on BMSCs that were stained for CD44, CD105, CD34, CD45 and CD11b. The following antibodies specific for human molecules were used: PC5-CD11b (Beckman Coulter, Brea, CA, USA), FITC-CD44 (Beckman Coulter), PC7-CD45 (Beckman Coulter, CA, USA), ECD-CD34 (Beckman Coulter, Brea, CA, USA) and PE-CD105 (eBioscience, CA, USA). Flow cytometry was performed on the Moflo XDP (Beckman Coulter, Brea, CA, USA). The corresponding isotype control monoclonal antibodies were from Beckman Coulter.
## Induction of adipogenic and osteogenic differentiation
After induction with differentiation media or hypoxia treatment, BMSCs were fixed in phosphate-buffered saline containing 4% Paraformaldehyde and stained with Oil Red O (cyagen, Guangzhou, China) or Alizarin red (cyagen, Guangzhou, China). Alkaline phosphatase (ALP) activity was determined using an ALP Staining Kit (beyotime, Shanghai, China).
## Quantitative pcr and rt 2 profiler arrays
Total RNA was extracted from cells and samples using Tri-zol® reagent (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using the Bio-Rad IQ™5 Multicolor Real-Time PCR detection System (Bio-Rad, Shanghai, China). The data were analyzed using iQ5 software (Bio-Rad, Shanghai, China). The relative gene expression was quantified on the basis of the threshold cycle value and normalized using the housekeeping gene β-actin. Data are representative of the means of three experiments. Student's t test was applied to compare two or more values; P <0.05 indicated that there was a significant difference. The quantitative PCR protocol was 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. A final melting curve analysis (60 to 95°C) was conducted. The standard curve was produced using a slope of approximately -3.32 (~100% efficiency). Primer sequences are presented in [fig_ref] Table 2: PCR primers used for gene expression [/fig_ref]. The human adipogenesis RT 2 Profiler PCR Arrays (Sabioscience, Shanghai, China) were used to profile the expression of 84 genes related to adipogenesis. RT 2 Profiler PCR Arrays were performed to quantify mRNAs of multiple genes, including housekeeping genes, according to the manufacturer's protocol. Total RNA was extracted from BMSCs in normoxic and hypoxic conditions with TRIzol. Single-strand cDNA was synthesized from 2 μg total RNA using the RT 2 first-strand cDNA synthesis kit (Sabioscience, Shanghai, China). The cDNAs were mixed with RT 2 Real Time SYBR Green/ROX PCR master mix (Sabioscience, Shanghai, China) and real-time PCR was performed in accordance with the manufacturer's instructions. Thermal cycling and fluorescence detection were performed using the Bio-Rad IQ™5 Multicolor Real-Time PCR detection System, and expressions of adipogenesisregulated transcripts were compared between the groups. Gene expressions with absolute fold change >2 were taken as differentially expressed genes between groups. The RT-PCR array data have been deposited in the public repository Gene Expression Omnibus [GEO:GSE65842]. The interactive network during the adipogenesis was
[formula] LPL 5′-TACTCGAGATGTGCATGCCTCTTA-3′ 5′-ATAAGCTTCAGGGCTTTGCTCTCC-3′ 1,179 CFD 5′-TCCTCGAGTGACTCTGTTCATCAGAAC-3′ 5′-GTAAGCTTCTACACAGCCCTGTCCCTC-3′ 989 HIG2 5′-TCCTCGAGTCTTTAGTTC AAGCCG-3′ 5′-ATAAGCTTCCGGAGGAAA GTCGGT-3′ 1,008 PGAR 5′-GACTCGAGAAAGTCTCTCCTGGTC-3′ 5′-ACAAGCTTGTTCCAGGTGCGAGGA-3′ 969 C/EBPδ 5′-TGCTCGAGATCTGCTCTGCTTT-3′ 5′-TCAAGCTTTGGAGTCGATGTAGGCG-3′ 1,001 Leptin 5′-TACTCGAGATCATGTAAA GCGGGG-3′ 5′-GCAAGCTTCAAGAAAGACCAGAGA-3′ 918OPN 5′-TTGCAGTGATTTGCTTTTGC-3′ 5′-GCCACAGCATCTGGGTATTT-3′ OCN 5′-GACTGTGACGAGTTGGCTGA-3′ 5′-CTGGAGAGGAGCAGAACTGG-3′ ALP 5′-CCACGTCTTCACATTTGGTG-3′ 5′-GCAGTGAAGGGCTTCTTGTC-3′ PGAR 5′-TGCAAGATGACCTCAGATGG-3′ 5′-CCATGATGCTATGCACCTTC-3′ HIG2 5′-CCACAGTGCAAGACTCCATC-3′ 5′-GCCATACTGCTGAGGAAAGC-3′ PPARγ 5′-GAGCCCAAGTTTGAGTTTGC-3′ 5′-CTGTGAGGACTCAGGGTGGT-3′ LPL 5′-AGTGGCCAAATAGCACATCC-3′ 5′-CCGAAAGATCCAGAATTCCA-3′ β-actin 5′-ACTGGAACGGTGAAGGTGAC-3′ 5′-AGAGAAGTGGGGTGGCTTTT-3′ [/formula]
constructed by Gene Network Central Pro™ (Sabioscience, Shanghai, China).
## Western blot
The protein used for western blotting was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer's instructions. Rabbitanti-human Hif-1A antibody was purchased from Santa Cruz (Santa Cruz, CA, USA); rabbit-anti-human Hif-1A antibody for chromatin immunoprecipitation was purchased from Abcam (Shanghai, China). β-actin antibody was purchased from Sigma. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1,000. Primary antibody was incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibody marked by horseradish peroxidase for 1 hour at room temperature. After rinsing, the Polyvinylidene Difluoride (PVDF) membrane carried blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system, and then 200 μl Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China).
## Luciferase activity assay
The firefly luciferase is widely used as a reporter of promoter activities by cloning interested promoters to the upstream of the firefly luciferase coding gene. The activity of experimental reporter (firefly) is normalized by the activity of the internal control (renilla). HEK293 cells were transfected with constructed reporter vectors simultaneously with plasmid vector (Promega, Madison, WI, USA) containing cDNA coding renilla luciferase, which is driven by CMV promoter. Forty-eight hours after transfection, cells were harvested with passive Lysis buffer supplied by the Dual-Luciferase Reporter (DLR™) Assay System (Promega), referring to the manufacturer's instructions. An appropriate volume of cell lysate was added into a well of the 96 MicroWell™ Plates (NUNC, Roskilde, Denmark), followed by 25 μl LARII. The firefly luciferase activities were measured with a luminometer (TECAN, Männedorf, Swiss). The renilla luciferase activities were also measured. The relative luciferase activity was represented by the ratio of firefly luciferase activity to renilla luciferase activity. Each experimental group included three repeats and data are shown as means.
## Chromatin precipitation
The chromatin immunoprecipitation assay was performed using the Chromatin Immunoprecipitation Assay kit (EZ ChIP; Millipore) according to the manufacturer's instructions. Briefly, HEK293 cells were transfected with pCDNA3.1-HIF-1A or human C/EBPδ. C/EBPδ plasmid construct was engineered to express the complete open reading frame with an expression Flag tag. Forty-eight hours after transfection, the cells were then cross-linked by 1% formaldehyde for 10 minutes. The formaldehyde was quenched using 2 M glycine for 5 minutes at room temperature before harvest. Cells were collected by centrifugation in phosphate-buffered saline containing protease inhibitors and were lysed in SDS-lysis buffer. Soluble chromatin was prepared after sonication to an average DNA length of 200 to 500 base pairs. Fragmented chromatin was immunoprecipitated using antibodies against HIF-1α (Millipore) or Flag together with Protein A/G PLUS-Agarose overnight at 4°C on a rotating platform. The agarose beads were washed, chromatin extracted and protein-DNA cross-links reversed. DNA was purified and was analyzed by PCR using the specific primers. Normal immunoglobulin G was used as a negative control. Anti-RNA Polymerase II was used as positive control. The total input was the supernatant from the no-antibody control.
# Statistical analysis
Statistical analysis was performed using the Statistical Package for Social Science-10 software (SPSS, Chicago, IL, USA) and GraphPad (San Diego, CA, USA). Data are presented as mean ± standard deviation of the results from three independent experiments. Results of gene expressions were analyzed by two-tail Student's t tests. P <0.05 was considered statistically significant.
# Results
## Mesenchymal stem cells under hypoxic conditions showed the potential to differentiate into adipocytes
BMSCs isolated from the ribs of patients undergoing thoracic surgery or bone marrow aspiration were screened by surface marker expression using flow cytometry. Immunophenotyping confirmed that the BMSCs displayed mesenchymal markers such as CD44 and CD105 and did not display the hematopoietic progenitor markers CD34, CD45 and CD11b [fig_ref] Figure 1: Phenotypic analysis of bone marrow-derived mesenchymal stem cells [/fig_ref]. The capability of these cells to differentiate into adipogenic and osteogenic lineages in vitro is displayed in [fig_ref] Figure 1: Phenotypic analysis of bone marrow-derived mesenchymal stem cells [/fig_ref]. Osteogenic or adipogenic media induced the osteocalcin production or lipid droplet accumulation, respectively.
To evaluate the effects of low oxygen levels, BMSCs were cultured under normoxia (21% oxygen) or hypoxia (0.2% oxygen). Three days after hypoxic treatment, ALP staining, Oil red O staining and real-time RT-PCR were used to assess the effects of hypoxia on osteogenic and adipogenic differentiation in BMSCs. BMSCs under hypoxic conditions showed less intense ALP staining and more intense Oil red O staining in normal media or differentiation media . Oil red O staining revealed that the lipid accumulation in BMSCs was significantly higher under hypoxic conditions than normoxic conditions, even with short-term hypoxic treatment .
As shown in ,D, hypoxia decreased mRNA expression of osteoblast marker genes ALP, OPN and osteocalcin and increased mRNA expression of adipocyte-associated genes such as LPL, leptin, HIG2, CFD and PGAR. The results showed that extreme hypoxia enhanced the adipogenic differentiation of BMSCs and inhibited the osteogenic differentiation of BMSCs. We also performed Oil red O staining and real-time PCR to evaluate the adipogenic differentiation of BMSCs under hypoxic conditions for an additional period of 7 and 14 days . BMSCs under hypoxic conditions showed more lipid accumulation than BMSCs under normoxic conditions. Treatment with complete media did not result in lipid accumulation under normoxia. After long-term hypoxic treatment, BMSCs expressed mature adipocyte markers such as leptin, HIG2 and PGAR. We noticed that when treated with adipogenic differentiation media, BMSCs under normoxia expressed more LPL compared with those under long-term hypoxia. This suggested that the early adipocyte marker LPL acts on preadipocytes during the early stage of BMSC differentiation. Hypoxia promoted the differentiation of BMSCs, and these BMSCs showed characteristics of mature adipocytes.
## Hif-1a transcriptionally regulated the adipogenic differentiation of bmscs
HIF-1A plays an important role in the adaptive response of cells to hypoxia. We then investigated the effect of HIF-1A on the adipogenesis of BMSCs under hypoxic conditions. Stable HIF-1A suppression in BMSCs was established using lentivirus-based delivery [fig_ref] Figure 3: HIF-1A regulates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells [/fig_ref]. HIF-1A knockdown BMSCs under hypoxic conditions showed less lipid droplet accumulation, indicating that HIF-1A may be involved in BMSC adipogenesis under hypoxic conditions [fig_ref] Figure 3: HIF-1A regulates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells [/fig_ref]. In the HIF-1A knockdown BMSCs, hypoxia induced decreased HIG2 and PGAR expression compared with BMSCs expressing wild-type HIF-1A [fig_ref] Figure 3: HIF-1A regulates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells [/fig_ref]. Electroporationmediated HIF-1A overexpression in BMSCs induced lipid droplet accumulation [fig_ref] Figure 3: HIF-1A regulates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells [/fig_ref].
## Gene expression profiling of bmscs under hypoxic conditions
To identify proteins involved in the regulation of adipogenic differentiation under hypoxic conditions, an RT 2 Profiler PCR Array was used to screen a panel of 84 genes associated with human adipogenesis in BMSCs under normal and hypoxic conditions. Fold changes in gene expression were calculated using the ΔΔ Ct method. The RT-PCR array data have been deposited in the public repository Gene Expression Omnibus [GEO:GSE65842]. As a result, 10 genes were upregulated, and 11 genes were downregulated by twofold in BMSCs under 0.2% oxygen compared with those under 21% oxygen . Expression of the genes encoding leptin, LPL and CFD was dramatically increased under 0.2% oxygen. Expression of the genes encoding PRDM16 and BMP2 was decreased under 0.2% oxygen . The mRNA levels of the adipogenic transcription factors C/EBPs, such as C/EBPα (fold change 1.87), C/ EBPβ (fold change 1.79) and C/EBPδ (fold change 6.15), were significantly higher in BMSCs under hypoxic conditions compared with cells under normoxic conditions. However, the mRNA levels of PPARγ were decreased under hypoxic conditions (downregulated by 3.36-fold; . The interactive network during the adipogenesis is shown in . As mentioned, extreme hypoxia induces a proadipogenic effect. Adipogenesis is driven by a complex transcriptional cascade that involves the activation of C/ EBPs, which are rapidly expressed after hypoxic treatment. However, PPARγthe master adipogenic transcriptional regulatormay inhibit the differentiation of BMSCs into adipocytes under extreme hypoxic conditions.
## Adipogenic differentiation of mscs is regulated by a c/ebp-mediated pathway
As indicated by the PCR array data, under extreme hypoxic conditions BMSCs differentiate into adipocytes in a C/EBP-dependent manner, and PPARγ may inhibit this adipogenic differentiation. We then used the PPARγ inhibitor GW9662 and the C/EBP inhibitor betulinic acid to treat the cells under hypoxic conditions, and Oil red O staining and real-time RT-PCR for adipocyte-specific genes were performed. These results showed that inhibition of PPARγ using GW9662 promoted the intracellular accumulation of lipid droplets and promoted the expression of the adipocyte-specific genes LPL and HIG2 . The inhibition of C/EBPs using betulinic acid resulted in less intracellular lipid droplet accumulation . Betulinic acid inhibited the expression of the adipocyte-specific genes CFD and leptin expression under hypoxic conditions, indicating that C/EBPs play crucial roles in the adipogenesis of BMSCs under extreme hypoxic conditions . Loss-of-function studies showed that C/EBPs, but not PPARγ, are necessary to promote the adipogenesis of BMSCs under extreme hypoxia.
From the above-described data, we concluded that HIF-1A and C/EBPs play roles in the adipogenesis of BMSCs under hypoxic conditions. We then investigated whether HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes, including leptin, LPL, CFD, PGAR and HIG2. The sequences approximately 1 kb upstream of the transcription start sites of these genes were cloned into pGL3 basic luciferase plasmids. The expected binding sites for HIF-1A and C/ EBPs in the promoters of these genes were searched for using TFSEARCH [fig_ref] Figure 6: HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes [/fig_ref]. The clones were cotransfected with the HIF-1A expression vector and the C/EBPα, C/EBPβ or C/EBPδ expression vector. Luciferase activity was measured 48 hours after transfection. Co-transfection with the HIF-1A expression vector markedly increased the reporter expression rate of leptin (46-fold), HIG2 (1.88-fold) and PGAR (2.43-fold). Cotransfection with the C/EBPδ expression vector markedly increased the reporter expression rate of leptin (71-fold), LPL (58-fold), PGAR (2.98-fold) and CFD (2.62-fold). HIF-1A and C/EBPβ coordinately upregulated the promoter activities of leptin and HIG2. The expression of leptin was also coordinately upregulated by HIF-1A and C/EBPδ. In conclusion, HIF-1A transcriptionally regulates the expression of leptin, HIG2 and PGAR; C/ EBPs, especially C/EBPδ, transcriptionally regulate the expression of LPL, CFD, leptin and PGAR [fig_ref] Figure 6: HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes [/fig_ref].
To confirm the binding of HIF-1A or C/EBPδ to the promoters of these adipocyte-specific genes, the chromatin immunoprecipitation assay was performed. A HIF-1A plasmid or a C/EBPδ plasmid was transfected into HEK293 cells. Corresponding antibodies were used to precipitate protein/chromatin complexes from sonicated samples. PCR data were obtained. As shown in [fig_ref] Figure 6: HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes [/fig_ref] , the binding of HIF-1A to promoter region of leptin, HIG2 and PGAR was observed. C/EBPδ has been shown to bind to the promoter region of LPL, leptin, CFD and PGAR.
# Discussion
The presence of hypoxic regions in a solid tumor has long been known. Compared with normal tissue, oxygen in human tumors can drop to very low concentrations or even to zero. For example, the oxygen pressure level in pancreatic cancer is 2.7 mmHg (0.3%) [bib_ref] Hypoxia-inducible factors as key regulators of tumor inflammation, Mamlouk [/bib_ref]. Thus, it is reasonable to investigate tumor biology under hypoxic conditions. In cancer biology, hypoxia in the tumor microenvironment is linked to angiogenesis, proliferation, cancer stem cell niche, immune escape and metastasis. The adipogenic differentiation of BMSCs under hypoxia can be considered as a molecular adaptation to help cancer cells survive the loss of vital molecules such as oxygen or energy. The roles of hypoxia in the differentiation of MSCs remain controversial. MSCs under reduced oxygen conditions were believed to preserve their stemness and remain undifferentiated [bib_ref] Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and..., Fehrer [/bib_ref] [bib_ref] Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal..., Holzwarth [/bib_ref]. However, it was also reported that hypoxia enhances mesoderm lineage differentiation, including adipogenic, osteogenic or chondrogenic differentiation [bib_ref] Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic..., Valorani [/bib_ref]. Hypoxia has been shown to promote or inhibit adipogenesis. MSCs showed reduced adipogenic differentiation under 1% oxygen pressure [bib_ref] Prevention of CCAAT/enhancer-binding protein beta DNA binding by hypoxia during adipogenesis, Park [/bib_ref]. However, when MSCs were exposed to an atmosphere containing 1% of oxygen, the formation of an adipocyte-like phenotype with cytoplasmic lipid droplet accumulation was observed. However, in that case, the expression of neither the mature adipocyte-specific genes leptin and adipophilin nor the early marker gene LPL was induced under the hypoxic environment, indicating that despite the accumulation of the lipid droplets, true adipogenic differentiation did not occur [bib_ref] Induction of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia, Fink [/bib_ref]. The discrepancy may be due to the degree of oxygen deprivation and the heterogeneous nature of MSCs. In our study, to investigate the roles of BMSCs in the cancer microenvironment, we set the oxygen concentration at 0.2%, which is much lower than the oxygen concentration used in most studies. When exposed to an atmosphere containing only 0.2% oxygen, MSCs underwent obvious adipogenic differentiation, displaying not only the accumulation of lipid droplets but also the expression of the early marker gene LPL and the mature adipocyte-specific gene leptin. The expression of LPL decreased after long-term hypoxia treatment, indicating that LPL acts on preadipocytes during the early stage of BMSC differentiation and that hypoxia can initiate differentiation more efficiently than normoxia.
The transcription factor nuclear receptor PPARγ, the family of C/EBP and the sterol regulatory element binding protein SREBP or SREBF are believed to be crucial for conversion of precursor cells to adipocytes [bib_ref] Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination..., Fajas [/bib_ref]. Several studies support the important role of PPARγ in adipocyte differentiation, identifying PPARγ as an essential and sufficient factor to induce adipocyte differentiation. However, in this study, in BMSCs under extreme hypoxic conditions of 0.2% oxygen that showed potent adipogenic differentiation, PPARγ and SREBF1 expression was obviously downregulated. The PPARγ inhibitor GW9662 enhanced the adipogenic differentiation potential of BMSCs under hypoxic conditions, indicating that the regulation of adipogenic differentiation of BMSCs under extreme hypoxic conditions is different than that under normoxia. It was reported that the expression of PPARγ2 is repressed in a hypoxic environment [bib_ref] Prevention of CCAAT/enhancer-binding protein beta DNA binding by hypoxia during adipogenesis, Park [/bib_ref]. The adipocyte-associated genes such as PGAR in white fat can be upregulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. Under extreme hypoxic conditions, rather than peroxisome proliferator activated receptor, HIF-1A and C/EBPs bind to gene promoters and increase gene expression. Hypoxia appears to exert a potent lipogenic effect that is independent of the PPARγ-regulated maturation pathway. Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor HIF, a heterodimer of HIF-α and the aryl hydrocarbon receptor nuclear translocator subunit [bib_ref] Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene..., Hu [/bib_ref]. Hypoxia inhibits Rb phosphorylation and blocks the DNA-binding capability of C/EBPβ to PPARγ2 in both a HIF-1A-dependent mechanism that induces p27Kip1 and a HIF-1A/p27-independent mechanism [bib_ref] Prevention of CCAAT/enhancer-binding protein beta DNA binding by hypoxia during adipogenesis, Park [/bib_ref]. The ob gene product leptin is exclusively expressed in adipose tissue and is a signaling factor that regulates body weight homeostasis and energy balance [bib_ref] The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression, Miller [/bib_ref]. Leptin can be considered a protein marker of terminally differentiated adipocytes [bib_ref] Leptin gene expression in human preadipocytes is switched on by maturation-induced demethylation..., Melzner [/bib_ref]. White adipose tissue is now recognized to be a multifunctional organ; in addition to the central role of lipid storage, it has a major endocrine function, secreting several hormones, notably leptin and adiponectin, and a diverse range of other proteins. The C/EBP family is widely expressed, and its members play critical roles in the regulation energy metabolism, inflammation, hematopoiesis, cellular proliferation and differentiation. C/EBP binding sites are located within upstream regions of the human ob gene and drive the highlevel expression of ob genes in adipocytes [bib_ref] The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression, Miller [/bib_ref].
In our human adipogenesis PCR array assay, C/EBPsespecially C/EBPδwere dramatically upregulated under the 0.2% oxygen concentration, suggesting that the C/EBP family plays roles in the adipogenic differentiation of BMSCs. The C/EBP inhibitor betulinic acid reduced lipid droplet accumulation and leptin, LPL, CFD, PGAR and HIG2 expression under hypoxic conditions, indicating that C/EBPs transcriptionally regulate the adipogenic differentiation of BMSCs under extreme hypoxic conditions. C/ EBPδ is considered an acute phase response transcription factor [bib_ref] Loss of CCAAT/ enhancer binding protein delta promotes chromosomal instability, Huang [/bib_ref]. The expression of C/EBPδ is low or undetectable in most cell and tissue types. However, expression is rapidly induced by a variety of extracellular stimuli, such as growth hormone, insulin, interferon gamma, interleukin-1, interleukniin-6, lipopolysaccharide, TNFα and dexamethasone [bib_ref] CCAAT/enhancer-binding proteins: structure, function and regulation, Ramji [/bib_ref]. In the lipopolysaccharide-induced inflammatory response, C/EBPδ induced by nuclear factor-κB promotes the production of proinflammatory cytokines [bib_ref] Suppression of inflammation and acute lung injury by Miz1 via repression of..., Do-Umehara [/bib_ref].
# Conclusions
BMSCs under extreme hypoxia show the potential to differentiate into adipocytes and high adipokine expression. HIF-1A and C/EBP, especially C/EBPδ, play important regulatory roles in the process of differentiation.
[fig] Figure 1: Phenotypic analysis of bone marrow-derived mesenchymal stem cells. (A) Bone marrow-derived mesenchymal stem cells (BMSCs) were analyzed by flow cytometry analysis for the surface expression of CD34, CD44, CD45, CD105 and CD11B. (B) BMSCs were tested for their differentiation potential into osteocytes and adipocytes when cultured in differentiation media. Alizarin red staining and Oil red O staining were performed. (I) Alizarin red S staining showed calcium deposition (red). (II) Oil red O staining revealed lipid droplets (red) in adipocyte-differentiated mesenchymal stem cells. Magnification: 200 ×. [/fig]
[fig] Figure 3: HIF-1A regulates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells. (A) Stable hypoxia-inducible factor 1 alpha (HIF-1A) suppression in bone marrow-derived mesenchymal stem cells (BMSCs) was established using lentivirus-based delivery. (B) Oil red O staining revealed that BMSCs with knockdown of HIF-1A under hypoxic conditions showed less formation of lipid oil (magnification: 100×). (C) Real-time PCR results showed that hypoxia in the HIF-1A knockdown BMSCs induced decreased expression of HIG2 and PGAR compared with BMSCs expressing wild-type HIF-1A. (D) Oil red O staining showed intracellular lipid droplet accumulation after electroporation of a HIF-1A construct (magnification: 100×). (E) Real-time PCR assays showed that electroporation-mediated HIF-1A overexpression in mesenchymal stem cells induced the expression of HIG2 and PGAR, but not LPL. **p <0.01; ***p <0.001. [/fig]
[fig] Figure 6: HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes. (A) Predicted binding sites of hypoxia-inducible factor 1 alpha (HIF-1A; blue boxes) and CCAAT enhancer-binding proteins (C/EBPs; red boxes) in the sequence approximately 1 kb upstream of the transcription start sites (TSSs). Luciferase activity was measured 48 hours after transfection. Co-transfection with a HIF-1A expression vector markedly increased the reporter expression rate of leptin, HIG2 and PGAR. Co-transfection with a C/EBPδ expression vector markedly increased the reporter expression rate of leptin, PGAR, CFD and LPL. HIF-1A and C/EBPβ coordinately upregulated the promoter activities of leptin and HIG2. The expression of leptin was also coordinately upregulated by HIF-1A and C/EBPδ. (B) Chromatin immunoprecipitation analysis of HIF-1A or C/EBPδ binding to the promoters was conducted. DNA released from the precipitated complexes was amplified by PCR and the PCR products were separated by agarose gel electrophoresis. Lane 1, DNA ladder; lanes 2 to 6, HIF-1A antibody or C/EBPδ antibody pull-down; lane 7, anti-RNA polymerase II; lane 8, immunoglobulin G control; lanes 9 to 13, input. [/fig]
[table] Table 1: PCR primers used for luciferase constructs [/table]
[table] Table 2: PCR primers used for gene expression [/table]
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Gut Microbiota and Chemical-Induced Acute Liver Injury
Background: Drug overdose or chemical exposures are the main causes of acute liver injury (ALI). Severe liver injury can develop into liver failure that is an important cause of liver-related mortality in intensive care units in most countries. Pharmacological studies have utilized a variety of comprehensive chemical induction models that recapitulate the natural pathogenesis of acute liver injury. Their mechanism is always based on redox imbalance-induced direct hepatotoxicity and massive hepatocyte cell death, which can trigger immune cell activation and recruitment to the liver. However, the pathogenesis of these models has not been fully stated. Many studies showed that gut microbiota plays a crucial role in chemical-induced liver injury. Hepatotoxicity is likely induced by imbalanced microbiota homeostasis, gut mucosal barrier damage, systemic immune activation, microbial-associated molecular patterns, and bacterial metabolites. Meanwhile, many preclinical studies have shown that supplementation with probiotics can improve chemical-induced liver injury. In this review, we highlight the pathogenesis of gut microorganisms in chemical-induced acute liver injury animal models and explore the protective mechanism of exogenous microbial supplements on acute liver injury.
# Introduction
Acute liver injury (ALI) is a common disease that seriously threatens the life and health of the patients. The main pathological manifestation is a sharp decline in liver function caused by the necrosis of a large number of hepatocytes. ALI caused by multiple etiologies has become a crucial public-health issue at both regional and global scales. The most common causes of ALI in developed countries are acetaminophen (APAP) induced hepatotoxicity, drug induced liver damage, autoimmunity, and viral hepatitis B, which accounts for about 70% of the cases; on the other hand, the most common causes in developing countries are viral hepatitis A, B, and E. Currently, liver transplantation (LT) is the only clinically essential therapy for the treatment of acute liver failure (ALF). However, the rapid progression of ALI, the scarcity of liver sources, and the high medical costs limit its application. Therefore, it is necessary to find other effective interventions for severe acute liver injury patients. Recently, there is increasing evidence points to the intimate connection between gut microbiota and liver injury. However, compared with ALI, these studies mostly focused on chronic liver diseases, such as non-alcoholic fatty liver disease (NAFLD), autoimmune hepatitis, liver cirrhosis, and hepatocellular carcinoma. In recent years, an increasing number of preventive or adjunct therapeutic measures for ALI have been proposed based on the regulation of gut microbiota. The gut contains around 1,000 different species of bacteria, which play a crucial role in the survival of organisms. The crosstalk between gut and liver is increasingly recognized. Gut microbiota has been defined as a significant micro-ecosystem, which is symbiotic with the organism and participates in a variety of physiological and pathological processes.
In this review, we focused on the current knowledge of the gut microbiota's contribution and the protective mechanism of exogenous microbial supplements on chemically induced ALI.
## Role of the microbiota in the immune homeostasis of the gut, liver and liver disease
Immune Homeostasis of the Gut and Liver
The immune system plays a critical role in maintaining the symbiotic relationship between the microbiome and the host. Intestinal mucosa-associated lymphoid tissue contains a variety of immune cells, such as antigen-presenting cells (APCs), innate lymphocytes, T cells, and B cells, which play a vital role in the host immune response. Gut bacteria regulate the maturation of the mucosal immune system, which in turn affects the composition of the gut microbiota. Studies have shown that microbes activated the immune cells to produce cytokines and initiated the host's immune response to balance the intestinal tolerance and immunity. Thus, the homeostasis of the gut microbiota is essential for the maintenance of gut and liver metabolism and immune homeostasis . For example, intestinal symbiotic bacteria help induce a stable production of Th17 cells in the lamina propria of the small intestine and protect the intestinal mucosal barrier, and some bacteria also play an immunomodulatory role of macrophage polarization.
Dysregulation of the gut microbiota leads to an inability of the intestinal mucosal barrier to resist an infection with pathogenic bacteria and facilitates the occurrence of diseases affecting the normal physiological function of the host. Meanwhile, gut metabolites, such as short-chain fatty acids (SCFAs), can also modulate local or systemic immune responses. Studies have demonstrated that SCFAs can affect the differentiation of B cells and maintain the homeostasis of intestinal T cells. Butyric acid, a representative SCFAs, is necessary for the colonization of intestinal bacteria.
Recent studies have suggested that yeast β-glucan, a polysaccharide produced by fungi, can contribute to the differentiation of Th2 cells and maintain the steady-state of the intestinal immune system. Additionally, the FIGURE 1 | The main role of gut microbiota in gut-liver-immune axis under physiological condition. First, by utilizing the nutrients and metabolic substrates (such as primary bile acids from liver), gut microbiota can produce various bioactive metabolites (such as acetate, propionate, butyrate, secondary bile acids, and amino acids) which were absorbed through the portal vein into the liver, to regulate hepatic function. Second, gut microbiota can also directly or indirectly establish and balance the hepatic immune response through metabolites, secondary bile acids and MAMPs. SCFAs, short chain fatty acids. MAMPs, microbial-related molecular patterns. Figure was partly adapted from Smart Servier Medical Art (https://smart.servier.com/), which is licensed under a Creative Common Attribution 3.0 Generic License. gut microbiota maintains the immune homeostasis of the liver through the gut-liver axis. The liver proposed as an important innate immune organ contains a large number of immune cells, such as NK, Kupffer, and T cells. The activation of Kupffer and T cells can trigger the innate and adaptive immune response when the liver is continuously stimulated by antigens, pathogens, and endotoxins produced by the gut microbiota. This process can affect the tolerance of liver immunity.
## Liver disease
In recent years, the gut microbiota has received increasing attention because of its central role in host-microbiota interaction and its impact on liver diseases . A healthy gut microbiota can maintain liver metabolism and immune homeostasis. Meanwhile, this direct connection can cause several "negative effects." Bacteria-derived products or toxins can get into the liver and disrupt its homeostasis if the liver immune system is pre-imbalanced or the natural barrier of the intestine is disrupted. A large number of intestinal-derived toxic products enter the liver and are recognized by specific receptors, such as TLR. This can promote an inflammatory response, direct liver cell death, or chronic liver damage.
Bile acids and many other bioactive substances released by the liver enter the gut to be processed and metabolized by intestinal bacteria. On the other hand, the liver receives 70% of its blood supply from the gut via the portal vein, which contains metabolites, endotoxins, peptidoglycan and even microbiota. Thus, various types of liver cells, including hepatocytes, kupffer cells, hepatic stellate cells, sinusoidal cells, and biliary epithelial cells can respond to signals from the gut and affect liver function. The interaction between the gut microbiota and the liver involves multiple components, including metabolism, immunity, and neuroendocrine signals, and constitutes a complicated interaction network. The gut-liver axis plays an essential role in the pathogenesis of numerous liver diseases, such as alcoholic liver disease (ALD), chronic hepatitis, NAFLD, liver cancer, and drug-induced liver injury.
In general, increased intestinal permeability and bacterial translocation promote the arrival of microbial metabolites to the liver and aggravate the liver injury. This leads to dysfunctions in bile acid metabolism and intestinal peristalsis, systemic inflammation, and liver damage. The level of liver damage is closely related to the severity of intestinal disorders. Alteration of the bacteria that produce SCFAs, which dominate the composition of gut microbiota, has already been reported to affect intestinal homeostasis. Thus, a treatment based on microbial intervention is a promising method to improve the pathological process in the liver.
At present, many studies have shown that supplementing with probiotics can balance the gut homoestasis, reduce intestinal bacterial translocation, inhibit liver inflammation, and improve acute liver damage Table 1. Recent studies have also demonstrated that differences of distribution, specific composition and metabolites produced by the gut microbiota constitute the risk for acute liver injury, the major ways for the gut microbiota participate in ALI. Thus, the specific mechanisms of gut microbiota in the onset and progression of ALI deserve to further study. Considering the different types of ALI, we summarized the currently reported four chemical-induced acute liver injury model that modulated by gut microbiota.
## The gut microbiota and apap-induced liver injury
Drug-induced liver injury (DILI) is the main cause of clinical liver injury in developed countries. For example, in the United States, nearly 46% of ALI cases were caused by overdose or abuse of N-acetaminophen (APAP). APAP has been the focus of the Food and Drug Administration (FDA) advisory committees over the past several decades. The degree of liver injury is the determining factor in acute liver failure survival without transplantation. During the past few decades, around three-quarters of APAP-induced ALI patients survive with their naïve liver. Generally, APAP is a safe drug when used at therapeutic doses for the treatment of fever and pain (1-4 g/days). Its safety margin is, however, relatively narrow. A randomized study reported that a maximum dose of APAP for 5 days in healthy adults increased the level of serum transaminase. Meanwhile, the susceptibility to APAP-induced liver injury may also be due to several risk factors, such as alcohol use, obesity, nutritional depletion, and the use of drugs that stimulate the cytochrome P450 (CYP) system.
Typically, APAP is cleared in the liver primarily where it binds to O-sulfuric acid or glucuronic acid and is excreted into the bile or urine. At therapeutic doses, only a small amount of APAP is metabolized by the cytochrome P450 enzyme (CYPs) to N-acetyl-p-phenyl quinone imine (NAPQI). With high doses of APAP overdose or if the ability of glucuronide and sulfate esterification is saturated, CYPs (mainly CYP-2E1 and CYP-3A4 in mammals) become the major APAP metabolic enzyme and lead to the massive production of NAPQI, a highly active intermediate compound that leads to cytotoxicity. In the early stage, the majority of NAPQI is rapidly detoxified and excreted into the urine by binding with glutathione (GSH). Once the GSH storage is depleted, the remaining NAPQIs accumulate in hepatocytes and bind covalently to proteins sulfhydryl groups, producing harmful APAP protein adduct (APAP-ADs) that irreparably lead to liver cell necrosis.
N-acetylcysteine (NAC), the only approved therapeutic drug for APAP-induced hepatotoxicity, can protect the liver from damage by providing cysteine precursors and restore the hepatocytes GSH stores. However, the treatment window for NAC is narrow and requires a controlled use of APAP (no more than 24 h). The accumulation of NAPQIs can destroy cytoplasmic membranes and induce severe mitochondrial dysfunctions, an overproduction of reactive oxygen species (ROS), ATP depletion, a fragmentation of nuclear DNA, and lipid peroxidation. Therefore, massive hepatocellular necrosis subsequently leads to several damageassociated molecular patterns (DAMPs) releases, which activate macrophages and induce a sterile inflammation . Activated macrophages produce several chemokines and cytokines, which induce the intrahepatic aggregation of neutrophils, monocytes, and other immune cells and trigger intrahepatic and systemic inflammatory responses. Notably, sterile inflammatory responses not only clear necrotic cell debris and promote tissue repair, but also aggravate liver injury . Finally, when the degree of hepatocyte damage exceeds regeneration, the liver function rapidly fails.
The hepatotoxic effect of paracetamol is still considered to be the most important part of ALI. However, recent evidence suggests that changes in the gut microbiota, including their abundance, diversity, metabolites, intestinal permeability, and bacterial translocation also play profound roles in APAP-induced hepatotoxicity.
Several studies have shown that the presence of gut microbiota is important for APAP-induced hepatotoxicity. found that the degree of APAP liver injury in germ-free (GF) Wistar rats LPS / D-GalN, intraperitoneal injection of 50 µg/kg LPS and 300 mg/kg D-GalN and sacrificed after 8 h.
Modulation of the TLR-MAPK-PPAR-γ pathways to reduce pro-inflammatory cytokines and hepatic inflammationLactobacillus rhamnosus GG (mixed with drinking water approximately 10 9 CFU /daily for 5 days)
C57BL/6N mice Alcohol, oral gavage of 6 g/kg bodyweight and mice were sacrificed after alcohol gavage 1.5 or 6 h.
Activating HIF signaling to decrease the damage of alcohol-induced increased intestinal permeability and endotoxemia.
D-GaIN, D-galactosamine; APAP, acetaminophen; 5-MIAA, 5-methoxyindoleacetic acid; Con A, Concanavalin A.
mice or mice cleared of gut microbiota was lower than in SPF mice. The expression of the P450 enzyme, which is involved in APAP metabolism in the liver, is closely linked to intestinal microorganisms. The expression levels of CYP-1A2 and CYP-3A4 in the liver of GF mice were significantly lower than in SPF mice, which may explain the significant reduction of APAP-induced hepatotoxicity in GF mice. Meanwhile, single-cell sequencing data showed that there were significantly fewer LY6C-positive monocytes and a lower proportion of activated non-parenchymal cells (hepatic stellate cells, sinusoidal endothelial cells, and Kupffer cells) in the liver of antibiotic-treated mice compared with SPF mice.
The key mechanism depends on the clearance of intestinal microbiota, which dramatically reduces the microbial-associated molecular patterns (MAMPs) entering the liver via the portal vein. Blocking the TLRs-MYC signaling pathway of MAMPs can downregulate the activation of stellate, endothelial, and Kupffer cells and significantly decrease the expression of chemokines and pro-inflammatory mediators, thus reducing the intrahepatic inflammatory response in APAP-induced model. These results suggested that single cell sequencing is an important method to study the direct relationship between gut microbiota and liver. Similarly, the change in diversity and abundance of the intestinal microbiota can also participate in the transformation and detoxification of APAP. Zheng et al. changed the composition of the gut microbiota in mice using vancomycin, which reduced the abundance of Grampositive bacteria in the intestine and increased the level of 2-hydroxybutyric acid in the cecum and serum. The bioavailability of APAP was also decreased and the level of GSH in the liver was increased, which improved the APAP-induced liver injury in mice. The pharmacokinetics of APAP is also affected by changing the composition of gut bacteria in mice. Several researchers showed that the degradation of APAP increased by 68% in Frontiers in Physiology | www.frontiersin.org mice treated with Lactobacillus reuteri, while treatment with Lactobacillus rhamnosus did not show a similar effect.
The susceptibility to APAP toxicity varies considerably among different individuals. Endogenous p-cresol, a protein residue in the intestinal cavity, is mainly produced by intestinal microorganisms, such as Clostridium difficile. Pcresol absorbed into the liver is transformed into p-cresol sulfate by sulfotransferases in hepatocytes. Likewise, acetaminophen is a substrate of sulfotransferases and p-cresol can reduce the ability of sulfotransferases to sulfonate acetaminophen. The content of p-cresol varies greatly in different individuals, which may explain the different sensitivity of individuals to APAP hepatotoxicity. Furthermore, individuals with intestinal dysbiosis are more sensitive to APAP-induced liver injury. Schneider KM et al. analyzed a cohort of 500,000 participants in the British Biobank and found that proton pump inhibitors (PPI) or long-term antibiotics (ABX) can cause intestinal microbial dysbiosis. The risk of ALF induced by APAP was significantly increased in participants with intestinal microbial dysbiosis. Similarly, compared with wild-type mice, Nlrp6 −/− mice (an intestinal dysbiosis model) also showed that microbial dysbiosis could aggravate APAP-induced liver injury. This phenotype was reproduced after fecal bacteria transplantation.
Microbial communities are altered by the circadian rhythm system and affect the host metabolism, energy homeostasis, and immune system. Disruptions in the rhythm function of microbe-host interactions can seriously affect the pathology and severity of the disease. Interestingly, changes in the gut microbial circadian rhythm can affect APAP-induced liver toxicity. 16S rRNA gene sequencing showed that changes in the daily rhythm were associated with changes in the relative abundance of gut microbiota. Compared with ZT0, the ratio of Firmicutes/Bacteroides in ZT12 was significantly reduced. The hepatotoxicity of APAP was more severe at night (ZT12) than in the morning (ZT0). This was likely due to the increased abundance of the metabolite 1-phenyl-1,2-propanedione (PPD), which can consume GSH in hepatocytes after being ingested by the liver through the portal vein at ZT12. Therefore, early consumption of GSH can cause the accumulation of a large amount of NAPQI in the liver of mice with an excessive APAP metabolism, which can aggravate the liver injury. The oral gavage of APAP weakens the intestinal mucosal barrier function during intestinal absorption and allows plasma albumin to seep into the intestinal cavity. Similarly, a higher intestinal permeability can also allow abundant harmful substances to enter the liver, which aggravates the inflammatory reaction and the hepatotoxicity of APAP. Niu et al. confirmed that the disruption of the intestinal barrier integrity, which may be mediated by intestinal immune microenvironments, can aggravate APAP-induced hepatotoxicity.
A high concentration of APAP can rapidly induce the apoptosis of Lgr5 + crypt basal stem cells in the small intestine. Although apoptotic cells are completely removed within 24 h, the potential consequence is a long-term defect in the intestinal barrier function. Apoptosis of Lgr5 + crypt basal stem cells may partly explain why the low 30-days survival rate of ALF caused by APAP toxicity in liver transplant recipients compared with ALF caused by non-APAP-related causes.
Probiotics restore the balance of the intestinal microbiota (symbiotic and pathogenic bacteria), maintain the integrity of the intestinal barrier, reduce the production of toxic products, and improve liver function. We wondered, however, how probiotics could affect the APAP-induced hepatotoxicity. It is a known fact that gut microbiota and its metabolites are involved in the regulation of oxidative stress and inflammation which play key roles in drug-induced hepatotoxicity. Many studies showed that, compared with GF mice, normal mice displayed an upregulation of the transcription factor Nrf2 in liver that improved the antioxidant and xenobiotic response to protect the liver from acute acetaminophen; this signaling upregulation is enhanced by supplementary of human commensal Lactobacillus rhamnosus GG. Meanwhile, Sharma et al. reported that probiotic Enterococcus lactis IITRHR1 and Lactobacillus acidophilus MTCC447 protect against APAP-induced liver injury by modulating the antioxidant capacity of the liver and the expression of key apoptotic/antiapoptotic proteins. MegaSporeBioticTM probiotic capsules are composed of a probiotic blend of spores from five Bacillus species that improve the histopathological hepatic injury and decreased the level of proinflammatory cytokines, indicating that Bacillus spa spores have a protective effect on acute hepatic injury induced by APAP. This data is significant for the treatment of ALI and we also need to further explore whether the pretreatment of probiotics could improve the detoxification ability and antioxidant capacity of hepatocytes.
## The gut microbiota and con a-induced autoimmune hepatitis
Autoimmune hepatitis (AIH) is a complicated immune-mediated liver disease with a variable clinical phenotype that occurs worldwide. In the UK, the incidence rate of AIH reaches nearly 1.94 cases per 100,000 people. The histological diagnosis of AIH comprises interfacial hepatitis, increased serum alanine aminotransferase (ALT), increased aspartate aminotransferase (AST), elevated immunoglobulin G (IgG) levels, and the presence of autoantibodies. Additionally, patients with acute severe autoimmune liver disease are more likely to develop acute liver failure and may need LT. However, autoimmune liver disease can still develop or recur in allografts with a 5-years recurrence rate of 36% to 68% after LT. The mechanism of AIH recurrence after LT is still unclear. However, several extrahepatic factors may play an important role in the recurrence of AIH. Mounting evidence showed that the mechanisms of AIH were related to gut dysbiosis. Moreover, microbiome restoration therapies, such as probiotics, prebiotics, and fecal microbiota transplantation (FMT), can effectively improve AIH. The study of gut microbiota will provide new insights into the mechanism of AIH.
Due to the lack of widely accepted and valid mouse models for AIH, the research on the pathogenesis of AIH is still very limited. Concanavalin A (Con A) is a lectin originally extracted from jack-bean. In early 1992, it was first used to establish an immune hepatitis model. Con A-induced hepatitis is a typical chemical model that is used to investigate the cellular and molecular mechanism of immune-mediated liver injury. The model is acute and the injury caused by Con A usually lasts for only 48 h. Con A can partly simulate the pathogenesis of human acute autoimmune liver diseases as it can rapidly induce the activation of natural killer T (NKT) cells and CD4 positive T cells in the mice liver and release a mass of cytokines to cause liver damage. Here, we mainly summarize the role of gut microbiota in Con A-induced hepatitis and summarize the discovery and progress of immunotherapy related to the gut-liver axis.
NKT cell activation plays a critical role in Con A-induced hepatitis and the gut microbiota regulates the activation of NKT cells. shown that GF mice are not sensitive to Con A-induced liver injury when compared with SPF mice. This is mainly because NKT cells in the liver of GF mice are not activated after Con A treatment. Meanwhile, compared with GF mice, Con A treatment can significantly increase the circulation of LPS and the level of glycolipid antigen presented by CD1d in SPF mice. Intestinal microbial-derived antigens (glycolipids) are also important activators of liver NKT cells as they can activate them to mediate Con A-induced ALF.
Furthermore, Chen et al. showed that exogenous pathogenic bacteria exposed to the gut can exacerbate Con A-induced liver injury. This may be due to the increase of DCs activation, which subsequently augments the cytotoxicity of hepatic NKT cells against the liver parenchyma cells. In contrast, gentamicin treatment, which is bactericidal mainly against gram-negative (G − ) organisms in the gut, can alleviate Con A-induced ALI. Meanwhile, Celaj et al. demonstrated that differences in the gut microbiota could determine the sensitivity to Con A-induced acute liver injury. They found that mice from the Taconic Farms (TAC), and the Jackson Laboratory (JAX) exhibited different levels of liver damage induced by Con A. This difference in sensitivity is caused by the regulation of the Fas response pathway in the gut microbiota. Interestingly, this difference in susceptibility disappeared after co-housing (a way for minimizing the discrepancy of gut microbiota from the different background). By analyzing the fecal microbe by 16S rRNA gene sequencing, they found that among the identified OTUs, 8 genera abundance exhibited statistically significant differences in the two company's mice. Furthermore, they found the abundance of Ruminococcaceae was positively associated with the degree of liver injury. Dopamine is an important neurotransmitter and also an immune modulator.
It participates in the regulation of the T cell function through the D1-like receptor. Xue et al. demonstrated that gut microbes can improve the synthesis of peripheral dopamine to inhibit the activation of hepatic iNKT cells and alleviate Con A-induced liver injury. Different with in GF mice, the depletion of the gut microbiota by antibiotics reduced the synthesis of dopamine and exacerbated Con A-induced liver injury. These conflict phenotypes may be resulted from the difference of microbiota abundance between GF and antibiotics treated mice. Nobuhito et al. showed that inducing colitis with DSS for 7 days led to intestinal leakage, exposed intestinal microbes to the systemic immune cells and the liver through the portal vein, induced a systemic immune tolerance, and thereby reduced Con A-induced liver damage.
Due to the high plasticity of the intestinal microbiota, the research of exogenous probiotics or prebiotics in autoimmune liver disease is progressing substantially. However, there are few reports about the impact of probiotics or prebiotics on Con A-induced immune liver injury. The gut microbiota may influence the susceptibility and severity of acute liver injuries caused by Con A in mice based on current evidence. Gabriela et al. reported that Propionibacterium acidipropionici CRL 1,198 decreased the proliferative effects of lectins in adenocarcinoma cells, inhibited the fermentative activity of colonic microbiota, and avoided undesirable lectin-epithelia-microbiota interactions. As a promising probiotic with beneficial effects on liver diseases, Akkermansia muciniphila pre-treatment can also alleviate liver damage by altering transaminase activities and attenuate systemic inflammation by suppressing cytokines (including IFN-γ, IL-1β, IL-2, and IL-12p40) in Con A-induced liver injury. The beneficial effects of A. muciniphila were partly dependent on improved intestinal barrier and restored composition and function of gut microbiota.
Therefore, the study of the relationship between intestinal microorganisms and liver immune homeostasis is helpful for the diagnosis and treatment of acute autoimmune liver injuries.
## The gut microbiota and alcohol-induced acute liver injury
Alcohol (ethanol) abuse is a common risk factor for multiple diseases including alcoholic liver diseases (ALD), cardiovascular diseases, and inflammatory bowel diseases. Every year, nearly 4% of adults get sick because of drinking and the harmfulness of alcohol even exceeds the perniciousness of smoking. Alcohol is transformed by alcohol dehydrogenases and cytochrome CYP-2E1 to acetaldehyde in the endoplasmic reticulum of hepatocytes. Acetaldehyde is converted into acetic acid by acetaldehyde dehydrogenases and is finally excreted in urine. However, the gut microbiota also have a crucial part to play in the metabolism of ethanol because a large number of bacteria can express alcohol dehydrogenases in the colon. Evidence showed that a leaky gut, bacterial translocation, and intestinal inflammation modulate the susceptibility of acute ALD. showed that compared with GF mice, administration of alcohol for 7 days can cause significant liver injury and increase the level of neutrophil infiltration and proinflammatory cytokines (CXCL-1 and interleukin (IL)-6) in SPF mice. This implied that the gut microbiota was directly or indirectly involved in acute ALD. However, our data showed that a single gavage of a high concentration of alcohol (300 µl of 30% (vol/vol) EtOH at a dose of 3 g/kg) caused more severe liver damage, inflammation, hepatic steatosis, and higher levels of CYP-2E1 in GF mice than in SPF mice, which also indicated that the intestinal microbiota was directly or indirectly involved in alcohol-induced liver damage. According to these results, the time and concentration of alcohol exposure may explain the inconsistencies between the two programs.
To date, several studies showed that changes in the enterohepatic axis, such as increased permeability of the intestinal barrier, a thinning of the protective mucosal layer, and changes in the gut microbiota, occurred after alcohol consumption. Akkermansia muciniphila, a Gram-negative bacteria, can enhance mucus production to improve intestinal barrier function. Studies have documented that A. muciniphila abundance is significantly decreased in patients with alcoholic hepatitis and mice exposed to alcohol. Interestingly, the presence of alcohol did not affect the commensal A. muciniphila growth in vitro. The mechanism by which ethanol depletes A. muciniphila remains unclear. Lee et al. demonstrated that a low-dose (0.8 g/kg/days) of alcohol for 7 days can change the fecal microbiota composition, while fermented rice liquor can restore the microbial composition and inhibit intestinal inflammation .
The regulation of the gut microbiota appears to be a promising strategy to improve acute ALD. Pharmacological research showed that several drugs could regulate the composition of the gut microbiota and treat acute ALD. Audrey et al. proved that rhubarb extracts increased the abundance of mucus A. muciniphila and Parabacteroides goldsteinii by decreasing the activation of the TLR4 pathway and reducing the levels of inflammation and oxidative stress in the liver tissue, which improved acute liver injury caused by alcohol (30% w/v, 6 g/kg body weight). Meanwhile, there have been reports on the intervention of the gut microbiota in acute alcoholic liver disease. The supplementation of Pediococcus pentosaceus CGMCC 7,049 improved the intestinal barrier function and reversed gut microbiota dysbiosis by reducing the level of circulating endotoxin and proinflammatory cytokines .
Animal studies have shown that probiotics and synbiotics improved gut microbiota disorders, enhanced the mucosal barrier, and protected hepatocyte from acute and chronic ethanol injury. However, clinical studies data is still insufficient and we need further investigation in the future.
## The gut microbiota and d-galactosamine-induced acute liver injury
D-galactosamine (D-GalN), a hexosamine derived from galactose, is a component of various glycoprotein hormones and a hepatotoxic agent. Intraperitoneal injection of D-GalN can cause diffuse hepatocyte necrosis and inflammation analogous to the changes of the liver pathology after clinical viral hepatitis. In general, the co-administration of a sublethal dose D-GalN and lipopolysaccharide (LPS) has been widely used in repetitive experimental animal models of fulminant liver failure in the clinic. The hepatotoxicity induced by D-GalN is mainly due to a decrease in the concentration of uridine diphosphate in hepatocytes and the methylation of ribosomal RNA, which can affect the normal translation of proteins. This results in the inhibition of RNA and protein synthesis and the disruption of the normal hepatocytes metabolism, which leads to cell necrosis and inflammatory infiltration. It has been reported that D-GalN also affects the synthesis of cell membrane components and eventually causes hepatocyte death.
We recently found LPS/D-GalN administration could rapidly change gut microbial function which may further influence intestinal soyasaponin II level. Soyasaponin II exerts antiinflammatory effect by targeting Y-Box Binding Protein 1 and Nlrp3 inflammasome. Beside our work, accumulating evidence suggests that D-GalN treatment not just induces hepatocyte damage but also destroys the gut microbiota homeostasis and the intestinal barrier structure by allowing MAMPs, such as LPS, to enter the liver and peripheral blood. Conversely, pretreatment with probiotic Lactobacillus reuteri DSM 17938 can relieve the gut dysbiosis, reduce the transcription of inflammatory factors, and alleviate D-GalN-induced liver injury in rats. Probiotic Lactobacillus casei Zhang can also reduce LPS/D-GalN-induced pro-inflammatory cytokines and hepatic inflammation through the modulation of the TLR-MAPK-PPARγ signaling pathways. showed that the oral gavage of Bacillus cereus for 15 days before D-GalN administration significantly improved liver injury and inflammatory processes by decreasing plasma endotoxin levels, reinforcing the gut barrier function, and improving the gut microbiota. Wang et al. demonstrated that Sprague-Dawley rats pretreated with Lactobacillus helveticus R0052 for 7 days showed a significant reduction in the levels of ALT, bilirubin, and total bile acid that were changed by D-GalN. Additionally, R0052 exhibits anti-inflammatory properties by down-regulating the transcription of Toll-like receptors, tumor necrosis factor-α, and nuclear factor-kappa beta (NFκβ) in liver tissues. R0052 can also improve intestinal lactic acid bacteria and Bacteroides. Similarly, Bifidobacterium FIGURE 2 | The mechanism of gut microbiota in ALI can be summarized by three points: x Chemicals transformation and metabolites of gut microbiota can influence the hepatocyte's ability to metabolize toxic substances and affect hepatic function; y Gut harmful bacteria and MAMPs activate the systemic immune response, release a large number of chemokines and pro-inflammatory cytokines into the liver, leading to liver inflammation; z Intestinal mucosal barrier destroy (containing the weakening of the mucus barrier, the destruction of cell tight junction, and the necrosis of intestinal epithelial cells) increases bacterial translocation and the entry of MAMPs into the liver, which mediates liver damage. PRRs, Pattern Recognition Receptors. Figure was partly adapted from Smart Servier Medical Art (https://smart. servier.com/), which is licensed under a Creative Common Attribution 3.0 Generic License. adolescents CGMCC15058, Bifidobacterium longum R0175, and Saccharomyces boulardii reduce the increase of cytotoxic factors and inflammatory cytokines in D-GalN-induced liver injury in rats. In brief, D-GalN is a widely used model to examine the protective mechanism of exogenous probiotics, prebiotics on fulminant liver failure.
# Conclusion
Acute liver injury is a life-threatening disease with various causes and rapid progress. The application of chemical-induced liver injury models is of great significance for the study of the pathophysiological mechanism of ALI. In recent years, with the development of rapid, sensitive, and cheap gene sequencing technology and omics technology, the gut microbiota was being found to play a key role in host liver immunity, metabolism, and detoxification. The role of gut microbiota in ALI has also been gradually revealed. In the chemical-induced ALI models, the main mechanisms of gut microbiota in regulating ALI can be summarized by three points . Firstly, the intestinal microbes directly affect the detoxification ability of hepatocyte through the changes of bacterial metabolites and the transformation of chemical toxins; secondly, microbes and MAMPs are exposed to the systemic immune cells through the damaged intestinal mucosal barrier. Then the immune cells were activated, which release a large number of chemokines and pro-inflammatory cytokines into the liver, and induce liver inflammation. Finally, gut microbiota dysbiosis or chemical poisons directly induced the intestinal mucosal barrier damage (including weakened mucus barrier, destructed cellular tight junctions and the necrosis of intestinal epithelial cells), increase bacteria and MAMPs translocation into the liver, and mediate liver injury. In future, using single-cell RNA sequencing might help to further study how the gut microbiota directly affects hepatocytes and liver non-parenchymal cells. Numerous studies have shown that exogenous supplementation with prebiotics and probiotics can improve various chemical-induced ALI models. These data suggest that modulation of the gut microbiota for applying to clinical treatment of ALI is one possibility. The gut microbiota is a multifaceted community but nearly all of the research have focused on the role of bacterial disturbance in ALI models. The progress of fungi and viruses in this field is very limited. Future studies are required to better understand the systemic role of the gut microbiota; including viruses, fungi, and parasites, in the occurrence and development of ALI.
# Author contributions
TC and RL draft the manuscript. PC edit the manuscript and supervised the work. All authors contributed to the article and approved the submitted version.
# Funding
This study was supported by National Natural Science Foundation of China (81873926) to PC. |
A case of clonal seborrheic keratosis with characteristic dermoscopic features
[bib_ref] Different dermoscopic features of clonal seborrhoeic keratoses, Uzuncakmak [/bib_ref] [bib_ref] Different dermoscopic features of clonal seborrhoeic keratoses, Uzuncakmak [/bib_ref] [bib_ref] Dermoscopy of clonal seborrheic keratosis, Ramyead [/bib_ref] [bib_ref] Clonal seborrheic keratosis: a dermoscopic pitfall, Zalaudek [/bib_ref] [bib_ref] Globulelike" dermoscopic structures in pigmented seborrheic keratosis, Hirata [/bib_ref] [bib_ref] Clonal seborrheic keratosis: a dermoscopic pitfall, Zalaudek [/bib_ref] [bib_ref] Different dermoscopic features of clonal seborrhoeic keratoses, Uzuncakmak [/bib_ref] [bib_ref] Clonal seborrheic keratosis: a dermoscopic pitfall, Zalaudek [/bib_ref] [bib_ref] Seborrheic keratosis with unique dermoscopic features, Luo [/bib_ref]
## Declaration of patient consent
The authors certify that they have obtained the appropriate patient consent form. In the form, the patient provided her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initials will not be published and that due efforts will be made to conceal her identity but that anonymity cannot be guaranteed.
# Funding
This work was supported by grants from the Beijing Natural Science Foundation (No. 7182127) and the National Natural Science Foundation of China (No. 61871011).
Conflicts of interestNone. |
Inappropriate Sinus Tachycardia Diagnosed and Treated as Depression Successfully Treated by Radiofrequency Catheter Ablation
We experienced a man in his 20s with inappropriate sinus tachycardia (IST) initially diagnosed and treated as depression who was steadily treated with radiofrequency catheter ablation (RFCA) using an EnSite™ system. The patient has remained well without any symptoms or medications, including antidepressants, for two years since the RFCA. To avoid missing IST and treating it as an emotional problem and/or mental illness such as depression, physicians -including cardiologists -should be aware of these conditions when examining patients with multiple and incapacitating complaints including palpitations and general fatigue and/or tachycardia, especially characterized by an elevated resting heart rate or a disproportionate increase in the heart rate with minimal exertion.
# Introduction
Inappropriate sinus tachycardia (IST) is an uncommon clinical and pathological syndrome that is characterized by an elevated resting heart rate or disproportionate increase in the rate with minimal exertion [bib_ref] Inappropriate sinus tachycardia, Olshansky [/bib_ref]. Because not only many physicians but also some cardiologists do not recognize IST, it is sometimes misdiagnosed as an unpathological sinus tachycardia, emotional problem, and/or mental illness, such as depression [bib_ref] Three-dimensional nonfluoroscopic mapping and ablation of inappropriate sinus tachycardia. Procedural strategies and..., Marrouche [/bib_ref]. Indeed, we have experienced two patients with IST diagnosed and treated as an emotional disorder or depression. Radiofrequency catheter ablation (RFCA) is an acceptable treatment modality for drug-refractory IST [bib_ref] Inappropriate sinus tachycardia, Olshansky [/bib_ref] [bib_ref] Use of noncontact mapping and saline-cooled ablation catheter for sinus node modification..., Lin [/bib_ref] [bib_ref] How to manage patients with inappropriate sinus tachycardia, Shen [/bib_ref] [bib_ref] Usefulness of non-contact mapping for radiofrequency catheter ablation of inappropriate sinus tachycardia:..., Takemoto [/bib_ref]. We herein report a case of IST successfully treated by RFCA in a patient in whom the IST had been initially diagnosed and treated as depression.
## Case report
A man in his 20s presented to our hospital with a chief complaint of palpitations, dizziness, weakness, easy fatigability, and general fatigue. He was diagnosed with depression by his previous doctor, and antidepressants were prescribed for 14 months. His symptoms had not changed before or after the treatment with antidepressants. His physical examination, laboratory analysis, chest X-ray, and echocardiograms, yielded no evidence of clinically overt structural and/or organic heart disease, or a secondary pathological sinus tachycardia, such as anemia or hyperthyroidism. His Pwave axis during the tachycardia was similar to that during sinus rhythm, and tall P-waves were noted during the tachycardia, especially in leads II, III, and aVF [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref] and B) on the 12-lead electrocardiogram. His heart rate (HR) was greater than 100 beats per minute (bpm) with minimal exertion [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref] , the 24-h Holter monitoring demonstrated a mean HR of greater than 90 bpm with palpitations, and he exhibited an HR of greater than 130 bpm within the first 90 seconds of a standard Bruce protocol on the treadmill test . Given the above, he was diagnosed with IST.
His serum brain natriuretic peptide (BNP) concentration was elevated at 34 pg/dL, and his New York Heart Association (NYHA) functional class was class II on admission (Table). His IST was therefore deemed drug refractory, including to an oral administration of the beta-adrenergic blockers bisoprolol 5 mg/d and verapamil 120 mg/d. RFCA was performed guided by a multielectrode array catheter using the EnSite™ non-contact mapping system. An electrophysiological study was performed before and after the RFCA to verify the mechanism of the arrhythmia and to exclude the coexistence of other arrhythmias. Using a betaadrenergic blocker and agonist, the heart rate was controlled between 80 and 150 bpm before and after the RFCA. IST with tall P-waves during the tachycardia in leads II, III, and aVF was steadily induced by a beta-adrenergic agonist [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref]. Warm-up at the initiation and cool-down at the termination of the IST were observed under intravenous administration of a beta-adrenergic blocker and agonist, respectively. The activation maps from the EnSite™ images interestingly demonstrated that the breakout sites (BOSs) for HR of less or more than 100 bpm could easily and clearly be separated [fig_ref] Figure 2: Right anterior oblique view of the EnSite TM voltage [/fig_ref]. After delivering radiofrequency energy for 30 to 60 seconds with a preset temperature of 50 and power limit of 30 W at the BOSs during an HR of more than 100 bpm, the BOSs observed during an HR of more than 100 bpm completely moved to the sites for an HR of less than 100 bpm [fig_ref] Figure 2: Right anterior oblique view of the EnSite TM voltage [/fig_ref] , in accordance with the disappearance of the tall P-waves in leads II, III, and aVF [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref].
After treating the IST, his incapacitating symptoms steadily disappeared. His 24-h Holter monitoring and standard Bruce protocol on the treadmill test demonstrated a mean HR of less than 90 bpm without any symptoms and an HR of greater than 130 bpm over the first 90 seconds . His serum BNP concentration and NYHA functional class normalized to 8 pg/dL and class I, respectively . He has remained well without any symptoms or medications, including antidepressants, for two years since the RFCA.
# Discussion
Although any relationship with IST is uncertain, associ- [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref] than 100 bpm could easily and clearly be separated (A, C). The area in the red circle with the dotted line was the target site for the radiofrequency catheter ablation . After the RFCA, the BOSs observed during an HR of more than 100 bpm [fig_ref] Figure 1: A 12-lead electrocardiogram at rest [/fig_ref] completely moved to the sites for an HR of less than 100 bpm (green circles with 80 and 90) (B, C). bpm: beats per minute ated emotional and psychiatric problems are often noted in patients with tachycardia (2), causing the IST to be misdiagnosed as an emotional problem or mental illness such as depression, as in the present case. Because the symptoms associated with those emotional and psychiatric problems sometimes resemble those associated with IST, IST can be difficult to diagnose accurately even by seasoned physicians, including cardiologists. Indeed, it has been reported that 100% of patients with IST had initially received some psychiatric diagnosis, such as depression, panic disorder, schizophrenia, or somatoform disorder (1). We may have been able to accurately diagnose IST in the present case because we've had experience diagnosing and treating such patients before. It may therefore be important to consult an expert on patients with those symptoms, especially when faced with laboratory findings of tall P-waves during the tachycardia, particularly in leads II, III, and aVF on the 12-lead electrocardiogram, an HR of greater than 100 bpm on minimal exertion, and/or a mean HR of greater than 90 bpm with palpitations of the 24-h Holter monitoring.
RFCA is reported to be an acceptable treatment modality for IST [bib_ref] Inappropriate sinus tachycardia, Olshansky [/bib_ref] [bib_ref] Use of noncontact mapping and saline-cooled ablation catheter for sinus node modification..., Lin [/bib_ref] [bib_ref] How to manage patients with inappropriate sinus tachycardia, Shen [/bib_ref] [bib_ref] Usefulness of non-contact mapping for radiofrequency catheter ablation of inappropriate sinus tachycardia:..., Takemoto [/bib_ref]. Because this patient was young and his IST was drug-refractory, we ultimately decided to perform RFCA using EnSite™ (5) after informed consent was obtained. As a result, his clinical status improved , and an acceptable long-term clinical outcome was achieved. The symptoms associated with certain emotional and psychiatric problems can sometimes resemble those associated with IST. Furthermore, the symptoms associated with IST are especially characterized by an elevated resting heart rate or a disproportionate increase in the heart rate with minimal exertion. To avoid missing IST and treating it as an emotional problem and/or mental illness such as depression, physicians -including cardiologists -should be aware of these conditions when examining patients with multiple and incapacitating complaints including palpitations and general fatigue and/or tachycardia.
## The authors state that they have no conflict of interest (coi).
[fig] Figure 1: A 12-lead electrocardiogram at rest (heart rate 90 bpm) (A), on minimal exertion (heart rate 120 bpm) (B), under intravenous administration of a beta-adrenergic agonist (heart rate 150 bpm) (C), and after delivering radiofrequency energy (heart rate 130 bpm) (D). bpm: beats per minute [/fig]
[fig] Figure 2: Right anterior oblique view of the EnSite TM voltage (A, B) and activation (C) maps showing the right atrium (RA). The breakout sites (BOSs) with a heart rate (HR) of less (green circle with 80 and 90) or more (yellow [/fig]
[table] Table: HR, Treadmill Test, BNP, and NYHA Functional Class. heart rate, bpm; beats per minute, BNP: brain natriuretic peptide, NYHA: New York Heart Association, RFCA: radiofrequency catheter ablation, Time-HR>130 bpm: heart rate of greater than 130 bpm during a standard Bruce protocol on the treadmill test [/table]
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Risk of selection bias in randomised trials
Background: Selection bias occurs when recruiters selectively enrol patients into the trial based on what the next treatment allocation is likely to be. This can occur even if appropriate allocation concealment is used if recruiters can guess the next treatment assignment with some degree of accuracy. This typically occurs in unblinded trials when restricted randomisation is implemented to force the number of patients in each arm or within each centre to be the same. Several methods to reduce the risk of selection bias have been suggested; however, it is unclear how often these techniques are used in practice. Methods: We performed a review of published trials which were not blinded to assess whether they utilised methods for reducing the risk of selection bias. We assessed the following techniques: (a) blinding of recruiters; (b) use of simple randomisation; (c) avoidance of stratification by site when restricted randomisation is used; (d) avoidance of permuted blocks if stratification by site is used; and (e) incorporation of prognostic covariates into the randomisation procedure when restricted randomisation is used. We included parallel group, individually randomised phase III trials published in four general medical journals (BMJ, Journal of the American Medical Association, The Lancet, and New England Journal of Medicine) in 2010. Results: We identified 152 eligible trials. Most trials (98 %) provided no information on whether recruiters were blind to previous treatment allocations. Only 3 % of trials used simple randomisation; 63 % used some form of restricted randomisation, and 35 % did not state the method of randomisation. Overall, 44 % of trials were stratified by site of recruitment; 27 % were not, and 29 % did not report this information. Most trials that did stratify by site of recruitment used permuted blocks (58 %), and only 15 % reported using random block sizes. Many trials that used restricted randomisation also included prognostic covariates in the randomisation procedure (56 %). Conclusions: The risk of selection bias could not be ascertained for most trials due to poor reporting. Many trials which did provide details on the randomisation procedure were at risk of selection bias due to a poorly chosen randomisation methods. Techniques to reduce the risk of selection bias should be more widely implemented.
# Background
Well-conducted randomised controlled trials (RCTs) are viewed as the 'gold standard' for comparing different interventions, as they are not subject to the same confounding as non-randomised studies. Randomisation (when performed correctly) guarantees that, on average, treatment groups will be well-balanced for both known and unknown factors, thus ensuring an unbiased estimate of the treatment effect. However, this guarantee of balance only occurs when randomisation is performed correctly; that is, when the probability of a patient being enrolled does not depend on the probability of them being assigned to a particular treatment group. Otherwise, the trial will be at risk of selection bias.
Selection bias occurs when those in charge of the recruitment or enrolment of patients (recruiters) selectively enrol patients into the trial based on what the next treatment allocation is likely to be. For example, if a recruiter believes the next allocation will be the intervention, they may wait to enrol a very sick patient, as they do not want to 'waste' an intervention allocation on a relatively healthy patient who is less likely to need it. This can lead to substantially biased estimates of treatment effect and misleading conclusions [bib_ref] Quantifying the magnitude of baseline covariate imbalances resulting from selection bias in..., Berger [/bib_ref] [bib_ref] Detecting selection bias in randomized clinical trials, Berger [/bib_ref] [bib_ref] The effect of estimation and biasing strategies on selection bias in clinical..., Follmann [/bib_ref] [bib_ref] The impact of selection bias on test decisions in randomized clinical trials, Kennes [/bib_ref] [bib_ref] Randomisation to protect against selection bias in healthcare trials, Odgaard-Jensen [/bib_ref] [bib_ref] Empirical evidence of bias. Dimensions of methodological quality associated with estimates of..., Schulz [/bib_ref] [bib_ref] Unequal group sizes in randomised trials: guarding against guessing, Schulz [/bib_ref] [bib_ref] Influence of selection bias on the test decision. A simulation study, Tamm [/bib_ref]. Selection bias is primarily an issue in RCTs where patients are enrolled sequentially (rather than all at once), and when recruiters can decide whether or not to enrol each eligible patient.
Selection bias depends on the ability of the recruiter to guess with greater than 50 % probability what the next treatment allocation will be. This could happen if, for example, the recruiter had access to the randomisation list, and knew what treatment each patient would be assigned to before enrolling them. For this reason, allocation concealment has been recommended as an essential tool for RCTs [bib_ref] Empirical evidence of bias. Dimensions of methodological quality associated with estimates of..., Schulz [/bib_ref] [bib_ref] The unpredictability paradox: review of empirical comparisons of randomised and non-randomised clinical..., Kunz [/bib_ref] [bib_ref] The importance of allocation concealment and patient blinding in osteoarthritis trials: a..., Nuesch [/bib_ref]. Allocation concealment is 'a technique used to prevent selection bias by concealing the allocation sequence from those assigning participants to intervention groups, until the moment of assignment' (http://www.consort-statement.org/resources/ glossary).
However, even if appropriate allocation concealment is used and recruiters do not have access to allocation list before enrolling patients, they still may be able to guess what the next allocation will be based on the method of randomisation. This is primarily a concern in unblinded trials, where investigators are aware of each patient's treatment assignment. However, this could also be an issue in blinded trials where there is a risk of unblinding: for example, because side effects may occur for patients in one arm only. Brown et al. [bib_ref] Minimization -reducing predictability for multi-centre trials whilst retaining balance within centre, Brown [/bib_ref] found that 16 % of the investigators they surveyed had admitted to keeping track of the number of allocations to each treatment group in an attempt to predict the next allocation. This type of behaviour has led to the identification of a number of RCTs which may have been affected by selection bias [bib_ref] Subverting randomization in controlled trials, Schulz [/bib_ref] [bib_ref] Ensuring the comparability of comparison groups: is randomization enough?, Berger [/bib_ref]. Because the possibility of selection bias is rarely reported in trial publications, it is likely that the overall problem is underreported. Selection bias can have serious implications for patient healthcare; the distortion of trial results could lead ineffective interventions appearing helpful or harmful interventions appearing safe. The purpose of this article is to highlight some simple methods to prevent selection bias, and assess how often these methods are being used in practice.
# Methods
We begin by describing the problem of selection bias in more detail, and then highlight some simple methods to reduce the risk (summarised in [fig_ref] Table 1: Methods to reduce the risk of selection bias in non-double-blind clinical trials* [/fig_ref]. We assume that the trial is open-label (unblinded), and that there is adequate allocation concealment.
## Selection bias
For trials that use adequate allocation concealment, selection bias can still be a problem if recruiters can guess the next allocation with greater than 50 % probability. This can occur when restricted randomisation is used. Restricted randomisation involves forcing the treatment groups to be similar in some way: for example, by requiring that a similar number of patients are assigned to each treatment group. The most common methods of restricted randomisation are permuted blocks and minimisation (described below) [bib_ref] Reporting and analysis of trials using stratified randomisation in leading medical journals:..., Kahan [/bib_ref].
Restricted randomisation can increase the risk of selection bias as follows; consider a trial in which patients are randomised using permuted blocks of size 4. This design forces the number of patients in each treatment group to be equal at the end of each block. This means that the imbalance between groups can never be more than two patients. When the imbalance between groups is 1, the recruiter will be able to correctly guess the next allocation with 83 % probability; when the imbalance is 2, this probability increases to 100 %. The probability of the allocation will depend on previous allocations at other sites, which recruiters are unlikely to have access to, making an accurate guess more difficult Risk of selection bias will be reduced, but not necessarily eliminated When randomisation is stratified by site, avoid permuted blocks Permuted blocks stratified by site will maximise the probability of correctly guessing the next allocation. Using alternative randomisation methods will reduce the probability of correctly guessing the next allocation Risk of selection bias will be reduced, but not necessarily eliminated When restricted randomisation is used, stratify by prognostic covariates as well
There is typically less variation in prognoses for patients with the same covariate pattern, making it more difficult for investigators to identify patients with a specific prognosis to enrol into the trial when their preferred treatment is more likely Risk of selection bias will be reduced, but not necessarily eliminated *This table assumes that allocation concealment has been appropriately implemented
## Methods to reduce the risk of selection bias simple randomisation
Simple randomisation (sometimes also referred to as 'complete' or 'unrestricted' randomisation) is both the simplest and most effective method to prevent selection bias. Simple randomisation works by assigning each patient to one of the treatment groups with a certain probability (usually 50 %); this probability is the same for every patient, regardless of previous allocations. For example, consider a trial where 15 of the first 20 patients were assigned to the intervention, and only 5 to the control; when the 21st patient presented for randomisation, they would still have an equal chance of being assigned to either treatment group, regardless of the imbalance in numbers. Because the probability is always the same, recruiters will not be able to guess with any accuracy which treatment the patient will be assigned to (as they would essentially be trying to guess the results of a coin-flip); therefore, selection bias cannot occur in this scenario.
In practice, simple randomisation is infrequently used [bib_ref] Reporting and analysis of trials using stratified randomisation in leading medical journals:..., Kahan [/bib_ref] [bib_ref] Is restricted randomisation necessary?, Hewitt [/bib_ref] , possibly because investigators prefer randomisation methods which provide balance in the number of patients assigned to each treatment group. However, provided the overall sample size is not too small, this lack of balance has only a very small impact on power, and should not be used as a reason to avoid simple randomisation. Therefore, we agree with others that simple randomisation should be used more frequently in practice [bib_ref] Unequal group sizes in randomised trials: guarding against guessing, Schulz [/bib_ref] [bib_ref] Is restricted randomisation necessary?, Hewitt [/bib_ref] [bib_ref] Potential for technical errors and subverted allocation can be reduced if certain..., Hewitt [/bib_ref].
## Do not stratify by site of recruitment if restricted randomisation is used
Despite the appeal of simple randomisation, some form of restricted randomisation is usually employed in practice. In trials with multiple sites of recruitment, the number of patients allocated to each arm can be forced to be similar in two different ways; either by forcing the number within each site to be the same (stratified by site), or by forcing the overall numbers to be the same, regardless of the numbers within each site (not stratified by site).
The risk of selection bias is most pronounced when randomisation is stratified by site of recruitment; that is, when the randomisation procedure is restricted to ensure an equal number of patients are allocated to each treatment group within each site. In this case, the probability of the next allocation depends solely on the previous allocations at that site, which the recruiters may have access to. However, if randomisation is not stratified by site, then the probability of the next allocation will also depend on the previous allocations at all the other sites, which the recruiter is unlikely to have access to. Therefore, even if restricted randomisation is used, the risk of selection bias can be reduced by not stratifying by site of recruitment. Investigators often stratify by site of recruitment as they are concerned that if betweencentre differences are large, a chance imbalance in the number of patients allocated to each treatment within a site could affect results. However, chance imbalances within sites can be accounted for during the analysis by adjusting for site-effects [bib_ref] Does clustering affect the usual test statistics of no treatment effect in..., Parzen [/bib_ref] [bib_ref] Accounting for center in the Early External Cephalic Version trials: an empirical..., Reitsma [/bib_ref] [bib_ref] Accounting for centre-effects in multicentre trials with a binary outcome -when, why,..., Kahan [/bib_ref] [bib_ref] Analysis of multicentre trials with continuous outcomes: when and how should we..., Kahan [/bib_ref] , and so stratification by site during randomisation is generally not necessary.
## If randomisation is stratified by site, avoid permuted blocks
Despite the increased risk of selection bias that accompanies stratification by site, it is still commonly used. In some cases, this may be due to administrative or practical reasons: for example when separate randomisation lists must be kept at each site, or when there is only one site and so any restriction on the number of patients assigned to each treatment group is equivalent to stratifying by site. However, it should be noted that in both of these cases, unrestricted randomisation could still be used (e.g. by generating the randomisation list for each site using simple randomisation).
If randomisation is stratified by site, the best method for reducing the risk of selection bias is to avoid the use of permuted blocks, as this has been shown to substantially increase the probability of correctly guessing the next allocation compared to other methods. Common suggestions for improving the performance of permuted blocks are to randomly vary the block size, and to use large block sizes. Although both approaches will help, neither will reduce the risk to an acceptable level [bib_ref] The impact of selection bias on test decisions in randomized clinical trials, Kennes [/bib_ref] [bib_ref] Varying the block size does not conceal the allocation, Berger [/bib_ref]. Therefore, permuted blocks stratified by site of recruitment should be avoided.
A preferable alternative is minimisation, which involves allocating the patient to whichever treatment arm minimises the imbalance in a set of baseline covariates. Minimisation can maintain some balance within centres, while also reducing the probability of correctly guessing the next allocation [bib_ref] Minimization -reducing predictability for multi-centre trials whilst retaining balance within centre, Brown [/bib_ref]. This is partly because the next allocation at each centre will depend on covariate information for patients from other centres, which recruiters are unlikely to know. Minimisation should only be used with a random element (that is, by assigning patients to the treatment group which minimises the imbalance with a degree of probability, rather than in a deterministic way) [bib_ref] Rank minimization with a two-step analysis should not replace randomization in clinical..., Kahan [/bib_ref]. Additionally, centre should be included as a minimisation factor like any other covariate (as opposed to using a stratified approach, where the minimisation procedure is performed separately within each site). Other alternatives to reduce the risk of selection bias when stratifying by site are also available, such as a stratified urn design [bib_ref] Properties of the urn randomization in clinical trials, Wei [/bib_ref]. However, although these techniques will reduce selection bias compared to permuted blocks, it is still preferable to avoid stratifying by site altogether.
## If restricted randomisation is used, balance on prognostic covariates as well
When restricted randomisation is used, balancing or stratifying on prognostic factors could help to combat selection bias. This works as follows: consider a trial using permuted blocks. If the recruiter knows that more patients have been allocated to the control, they can guess that the next allocation is likely to be to the intervention. Depending on their beliefs (conscious or subconscious), they may wish to enrol a relatively sick or relatively healthy patient for this allocation. The recruiter therefore needs to be able to find a patient who is relatively sick or healthy, compared to those who have been previously enrolled.
Imagine if the patient's age and their disease stage have been included as stratification factors during randomisation. Even if the recruiter can guess the next allocation, they still need to be able to find a patient to enrol who is much sicker or healthier than other patients of the same age and disease stage. This is clearly a much more difficult task, as there will typically be less variation in prognosis for patients with the same covariate patterns, making it more difficult to identify patients with specific prognoses. This becomes increasingly more difficult the more prognostic covariates that are added to the randomisation procedure (although it should be noted that increasing the number of stratification factors has implications for the analysis as well [bib_ref] Reporting and analysis of trials using stratified randomisation in leading medical journals:..., Kahan [/bib_ref] [bib_ref] The risks and rewards of covariate adjustment in randomized trials: an assessment..., Kahan [/bib_ref] [bib_ref] Improper analysis of trials randomised using stratified blocks or minimisation, Kahan [/bib_ref]. Therefore, when restricted randomisation is used, we would recommend also including prognostic covariates in the randomisation process. However, we caution that this is approach is not a valid reason to use a randomisation method that increases the risk of selection bias, such as stratification by site of recruitment; it is still preferable to use a method of randomisation that completely eliminates the risk of selection bias, such as simple randomisation.
## Ensure that those enrolling patients are blinded to previous treatment allocations
Even if a trial is unblinded, it may still be possible to blind recruiters (the people who recruit and enrol patients into the trial) [bib_ref] Bias in identifying and recruiting participants in cluster randomised trials: what can..., Eldridge [/bib_ref]. This could be done by using personnel who are not otherwise involved in patient care as recruiters. If they are blinded, they will not be aware of the previous trial allocations and, therefore, will not be able to predict the next allocation.
## Review of published trials
We performed a review of published trials to investigate whether investigators are taking adequate steps to reduce the risk of selection bias. We included parallel group, individually randomized, controlled trials which were not fully blinded. We defined a trial to be not fully blind when at least some trial personnel were not blinded to treatment allocation. This included (but was not limited to) participants, those administering the intervention, those providing medical care apart from the intervention, and those assessing outcomes. We excluded trials which were described as double-blind, stated that everyone involved in the study was blinded, or which used a placebo or sham treatment that was described as being identical to the intervention in terms of appearance. We also excluded pilot and phase I or II trials, as well as articles that reported only secondary analyses.
We included trials that were published in one of four major medical journals in 2010 (BMJ, Journal of the American Medical Association, The Lancet, and New England Journal of Medicine). Trials were identified from the electronic table of contents for each journal. One reviewer determined whether trials met the eligibility criteria for all trials identified; a second reviewer assessed this for a subset of trials, and agreement was found to be 100 %.
We extracted data onto a pre-piloted, standardised form. This included information on the blinding status of the trial, whether the randomisation procedure was stratified by site of recruitment (i.e. whether it would be possible for recruiters to keep track of each previous allocation at their site), whether the recruiters were blinded to previous allocations, and details on the randomisation method used. All trials were extracted by two independent reviewers, and disagreements were resolved by discussion.
# Results
We identified 152 eligible trials. General trial characteristics are shown in [fig_ref] Table 2: The use of methods to reduce the risk of selection bias Trials [/fig_ref]. Only 3 (2 %) stated whether those enrolling or recruiting patients were aware of previous allocations (2 were blinded, 1 was unblinded); 149 (98 %) gave no information on whether recruiters were blinded to previous treatment allocations. In most cases, the trial report did not state who was involved in recruiting patients, and whether this person had any other role in the trial, such as delivering the intervention, providing other aspects of patient care, or assessing outcomes.
Only 4 trials (3 %) used simple randomisation; the rest used either restricted randomisation (n = 95, 63 %), or did not state the method of randomisation (n = 53, 35 %). Of the trials using some form of restricted randomisation, most used permuted blocks (n = 72, 76 %) or minimisation (n = 21, 22 %). Overall, 67 (44 %) of trials stratified randomisation by site of recruitment; 41 (27 %) did not, and 44 (29 %) did not provide this information.
## Results for trials which stratified by site of recruitment
Amongst the 67 trials which stratified by site of recruitment, 65 (97 %) did not provide information on whether recruiters were blinded [fig_ref] Table 3: The use of methods to reduce the risk of selection bias in... [/fig_ref]. Most trials (n = 39, 58 %) used stratified permuted blocks; 13 (19 %) used minimisation, 3 (4 %) used another method of randomisation, and 12 (18 %) did not state which method they used.
Only 23 of the 39 trials (59 %) which used permuted blocks stated the block size(s), and only 6/39 (15 %) reported using random block sizes. Seventeen out of thirty-nine 17/39 (44 %) did not state whether block sizes were random or not. Most trials used small block sizes (median 8, interquartile range (IQR) 4 to 11). Of the 13 trials using minimisation, 12 (92 %) did not state whether it was deterministic or not.
# Discussion
Selection bias can subvert the randomisation process in RCTs, leading to biased estimates of treatment effect and misleading conclusions. For this reason, allocation concealment is regarded as an essential component of RCTs. However, selection bias can still occur even with adequate allocation concealment if the method of randomisation is poorly chosen.
Our review found that very few trials used techniques that would eliminate the risk of selection bias, such as simple randomisation (3 %) or blinding of recruiters (1 %). Most trials used some form of restricted randomisation (63 %), and many trials were stratified by site of recruitment (44 %). Furthermore, a substantial proportion of trials did not provide adequate information on whether randomisation was restricted (35 %) or whether it was stratified by site (29 %), effectively preventing readers from assessing the risk of selection bias. A comparison to Hewitt and Torgerson's review of trials published in 2002 [bib_ref] Is restricted randomisation necessary?, Hewitt [/bib_ref] indicates that reporting on the method of randomisation has not improved in the 8 years between reviews (method of randomisation unclear 34 % in 2002 versus 35 % in 2010), while the use of simple randomisation may have decreased (9 % in 2002 versus 3 % in 2010).
These findings indicate that a substantial proportion of unblinded trials are at risk of selection bias. This is surprising, given the relative simplicity with which methods to reduce this risk can be implemented. For example, simple randomisation is by far the easiest method of randomisation to implement, and has little effect on trial IQR, interquartile range organisation or the statistical analysis. Likewise, adjusting for site of recruitment in the analysis rather than stratifying on it during randomisation is also relatively straightforward, and typically has no adverse impact on results. Similarly, permuted blocks can often be replaced by allocation methods which do not substantially increase the probability of correctly guessing future allocations, such as minimisation with a random element or urn randomisation. We therefore suggest that investigators conducting unblinded trials (or double-blinded trials where the blinding may not be entirely effective) should choose randomisation methods that reduce the risk of selection bias. This primarily involves simple randomisation, or avoiding the use of stratification by site of recruitment if restricted randomisation is employed. As discussed in the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines, details of restricted randomisation schemes should not be shared with trial investigators, as this information could make it easier to correctly guess the next treatment allocation [bib_ref] Submit your next manuscript to BioMed Central and take full advantage of:..., Chan [/bib_ref]. However, it is important that the details of the randomisation procedure is fully reported in the trial publication, as this will enable readers to judge the risk of bias. This description should include whether stratification was employed, which stratification factors were used, the block sizes (and whether they were random) for permuted blocks, and whether a random component was used (and its size) for minimisation. It would also be helpful for investigators to report on who was involved in recruiting and enrolling patients into the trial, whether they were blinded to treatment allocation, and whether they had any other role in the trial, such as delivering the intervention, providing other aspects of medical care, or assessing outcomes.
# Conclusion
The risk of selection bias could not be ascertained for most trials due to poor reporting. Many trials which did provide details on the randomisation procedure were at risk of selection bias due to a poorly chosen randomisation methods. Investigators should choose randomisation methods which eliminate or reduce the risk of selection bias.
[table] Table 1: Methods to reduce the risk of selection bias in non-double-blind clinical trials* [/table]
[table] Table 2: The use of methods to reduce the risk of selection bias Trials (n = 152) Recruiters blinded?number (%) [/table]
[table] Table 3: The use of methods to reduce the risk of selection bias in trials that stratified by site of recruitment Number of prognostic factors balancedmedian (IQR) 2 (1 to 3) [/table]
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Colour-sensitive conjugated polymer inkjet-printed pixelated artificial retina model studied via a bio-hybrid photovoltaic device
In recent years, organic electronic materials have been shown to be a promising tool, even transplanted in vivo, for transducing light stimuli to non-functioning retinas. Here we developed a bio-hybrid optoelectronic device consisting of patterned organic polymer semiconductors interfaced with an electrolyte solution in a closed sandwich architecture in order to study the photo-response of photosensitive semiconducting layers or patterns in an environment imitating biological extracellular fluids. We demonstrate an artificial retina model composed of on an array of 42,100 pixels made of three different conjugated polymers via inkjet printing with 110 pixels/mm 2 packing density. Photosensing through three-colour pixelation allows to resolve incoming light spectrally and spatially. The compact colour sensitive optoelectronic device represents an easy-to-handle photosensitive platform for the study of the photo response of artificial retina systems.According to the World Health Organization around 285 million people worldwide are visually impaired, of whom 39 million are blind 1 . Retinal degenerative illness such as Retinitis Pigmentosa (RP) and Age-related Macular Degeneration (AMD) are the leading causes of partial or total vision loss. All retinal degenerative diseases present a damage to photoreceptor cells. Photoreceptors are light-sensitive specialized neurons found in the retina, the nervous tissue covering the inside-back of the eyeball. Human retina contains two kinds of photoreceptors, rods and cones, responsible for scotopic monochromatic and photopic trichromatic vision respectively (S-cones, M-cones and L-cones, often labelled as "blue", "green" and "red" cones sensing short, mid and long wavelengths respectively). Photoreceptors convert light into electrical signals that travel along the optical nerve to the brain where vision is perceived. Without a proper functionality of photoreceptors, vision is principally compromised because photons are not converted into electrical triggers needed to change the resting membrane potentials of retinal cells to let the visual process begin.With the development of biomedical engineering in the last decades, retinal prostheses, designed to partially restore vision, have seen progress 2,3 . These electronic devices are mainly based on silicon or metallic and rigid electrodes 4 and possess poor flexibility and biocompatibility 5 . Moreover they often require external power supplies, video camera and cables 6 . Alternatively, nowadays the fast-growing field of bio-electronics based on organic semiconductors is offering a novel scenario for biomedical devices 7,8 . Organic semiconductors can be interfaced with biological matter ranging from small biomolecules such as DNA 9-11 to cells and tissues 5 . In the last few years an exciting avenue of research has consisted in interfacing degenerated retinas with semiconducting polymers to elicit a response in the former upon photoexcitation of the latter by transduction of signals 12-15 . The effectiveness of interfacing conjugated polymers or quantum dots with retinas have been demonstrated even in vivo in blind rats either via implantation 12,13 or injection16,17. The flexible nature of organic electronic materials provides better bio-mechanical compatibility, making them suitable as biomedical implants. Undoubtedly their ability OPEN to conduct ions, in addition to electrons and holes, opens a new communication channel with biology due to the importance of ion fluxes in biological systems 18 . Furthermore, these materials in an engineered nanoscale, including carbon nanotubes 19,20 graphene 21,22 and conjugated polymers 23,24 , have the potential to interact with biological systems on a molecular scale, offering unprecedented levels of control over physiological activity25,26.Poly-3-hexylthiophene (P3HT) has been the polymer of choice in the vast majority of these studies 27,28 being used as optical mediator in biological in vitro and in vivo applications for retinal prosthetic devices 29 . Moreover, the synthesis of conjugated polymers with different spectral response enables absorption of light at different frequencies and this is a crucial feature for future multi-chromatic retinal prostheses 30,31 . Up to today, to the best of our knowledge, the colour response of these materials was only studied with a few types of living cells 32,33 . In addition, the spectral responsivity was considered individually for one polymer at a time while never for different polymers together in the same device. Furthermore, all these studies employed standard electrophysiological tools such as patch-clamps amplifiers and Multi-Electrode Arrays (MEAs) for their analysis, where one of the electrodes is a reference electrode immersed in a bath of electrolyte solution with a flat/electrode/polymer system2,3,12,13,[25][26][27]32,33.Here we present a novel closed type sandwich structure device consisting of two transparent electrodes separated by a spacer encapsulation layer which encloses a semiconducting photosensitive film over the bottom electrode interfaced with electrolyte imitating extracellular fluid. Furthermore, rather than using conventional microfabrication methods (e.g. spin coating and photolithographic patterning), in this work conjugated polymers were pixelated using inkjet printing technology, exploiting one of the main advantages of these types of materials, which is patterned additive printing, in order to prepare a simplified concentric model of a spectrally-sensitive retina which enables to investigate the geometrical and spectral response of the pixelated structure immersed in an biological electrolyte. The demonstration of a spectrally sensitive photo-response of these systems, depending locally on the absorption spectra of deposited polymer pixels, represents an important step towards a colour sensitive artificial retina.
# Results
The results section is organized following our research approach: first we designed and validated a bio-hybrid sandwich structure consisting of a polymer semiconductor and a physiological electrolyte solution; second we developed a simplified concentric model of a pixelated retina via inkjet printing of different conjugated polymers that mimic the three spectra of rods and two cones typical of mammalian retinas.
Bio-hybrid device: design, fabrication and biocompatibility. Here we present a bio-hybrid device composed of both biological (physiological electrolyte solution mimicking extracellular fluid) and non-biological components. The developed device architecture consists of two transparent electrodes separated by a thermoplastic encapsulant with the photosensitive polymer thin film interfaced with an electrolyte mediator. The electrolyte solution was a buffered isotonic solution (Phosphate buffered saline solution, PBS) commonly employed in a biological context to reproduce a physiological extracellular fluid [bib_ref] Effects of sintering temperatures on micro-morphology, mechanical properties, and bioactivity of bone..., Kaewsichan [/bib_ref]. The concept of the device is the result of the amalgamation of different set-ups from different fields, i.e. those from electrophysiology [bib_ref] A polymer optoelectronic interface provides visual cues to a blind retina, Gautam [/bib_ref] [bib_ref] A polymer optoelectronic interface restores light sensitivity in blind rat retinas, Ghezzi [/bib_ref] [bib_ref] A hybrid bioorganic interface for neuronal photoactivation, Ghezzi [/bib_ref] , organic photovoltaics (OPV) [bib_ref] Solar cells incorporating water/alcohol-soluble electron-extracting DNA nanolayers, Dagar [/bib_ref] [bib_ref] Organic photovoltaics over three decades, Inganäs [/bib_ref] and Dye-Sensitized Solar Cells (DSSC) [bib_ref] A perspective on the production of dye-sensitized solar modules, Fakharuddin [/bib_ref] [bib_ref] A low-cost, high-efficiency solar cell based on dye-sensitized colloidal TiO2 films, O'regan [/bib_ref] [bib_ref] The role of printing techniques for large-area dye sensitized solar cells, Mariani [/bib_ref]. ,b illustrates a schematic of the device structure. The working electrode (sensitive photo-electrode) is 100 nm-thick polymer semiconductor film, P3HT at first (see "Methods" section), spin-coated on a glass substrate covered with a transparent conducting oxide (fluorine tin oxide-FTO). P3HT is a prototype material for OPV applications [bib_ref] P3HT/PCBM bulk heterojunction organic photovoltaics: correlating efficiency and morphology, Chen [/bib_ref] , it works as an absorber . Bio-hybrid device layout for biological system/organic electronic interfaces and biocompatibility evaluated as cell adhesion, proliferation and viability. (a) Schematic of the device. It consists of two transparent conductive electrodes. The photo-electrode (polymer thin film on FTO) is grounded. The platinum layer deposited on FTO is the counter-electrode. A phosphate buffered saline solution (PBS) was used as electrolyte and it is enclosed in the inner chamber (60 µm thick) formed between the two electrodes (Image created by using Microsoft PowerPoint). (b) Picture of the fabricated device. Dimensions are also shown. (c) Phase contrast micrographs of randomly selected fields of SH-SY5Y cells cultured on standard polystyrene dish (CTRL) and on Glass|FTO|P3HT substrate (P3HT) for 3 days. Scale bar = 50 μm. (d) Cell adhesion evaluated at 16 h after seeding SH-SY5Y cells on polystyrene dish (CTRL) and on Glass|FTO|P3HT (P3HT). Data are expressed as percentage of the CTRL. Statistical difference vs CTRL **p < 0.01 (Data were analysed with GraphPad Prism software V7.0). (e) Cell proliferation evaluated after three days of culture and expressed as cell doubling time for SH-SY5Y cultured on polystyrene dish (CTRL) and on the P3HT layer (Image J software 1.49 V was used. Data were analysed with GraphPad Prism software V7.0). (f) Representative micrographs of SH-SY5Y cells cultured on polystyrene dish (CTRL) and on Glass|FTO|P3HT substrate (P3HT) and analysed via the Click-iT EdU proliferation assay after 3 days of culture (T3). White and red arrowheads indicate non-proliferating and proliferating (EdU positive) cells, respectively. Scale bar = 50 μm. (g) Quantification of cell proliferation measured by Click-iT EdU proliferation assay, expressed as percentage of positive cells on the total number of cells analysed at the beginning (T0) and at the end (T3) of culture (Data were analysed with GraphPad Prism software V7.0). (h) Representative micrographs of SH-SY5Y cells cultured on polystyrene dish (CTRL) and on Glass|FTO|P3HT substrate (P3HT) and analysed via the in-situ Cell Viability Imaging assay after three days of culture (T3). White and red arrowheads indicate live and dead (green) cells, respectively. Scale bar = 50 μm. (i) Quantification of cell death measured by in-situ Cell Viability Imaging assay, expressed as percentage of dead (green) cells on the total number of cells analysed at the beginning (T0) and at the end (T3) of culture (Image J software 1.49 V was used). www.nature.com/scientificreports/ and electron donor material. The counter-electrode is a platinum (Pt) layer screen printed through a precursor on FTO-coated substrate and then fired at 480 °C for 30 min 41 . The latter procedure, borrowed from DSSC technology [bib_ref] Blending CoS and Pt for amelioration of electrodeposited transparent counterelectrodes and the..., De Rossi [/bib_ref] , converts the used Pt precursor paste into catalytic Pt nanometre-sized islands that permit a facile exchange of electrons/ions at the electrode/electrolyte interface maintaining a high degree of transparency with a resulting transmittance of up to 79% between 400 and 700 nm [bib_ref] Pt-free counter electrodes based on modified screen-printed PEDOT: PSS catalytic layers for..., Gemeiner [/bib_ref]. Moreover platinum electrodes are widely used in bioelectronic applications because they can safely deliver charge densities ranging from 0.15 to 5.57 mC/cm 244 . PBS is contained in an inner chamber delimited by the thermoplastic sealant gasket (i.e. Surlyn). The presence of the biological electrolyte is important to provide a similar physiological aqueous environment for cells, mimicking eye conditions, and to test whether the polymer layer will retain its optoelectronic features in such biological/aqueous environment. The spacing between the two electrodes can be controlled via the thickness of the thermoplastic spacer. Here 60 µm thick gaskets were used representing a thickness which elicit measurable signals and potentially being able to accommodate smaller biological cells. Thicknesses greater than 60 µm can be used to place larger retinal tissues into the device for future studies. To address the issue of biocompatibility, human neuroblastoma cells SH-SY5Y were seeded on a P3HT layer and grown for up to 3 days, as described in "Methods" section. Cell adhesion, proliferation and viability were evaluated and compared with parallel cultures carried out with SH-SY5Y cells grown on tissue culture tested polystyrene dishes, as positive control (CTRL) . Adhesion was analysed 16 h after cell seeding (T0) and the results showed an adhesion rate of 50-60% on P3HT layer in comparison to control (CTRL = 100 ± 9.15%; P3HT = 54.07 ± 8.14%) .
For the analysis of cell proliferation, the number of cells seeded on P3HT was adjusted based on the calculated rate of adhesion to have the same number of adhered cells at the beginning of culture (T0) in CTRL and P3HT conditions. When the adhered cells (CTRL = 1.49 ± 0.38 × 10 4 cells/cm 2 ; P3HT = 1.40 ± 0.29 × 10 4 cells/cm 2 ) were allowed to grow for 3 days, no significant differences were observed between the two culture conditions in the proliferation rate expressed as cell doubling time (CTRL = 1.50 ± 0.02 day; P3HT = 1.50 ± 0.10 day) . Proliferation was also confirmed by the Click-iT EdU proliferation assay performed on the cells at the beginning (T0) and after 3 days (T3) of culture [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref] was shone on three different device architectures containing: (i) PBS, (ii) polymer, and (iii) the complete structure (polymer+PBS), to gauge the effectiveness of the concept. Without either polymer layer or electrolyte, no signals were elicited. Current densities at short circuit (J ph ) and photo-voltage at open circuit (V ph ) were instead generated upon photoexcitation for the complete device [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref]. We illuminated the device for 300 ms with a high-speed white LED (5000 K) as falling within the ranges used for in vitro retinas and living cells light stimulation studies [bib_ref] A polymer optoelectronic interface provides visual cues to a blind retina, Gautam [/bib_ref] [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref] [bib_ref] Bimodal functioning of a mesoporous, light sensitive polymer/electrolyte interface, Tullii [/bib_ref] [bib_ref] Conjugated polymers for the optical control of the electrical activity of living..., Vaquero [/bib_ref] [bib_ref] Photoactive poly(3-hexylthiophene) nanoweb for optoelectrical stimulation to enhance neurogenesis of human stem..., Yang [/bib_ref] [bib_ref] A fully organic retinal prosthesis restores vision in a rat model of..., Maya-Vetencourt [/bib_ref] and below the limit allowed for prosthetic application [bib_ref] Maintaining ocular safety with light exposure, focusing on devices for optogenetic stimulation, Yan [/bib_ref] [bib_ref] Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic..., Delori [/bib_ref]. The steady state photo-current after an initial spike is an evidence of a Faradaic process at the electrode/electrolyte interface [bib_ref] Band alignment engineers faradaic and capacitive photostimulation of neurons without surface modification, Srivastava [/bib_ref]. Similarly to the concept of a DSSC 38 charges are photogenerated in the semiconductor film, whilst the ions present in the biological liquid permit the exchange of charge at both the polymer/electrolyte and electrolyte/electrode interfaces enabling the presence of a sustained current. The platinum film catalyses charge transfer between the ions and the top transparent electrode. Current spikes visible when the system is perturbed 50,51 , i.e. when light is turned ON or OFF, are the result of a capacitive process due to electrostatic perturbation of accumulated carriers and ions with strong Coulomb interactions localized close to the polymer/electrolyte and electrolyte/metal interfaces (e.g. Helmholtz layers) [bib_ref] Band alignment engineers faradaic and capacitive photostimulation of neurons without surface modification, Srivastava [/bib_ref]. Capacitive coupling is one of the most biologically safe mechanism used for cells and tissues electrical stimulation [bib_ref] A hybrid bioorganic interface for neuronal photoactivation, Ghezzi [/bib_ref] [bib_ref] Optoelectronic control of single cells using organic photocapacitors, Jakešová [/bib_ref]. In this device the electrolyte maintains the physiological environment of biological cells and tissues (which would be present in an in vivo investigation or that could be potentially placed inside the device) and enables the movement and transport of charge carriers along the section of the device between photo-electrode and counter-electrode. J ph values, referred to the steady state (at the half of the light pulse width, namely at 150 ms from the light switched ON) current signals are in the order of 0.17 ± 0.10 µA/cm 2 . J ph spike values are in the order of 0.51 ± 0.28 µA/cm 2 and of − 0.29 ± 0.12 µA/cm 2 at the start and the end of the impulse of light respectively [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref]. Under the convention of positive J values typically used in physiology reporting, the negative V ph signals decrease as a function of time upon light stimulation with V ph values in the order of − 5.4 ± 1.8 mV at a radiant exposure of 53.4 J/m 2 corresponding to 17.8 mW/cm 2 for 300 ms [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref]. The elicited photo-responses were similar to those reported in patch-clamp electrophysiology [bib_ref] Bimodal functioning of a mesoporous, light sensitive polymer/electrolyte interface, Tullii [/bib_ref] , contact pad 14 , and MEA 12 experiments where the samples are immersed in an open electrolyte bath with a reference electrode. This demonstrates the successful operation of our bio-hybrid device architecture concept. The opto-electrical response of the device was tested at different light power levels [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref] ranging from 17.8 mW/cm 2 (∼10 × 10 3 lx) to 93.8 mW/cm 2 (∼60 × 10 3 lx) representing the intensity range typically experienced in human photopic vision outdoors 53 (from 10 to 100 × 10 3 lx) and the range of irradiance levels generally used for in vitro retinal stimulation experiments [bib_ref] A polymer optoelectronic interface provides visual cues to a blind retina, Gautam [/bib_ref] [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref] [bib_ref] Maintaining ocular safety with light exposure, focusing on devices for optogenetic stimulation, Yan [/bib_ref] [bib_ref] Cortical responses elicited by photovoltaic subretinal prostheses exhibit similarities to visually evoked..., Mandel [/bib_ref]. Currents (J ph first peaks, [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref] ; J ph second peaks Supplementary [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref] ; J ph steady state [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref] increase linearly with irradiance whereas V ph max (measured after 300 ms from light switched ON) as function of the increasing irradiance levels shows a quasilogarithmic trend [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref]. Considering possible future application of the bio-hybrid device for in vitro retinas stimulation, we also used shorter light pulse stimuli of 10 ms (optical pulse width for retinas stimulation [bib_ref] A polymer optoelectronic interface restores light sensitivity in blind rat retinas, Ghezzi [/bib_ref] [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref] , to opto-electrically characterize the device [fig_ref] Figure 3: Total charge measured upon photo-stimulation [/fig_ref]. Recorded values of current capacitive peaks are similar with good approximation for 10 and 300 ms light pulse duration [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref] We also examined the total charge generated by the device upon illumination. The total charge (calculated as the time integral of photocurrent) at different irradiance levels was 2.8 ± 0.2 nC/cm 2 and 10.4 ± 3.3 nC/cm 2 for 10 ms light pulse stimulation [fig_ref] Figure 3: Total charge measured upon photo-stimulation [/fig_ref] and 106.4 ± 23.7 nC/cm 2 and 331.9 ± 42.8 nC/cm 2 for 300 ms light pulse stimulation [fig_ref] Figure 3: Total charge measured upon photo-stimulation [/fig_ref] at 17.8 and 93.8 mW/cm 2 respectively. Thus, the device is able to generate a charge level in the range of few nC/cm 2 which is useful for neural stimulation and in particular for retinas stimulation (bipolar and ganglion cells) [bib_ref] An in vitro model of a retinal prosthesis, Ahuja [/bib_ref]. The total photo-generated charge consists of a capacitive and a faradaic component. While the capacitive charge corresponds to the current spikes generated during few ms after the light is switched ON and OFF and includes redistribution of charges at the polymer/electrolyte and electrolyte/metal interfaces, the faradaic component is related to the steady-state photocurrent and involves transfer of electrons across the electrode/electrolyte interface [bib_ref] Carbon nanotube-based multi electrode arrays for neuronal interfacing: progress and prospects, Hanein [/bib_ref]. As suggested by previous works, faradaic reactions implicate reduction/oxidation reactions involving the oxidation of the organic semiconductor interfaced with the electrolyte solutions. Oxygen reduction and hydrogen peroxide production or hydrogen evolution reactions have been implied [bib_ref] Tuning photoelectrochemical performance of poly(3-hexylthiophene) electrodes via surface structuring, Wei [/bib_ref] [bib_ref] Polymer-based photocatalytic hydrogen generation, Lanzarini [/bib_ref]. Transport of mobile ions present in the electrolyte solution across the device cross-section and charge-transfer at the electrodes are also processes that give rise to photo responses of this type [bib_ref] Diffusion in the electrolyte and charge-transfer reaction at the platinum electrode in..., Hauch [/bib_ref].
To gauge the lifetime of the device, we performed an accelerated ageing test. The sandwiched device containing PBS was subjected to continuous white light illumination at 17.8 mW/cm 2 (density power typically [fig_ref] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis [/fig_ref] up to 10 min, i.e. sufficient for testing the response of the devices, whilst the J ph max decreased by 15% [fig_ref] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis [/fig_ref]. At the very high optical powers of 93.8 mW/cm 2 degradation accelerated significantly: V ph max diminished by 12% [fig_ref] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis [/fig_ref] and J ph max by 40% [fig_ref] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis [/fig_ref] after 5 min. We continued the accelerated test at these high powers for 48 h noticing a reduction of the current by 64% from the fresh device and visible alteration of both the photo-electrode (formation of blisters and wrinkles) and counter-electrode (residues of the biological medium) as well as evaporation of the electrolyte [fig_ref] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis [/fig_ref]. This is not surprising as the test at the high irradiance of 93.8 mW/cm 2 is the equivalent of looking straight at the sun which is known to damage the eye even after few seconds [bib_ref] Solar retinopathy: a biophysical analysis, Sadun [/bib_ref] [bib_ref] Acute eclipse retinopathy: a small case series, Khatib [/bib_ref]. It would be interesting to carry out a long detailed study on lifetimes by varying systematically the irradiance levels and determine the threshold that leads to significant degradation on materials and electrolyte solutions in order to evaluate what changes are needed to improve the lifetimes of artificial retina systems.
## Pixelated polymer artificial retina via inkjet printing.
Once the biocompatibility and the optoelectronic operation of the devices presented in was demonstrated, the next step was to extend the singlepolymer-layer device to a pixelated, colour sensitive, three-polymer-based device towards an artificial retina model. The size of a human adult retina is typically around 22.0 mm in diameter [bib_ref] Variations in eyeball diameters of the healthy adults, Bekerman [/bib_ref]. Photoreceptors are organized in a well-defined mosaic [bib_ref] Count and density of human retinal photoreceptors. Graefe's Arch, Jonas [/bib_ref] where rod densities are predominant in the peripheral retinal region while cones in the central part (fovea). Out of all cones, about 64% are L type, 32% M type and only 2% S type [bib_ref] The arrangement of the three cone classes in the living human eye, Roorda [/bib_ref]. Relative ratio and position of cones varies greatly among different people even with regular vision [bib_ref] Packing arrangement of the three cone classes in primate retina, Roorda [/bib_ref]. Recently the concepts of pixelated visual implants have been exploited and retinal stimulating pixels (~ 100 µm in diameter) have been obtained by spin-coating, sputtering and photolithography techniques [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref]. We designed a simplified model to fabricate the pixelated artificial retina system using the resulting anatomical map of the photoreceptors mosaic and placing locally different polymer semiconductors with specific absorbance spectra via printing techniques [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref]. Conjugated polymers have been previously proposed as promising materials for colour detection 30,31,33 due to band gap and optical tunability. We firstly identified polymers with absorbance spectra similar to that of human photoreceptors (for human photoreceptors absorbance we referred to Bowmaker and Dartnall studies 66 ) [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref]. Regio-regular P3HT polymer (see "Methods" section) and a blend of P3HT with [bib_ref] Advances in retinal prosthesis systems, Bloch [/bib_ref] -Phenyl C61 butyric acid methyl ester (PC 61 BM) (P3HT:PCBM) were used, mimicking well the green cone and rods absorbance spectra, respectively. As a third photosensitive polymer we chose Poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO), to mimic S-cones. Its peak is shifted significantly from both those of P3HT and the P3HT:PCBM blend and thus they are all spectrally well distinguishable. Then the biocompatibility was evaluated for the P3HT:PCBM and PFO layers [fig_ref] Figure 6: Spectral responsivity of the inkjet-printed artificial retina [/fig_ref] , as previously described for P3HT [fig_ref] Figure 6: Spectral responsivity of the inkjet-printed artificial retina [/fig_ref] were observed on the polymer layers compared to control. Cell death analysed at T0 and T3 was very low, with no differences between CTRL, P3HT:PCBM and PFO [fig_ref] Figure 6: Spectral responsivity of the inkjet-printed artificial retina [/fig_ref]. These results indicated no toxicity of all the polymer layers analysed. Thus, a pixelated retina system based on M-cones (P3HT), S-cones (PFO) and rods (P3HT:PCBM) www.nature.com/scientificreports/ was printed, providing a dichromatic inkjet-printed artificial retina model. The three-polymer bio-hybrid printed device mimics the dichromatic vision of most mammals including rodents often used in biomedical studies [bib_ref] Number and distribution of mouse retinal cone photoreceptors: differences between an albino..., Ortín-Martínez [/bib_ref] such as mice that only possess S-and M-cones. Furthermore, it delineates a starting point for a future trichromatic human artificial retina system. Indeed, the device-concept can be easily extended to other polymers. For instance, a possible candidate to mimic human L-cones (maximal absorption at λ = 560 nm) could be Poly[N-9′-heptadecanyl-2,7-carbazole-alt-5,5-(4′,7′-di-2-thienyl-2′,1′,3′-benzothiadiazole)] (PCDTBT) which has an absorbance spectra maximum at 576 nm. In this study we assembled a simplified model of the anatomical retinal scheme consisting in a concentric ring design where each annulus represents a different retinal photoreceptor region. This simplification made it possible to selectively shadow-mask the different annuli in order to test the spatial and spectral response of our pixelated device. The outermost annulus of 360.5 mm 2 corresponds to rods (P3HT:PCBM), whilst the two central annuli correspond to the M-cones (P3HT) of 12.6 mm 2 and the S-cones (PFO) of 7.1 mm 2 areas respectively. Total diameter was 22.0 mm. Inkjet printing was used for selective deposition of the polymers [fig_ref] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated... [/fig_ref] being a powerful technique that offers excellent design flexibility and precise control in material deposition [bib_ref] Ink-jet printing of Cu−Ag-based highly conductive tracks on a transparent substrate, Woo [/bib_ref] , enabling contactless, mask-free and digital patterning minimising materials usage and waste produced.
Semiconducting polymer inks formulation (molecular weight, concentration, solvents, viscosity and surface tension) and inkjet deposition represented an important part of creating a complete colour-sensitive artificial retina system. High viscosity, nozzle clogging, agglomeration, precipitation, and uncontrollable drying patterns are among the frequently challenges encountered with inkjet printing [bib_ref] Construction of micro-patterned polymer structures by piezoelectric inkjet printing, Yun [/bib_ref] [bib_ref] Energy storage on demand: ultra-high-rate and highenergy-density inkjet-printed NiO micro-supercapacitors, Giannakou [/bib_ref]. PFO, P3HT and P3HT:PCBM were dissolved in a mixture of chlorobenzene (CB) and tetrahydronaphthalene (THN) (1:1) to obtain inkjet-printable polymer inks. The combination of CB:THN-medium/high boiling point solvent mixture-serves two purposes. CB was used to achieve homogeneous film formation owing to excellent solubility of the chosen polymers in CB. THN (b.p. = 270 °C) was used to prevent the nozzles clogging and to provide reliable jetting over time. Inkjet parameters (pulse voltages, cartridge temperature, substrate temperature, drop spacing, waveform) were carefully adjusted in order to obtain stable droplet formation and to achieve rounded-pixels layout (see "Methods" section). The artificial retina system was printed on Glass|FTO substrates. The inkjet-printed device consisted of ~ 42,100 pixels over the total area of 380.1 mm 2 . Optical microscope images show the concentric layout of The printed pixelated device is a starting point for higher pixel density artificial retina models. Indeed, to reduce the pixel size, it is possible to use additional layers to modify the wettability and surface energy of the substrate. An example is shown in , where a spin coated P3HT:PCBM layer was used as a layer on which to print P3HT and PFO pixels that enabled us to obtain small diameters of ~ 50 μm. A different underlying layer with negligible optical absorption in the visible range would be suitable for this purpose. In general, inkjet printing equipment with smaller nozzle orifices/small droplet volume and higher viscosity inks along with substrate chemical surface functionalisation, can enable the realisation of pixel sizes smaller than 30 μm 72 . During printing from a single nozzle, individual sub-nanoliter ink droplets are ejected. The resulting drop diameter is related to the size of the nozzle orifice (1 pL drop can create a 20-30 μm spot size) and final, dry, printed area size resulting from a single drop, can be adjusted significantly by the operating conditions 73 and the surface treatment of the substrate. In order to approach the 2-10 μm size, typical for human photoreceptors [bib_ref] Distribution of short-wavelength-sensitive cones in human fetal and postnatal retina: early development..., Cornish [/bib_ref] , alternative printing technologies can be adopted such as imprinting [bib_ref] Inkjet printing of high performance transistors with micron order chemically set gaps, Grubb [/bib_ref] [bib_ref] Fabrication of 60-nm transistors on 4-in wafer using nanoimprint at all lithography..., Zhang [/bib_ref] , flexographic printing 78 , laser patterning 79 or high resolution lithography techniques (optical and electronic).
To the best of our knowledge, no visual prosthetic devices can restore the natural colour vision to date. Our proposed concentric artificial retina model is simplified in order to enable demonstration of spatial resolution of the photo-response. Human cones are distributed radially from the fovea in a geometry that is unique from individual to individual. Even if it was not possible to achieve the ideal case of superimposing pixelated artificial photoreceptors with the same dimension and in the same position as that of the real retina it wants to mimic (e.g. for implantation), there remains possibilities for inducing and possibly restoring some trichromatic vision to the blind. For example, it would be convenient to identify local density variations of cone types across a region of the retina and to place the larger pixels of one type of polymer over the areas where there are larger concentrations of the homologous cones. The avenues for exploration and increasing understanding of the visual system are many.
The artificial retina device was completed by assembling a top transparent platinized counter-electrode with a 60 µm thick thermoplastic spacer and filling the space with PBS. It was then characterised by placing it in a customized black box and illuminating the whole active area (380.1 mm 2 ) via a train of 6 light pulses using a LED lamp, 6 mW/cm 2 at 5 cm distance (standard warm white light spectrum, . Chronoamperometric J ph curves (impulses of 2 s light ON, 2 s light OFF) are consistent with those measured for the prototypical single layer device [fig_ref] Figure 2: Bio-hybrid device optoelectrical characterization [/fig_ref] , showing a capacitive switch and a more steady contribution to the current with J ph ranging from 0.2 to 0.3 μA/cm 2 . On average responses were 7% greater . Photovoltage data were recorded with a train of white light pulses of 5 s duration followed by 60 s dark period. A significantly high V ph of 12.0 mV was recorded after a 5 s long light stimulus (it was ∼0.3 mV and ∼1.1 mV after 10 ms and 300 ms respectively, thus consistent with short light pulse stimuli).The darkness period was extended to 60 s to enable V ph to recover within 10% of its initial value. It would be interesting to evaluate the optoelectrical responses of a single polymer pixel in order to assess whether the one-polymer pixel/one-photoreceptor stimulation is possible. A low-level electrical measurement set-up with a current resolution of less than 1 pA would be required for this analysis. Thus, this will be part of a future investigation. With our current systems we were able to gauge the spectral and spatial responsivity of the different polymer regions of the pixelated device by masking/unmasking larger annuli regions (see "Methods" section). The resulting photocurrent (I ph ) as a function of wavelength of the light source is shown in [fig_ref] Figure 6: Spectral responsivity of the inkjet-printed artificial retina [/fig_ref] with the same measurements carried out on each of the single polymer (or blend) annulus of the printed artificial retina. Customized shadow masks were used to exclusively illuminate the polymer annulus of interest. Notwithstanding the geometrical domination of the P3HT:PCBM component (PFO 2.0%, P3HT 3.3%, P3HT:PCBM 94.7% of total inkjet-printed artificial retina area), each pixelated polymer annulus contributes to the overall I ph signal [fig_ref] Figure 6: Spectral responsivity of the inkjet-printed artificial retina [/fig_ref]. The pronounced red peak in the P3HT:PCBM spectrum can be due to various effects including layer thickness [bib_ref] Polymer photodetector with voltage-adjustable photocurrent spectrum, Chen [/bib_ref] , solvent used 40 , thermal treatment 81 or the interaction with materials such as dopants [bib_ref] Effect of boric acid doped PEDOT: PSS layer on the performance of..., Yagci [/bib_ref] as well with components of the electrolytic medium.
# Discussion
The main focus of this study was the development of a conjugated polymer-based bio-hybrid artificial retina model to study photo-response properties of different band-gap polymer pixels, interfaced with electrolyte medium, aiming to mimic the colour response of mammalian cones and rods, from the single-polymer-layer device concept to a fully pixelated system. The device layout consists of two transparent, planar and parallel electrodes: the P3HT-spin-coated photosensitive working electrode and the Pt-screen printed counter-electrode. Planar polymer layer for retinal opto-electrical stimulation have been already successfully demonstrated in literature, both as polymer thin films spin-coated on conductive glass substrates and as polymer-coated MEAs electrodes [bib_ref] A polymer optoelectronic interface provides visual cues to a blind retina, Gautam [/bib_ref] [bib_ref] A polymer optoelectronic interface restores light sensitivity in blind rat retinas, Ghezzi [/bib_ref] [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref] [bib_ref] A hybrid bioorganic interface for neuronal photoactivation, Ghezzi [/bib_ref]. In these works patch-clamp 27 , contact pad 14 , and MEA 12 electrodes are interfaced with electrolyte in an open container configuration with a metal counter-electrode immersed in the electrolyte. Distinctively, the presented bio-hybrid device enables the study of the spectral and spatial responses of the substrate/polymer system interfaced with biological electrolyte within a closed architecture. Consistently with other works on conjugated polymers [bib_ref] A polymer optoelectronic interface provides visual cues to a blind retina, Gautam [/bib_ref] [bib_ref] A polymer optoelectronic interface restores light sensitivity in blind rat retinas, Ghezzi [/bib_ref] [bib_ref] A hybrid bioorganic interface for neuronal photoactivation, Ghezzi [/bib_ref] [bib_ref] Characterization of a polymer-based, fully organic prosthesis for implantation into the subretinal..., Antognazza [/bib_ref] [bib_ref] Conjugated polymers for the optical control of the electrical activity of living..., Vaquero [/bib_ref] [bib_ref] Organic semiconductors for artificial vision, Martino [/bib_ref] [bib_ref] A fully organic retinal prosthesis restores vision in a rat model of..., Maya-Vetencourt [/bib_ref] , the fabricated bio-hybrid photo-electrode shows good biocompatibility. Thus, the device can represent a useful tool for studying light stimulation and recording of bio-electrical signals. The fabricated bio-hybrid device response to incident light elicits transient photo-voltage (mV) and photo-current (µA/cm 2 ) outputs that resemble those seen in patch-clamp and MEA experiments and that would suffice to elicit a response in a retina [bib_ref] Strength-duration relationship for extracellular neural stimulation: numerical and analytical models, Boinagrov [/bib_ref] or in neurons [bib_ref] Extracellular stimulation of central neurons: influence of stimulus waveform and frequency on..., Mcintyre [/bib_ref]. Even if capacitive coupling (0.51 ± 0.28 µA/cm 2 and of -0.29 ± 0.12 µA/ cm 2 spikes at the beginning and the end of light stimuli, 17.8 mW/cm 2 ) is the dominant process in the device upon illumination, a minor faradaic component (0.17 ± 0.10 µA/cm 2 ) is present. Clearly charge balancing is a vital process to ensure the electrode-tissue interface safety operation. However, we think that self-regulation of ionic www.nature.com/scientificreports/ gradients in living systems could help to prevent tissues and cells damage over time [bib_ref] Ion homeostasis, channels, and transporters: an update on cellular mechanisms, Dubyak [/bib_ref]. Thus, we can accept the recorded current density signals and faradaic component values with a good margin of tolerance also in respect to similar work found in literature where faradaic processes were not completely excluded [bib_ref] A polymer optoelectronic interface restores light sensitivity in blind rat retinas, Ghezzi [/bib_ref] [bib_ref] Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis, Ferlauto [/bib_ref] [bib_ref] A hybrid bioorganic interface for neuronal photoactivation, Ghezzi [/bib_ref]. The bio-hybrid device presents some advantages compared to electrophysiological investigative systems: easy-to-handle and transportable closed system, controllable size, and a required small amount of electrolyte. It permits the utilization of tools from an electronic engineering/physics/chemistry laboratory rather than the more sophisticated tools found in bio-medical laboratories. In addition, the device architecture opens the possibility of inserting biological materials (cells or tissues) in the chamber of a compact device filled with the biological electrolytic medium. Limitations of this device compared to the open immersions used in typical MEA or electrophysiology experiments, may be represented by the constraints in inserting fairly large tissues, such as retinas, in the small chamber for their study, as well as the large dimensions of the current electrodes used (see . The latter may need to be patterned in a smaller size (i.e. similar to those found in MEAs, i.e. of the order of ≤ 0.1 mm) and be more numerous for better efficiency to record bioelectrical signals from local areas of live retinas. It will also be critical to devise ways to keep living samples functioning in a restricted environment which may require the introduction of inlet and outlet orifices for perfusion. Until these evolutions are implemented and tested, the device can be useful to spatially and spectrally investigate photo-responses of semiconductor films or patterns immersed in physiological fluids that mimic those found in living organisms. The device was also fabricated via inkjet printing technique. Thus, an inkjet-printed colour-sensitive artificial retina device consisting of three different types of semiconducting round polymer pixels, with distinct absorption spectra, mimicking the chromatic sensitivity of photoreceptors in the eye and interfaced with a physiological medium was demonstrated. The number of pixels was 42,100, the density of the artificial photoreceptors was ∼11,000 pixels/cm 2 and the corresponding spatial resolution was 267 dpi (dots per inch), with pixel diameters of 95 ± 5 μm. Printing technologies enable placement of different materials in the locations of choice. In the future, with higher resolution techniques being developed (i.e. ≤ 10 µm), one could first scan/ image an individual retina and then print the pixels spatially where exactly one wants to place them and this can be done for each individual retina (more costly if one uses photolithographic techniques). Secondly, printing techniques enable deposition on uneven or curved surfaces which can be beneficial for artificial retina concepts. The polymer pixelation allows a light-sensing device to spectrally and spatially resolve incoming light providing photo-responses that are both chromatically and spatially sensitive. The optoelectrical characterization of a single polymer pixel awaits further investigation. Future studies can be devoted in replacing glass with flexible substrates such as PET, poly (dimethylsiloxane) (PDMS), and silk fibroin films, the latter of which are implantable in vivo [bib_ref] A fully organic retinal prosthesis restores vision in a rat model of..., Maya-Vetencourt [/bib_ref]. Moreover, additional studies on the device biocompatibility and its possible integration with healthy or degenerated explanted retinas from animal models are necessary to demonstrate future optoelectronic applications in the field of retinal prostheses.
# Methods
Substrates preparation for bio-hybrid interface. Transparent Glass|FTO substrates (2.2 mm thickness) were cut in 2.5 cm by 2.5 cm and cleaned by subsequent rinses in ultrasonic bath using deionized water, pure acetone, isopropyl alcohol (5 min each). Then dried with an air gun.
Polymer formulations for bio-hybrid interface. rr-P3HT (Rieke Metals, Inc.) (regio-regularity ≥ 95%, molecular weight: 51,000 g/mol) was used without any further purification. Polymer solutions in chlorobenzene (30 mg/mL) were prepared by heating polymer and solvent mixture at 100 °C for 5 min and then stirring at 50 °C overnight. rr-P3HT-chlorobenzene solution was spin-coated on FTO glass substrates (2000 r.p.m., rotation duration 60 s) inside the glovebox (Mbraun Inc.). Organic thin films were annealed on a hotplate (120 °C, 2 h). To obtain a square active area of 0.8 cm by 0.8 cm, the excess of polymer was removed using p-Xylene solvent and a polymer protective film mask.
Electrolyte compartment for bio-hybrid interface. Thermo-plastic sealant (Surlyn, 60 µm thickness, DU PONT) masks, creating the milli-volume chamber, were designed to cover the frame of the 0.8 cm by 0.8 cm active area on the device and cut using a cutting machine (Small-one, magicut cutting plotter). Two orifices placed in opposite sides were designed to fill electrolyte inside the device. Masks were pre-attached on the substrates via pneumatic heat press (model Special) (90 °C, 0.5 bar pressure).
## Counter-electrode for bio-hybrid interface. a transparent platinum layer was screen-printed on
Glass|FTO substrates (2.5 cm by 2.5 cm). After platinum paste precursor (3D-nano) deposition, the wet platinum layers were processed by a firing process (30 min, 480 °C).
## Electrolyte solution.
Phosphate buffered saline solution (Gibco) was used as electrolytic solution. PBS is a balanced salt solution containing: CaCl 2 (0.9 mM), MgCl 2 (0.5 mM), KCl (2.6 mM), KH 2 PO 4 (1.5 mM), NaCl (137.9 mM) and Na 2 HPO 4 (8.0 mM) dissolved in double distilled H 2 O. PBS was filled inside the device using a 1 mL syringe by the orifices present in the Surlyn masks. Devices were then closed using a biphasic glue.
Inkjet-printed artificial retina fabrication. FTO glass substrates (2.5 cm by 2.5 cm) were treated with a standard cleaning process as described before. They were then treated with Argon/Oxygen plasma (100 W, 10 min) and UV treatment (1000 W, 5 min) to improve wettability and hydrophilicity. A model design reproducing the photoreceptors distribution on the human retina was designed and drawn via a standard vector www.nature.com/scientificreports/ and Bonferroni post-analyses. The level significance was set at P < 0.05 (*, a), P < 0.01 (**, b), P < 0.001 (***, c) and P < 0.0001 (****, d).
## Measurement setup.
Three measurements set ups were used. ARKEO apparatus (Cicci Research s.r.l.) was used to opto-electrically characterize the bio-hybrid interface. Transient photovoltage (TPV) and charge extraction (CE) software routines were used. Light stimuli (from the bottom of the device) of 10 and 300 ms duration after 100 ms of dark were presented every 60 s with increasing light intensities , and 93.8 mW/cm 2 ). Each measurement was done immediately after the electrolyte injection in the device. Plotted signals are the mean over 3 samples. Each measurement represents an average of twenty consecutive sweeps (10 kHz and 1 kHz sampling rate for 10 ms and 300 ms light stimuli pulse duration).
In TPV recordings a baseline potential was removed to evaluate the potential difference (from dark to light). A 6 W LED lamp (6 mW/cm 2 , at 5 cm distance from the lamp, as measured with ThorLabs PM100D broadband power meter) driven by a microcontroller (schematics and connection in was used for pixelated device characterization. A train of 6 light pulses (alternating periods of 2 s light ON and 2 s OFF for J ph measurements, and 5 s ON and 60 s OFF for V ph measurements) was used. A Keysight precision currentvoltage analyser (Keysight technologies) was used to measure the J ph and V ph signals from the inkjet-printed artificial retina device (5 Hz sampling rate). I ph and V ph across different wavelengths were recorded by using a light source filtered through a monochromator before been focused on the polymer film/inkjet-printed artificial retina device. Data were collected at 2 nm intervals, with scans conducted from 300 to 800 nm. Monochromatic light was narrow-band, with approximately 3 nm FWHM. The light source was chopped by a mechanical chopper at 70 Hz, whose reference signal was fed to a lock-in amplifier (0 db gain). All measurements were taken at room temperature. To examine if the electrical signals drift over time, a device-tester with a P3HT thin film and PBS was stimulated via a 530 nm wavelength light chopped at 70 Hz for ca. 2 min. Monochromator/lock-in set up was used to record electrical signals. No evident dependence on time was observed. J ph , I ph and V ph generated were measured between platinum electrode (counter-electrode) and polymer electrode (photo-electrode) in short circuit (V applied = 0 mV) and open circuit (I applied = 0 mA) respectively. The photoelectrode was grounded.
The power and spectra of ARKEO fast speed LED (5000 K) were measured using a spectrometer (AVASPEC) and an integrating sphere (AVASPHERE-50-IRRAD). The 6 W LED lamp spectra were measured using an Ocean Optics Optics HR2000 + high resolution spectrometer (Ocean Optics, Inc.).
Data were analysed with OriginPro 2016.
## Data availability
Authors declare that all relevant data supporting the findings of this study are available in this paper and in its Supplementary Information file. Access to our raw data can be obtained from the corresponding authors upon reasonable request.
Scientific Reports | (2020) 10:21457 | https://doi.org/10.1038/s41598-020-77819-z www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
[fig] Figure 2: Bio-hybrid device optoelectrical characterization. (a) Transient current density generated by 300 ms white light stimulus (17.8 mW/cm 2 ). (b) Transient photo-voltage recorded from the device under 300 ms white light (17.8 mW/cm 2 ) stimulus. (a,b) black line: device containing only polymer thin film; dashed blue line: device containing only electrolyte solution; red line: device containing polymer (P3HT) and biological electrolyte (PBS). The shaded yellow area represents the duration of the light stimulus (300 ms). (c) Current capacitive peaks (J ph max) show a linear dependence with the light power intensity. Plot is referred to J ph first positive peaks. J ph values are the mean on samples n = 3. Y-error bar is also shown. (d) Photo-Voltage as function of increasing light intensities. V ph max values (in absolute value) are referred to the value reached after 300 ms from the light switched ON. Radiant intensities used for J ph and V ph : 17.8, 26.6, 35.3, 43.9, 52.4, 60.9, 69.3, 77.4, 85.7, and 93.8 mW/cm 2 . (OriginPro 2016 was used). Scientific Reports | (2020) 10:21457 | https://doi.org/10.1038/s41598-020-77819-z www.nature.com/scientificreports/ experienced in human photopic vision outdoors 53 ) and at close to 100 mW/cm 2 (density power used to evaluate electrical performance in solar cells) and the opto-electrical responses of the device (at light stimuli of 300 ms, power levels ranging from 17.8 up to 93.8 mW/cm 2 ) were monitored. Irradiance levels generally used for in vitro retinal stimulation experiments span from 1 to 94 mW/cm 212-14 . For continuous light irradiation at 17.8 mW/ cm 2 photo-voltage electrical outputs did not change substantially (Supplementary Fig. [/fig]
[fig] Figure 3: Total charge measured upon photo-stimulation. Photo-generated charge calculated from the time integral of the photocurrent response curves as a function of increasing light power for 10 ms (a) and 300 ms (b) light pulse duration. Samples n = 3. Y-error bar referred to the Standard Deviation (SD) are shown. (OriginPro 2016 was used). Scientific Reports | (2020) 10:21457 | https://doi.org/10.1038/s41598-020-77819-z [/fig]
[fig] Figure 4: Model, materials and inkjet printing technique for the fabrication of the pixelated polymer artificial retina system. (a) Schematic representation of photoreceptors distributed on a human retina based on anatomical model. The S-Cones (blue), M-Cones (green) and L-Cones (red) are mostly packed into the fovea, while rods (grey) are located mostly in the periphery of the retina. (b) A proposed simplified concentrically organized artificial retina model to mimic the spectral response of the M-cones, L-cones and rods from mammalian retina. Concentric geometry model was used for ease of study. Rods-P3HT:PCBM annulus = 360.5 mm 2 ; S-Cones annulus = 7.1 mm 2 ; M-Cones annulus = 12.5 mm 2 . ((a,b) images created by using Microsoft PowerPoint). (c) Absorbance spectra of human photoreceptors (dashed lines: Rods, S-Cones, M-Cones, L-Cones) compared with those of polymers used in the pixelated inkjet-printed artificial retina of this work (continuous lines). Human photoreceptors absorbance from Bowmaker and Dartnall studies (OriginPro 2016 was used). (d) Schematics showing the inkjet printing process for the printed artificial retina. Chemical structures of conjugated polymers used for the artificial retina fabrication are shown: step (a), PCBM; step (b), P3HT; step (c), PFO. Step (d) represent the thermal annealing (Images created by using Microsoft PowerPoint). Scientific Reports | (2020) 10:21457 | https://doi.org/10.1038/s41598-020-77819-z www.nature.com/scientificreports/ arrays of small round polymer pixels (Fig. 5) with an average diameter size of 95 ± 5 μm and of 50 to 60 nm thickness as measured by Atomic Force Microscopy (AFM) with an average roughness of 6 to 8 nm (underlying Glass|FTO roughness was around 4 nm) and a packing density of ~ 110 pixels/mm 2 . [/fig]
[fig] Figure 5: Inkjet-printed artificial retina device prototype and related optical microscopy analysis. (a) Device picture and (b) optical microscopy image of polymer inkjet-printed artificial retina showing, (c-e) different regions of the concentrically organized polymer pixels. (f-h) AFM images of photosensitive round polymer pixels. AFM characterisation was also used for thickness and roughness measurements of the pixels. was shone from the bottom of the polymer layer rather than through top platinized electrode, likely due to the absorption of the electrolyte and counter-electrode (Supplementary [/fig]
[fig] Figure 6: Spectral responsivity of the inkjet-printed artificial retina. Photocurrent measured from the pixelated retina compared to the photocurrent calculated as the weighted linear summation of the single polymer-annuli contributions. I ph originating from singular annuli were recorded by using a monochromator/lock-in set up and additional customized shadow masks so as to illuminate only the polymer annulus of interest by covering the remaining part of the printed artificial retina. (OriginPro 2016 was used). Scientific Reports | (2020) 10:21457 | https://doi.org/10.1038/s41598-020-77819-z [/fig]
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Prevention and Treatment of Side Effects of Immunotherapy for Bladder Cancer
# Introduction
BC is among the top ten most common cancer types in the world, according to an observatory in 2018, with approximately 55000 new cases and 200000 deaths annually. It ranks tenth in worldwide absolute incidence: sixth in men and seventeenth in women [bib_ref] Nivolumab Versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer, Brahmer [/bib_ref]. The worldwide Age Standardized Incidence Rate per year (ASR) is 9.6 per 100000 for males and 2.4 per 100000 for females [bib_ref] The Global Burden of Urinary Bladder Cancer: An Update, Richters [/bib_ref].
Smoking is the most significant risk factor of BC, associated with 50-65% of male cases and 20-30% of female cases. The incidence of BC is reportedly directly associated with the duration of smoking, and the number of cigarettes smoked per day. Occupational factors are the second most important risk factor for BC [bib_ref] Epidemiology of Bladder Cancer, Saginala [/bib_ref].
Uroepithelial carcinoma originating from the bladder is the most common histologic type of cancer. Over 70% of cases are diagnosed at the non-muscle invasive stage and managed by minimally invasive local treatment. Unfortunately, this disease has a high recurrence rate and may require further treatment with more than one modality. In contrast, the muscle-invasive and metastatic stage of the disease requires multimodal treatment strategies, including surgical treatment and chemotherapy in addition to neoadjuvant, adjuvant or palliative care [bib_ref] Role of Microtubule-Associated Protein 1b in Urothelial Carcinoma: Overexpression Predicts Poor Prognosis, Chien [/bib_ref].
Cancer therapies that alter the immune status have gained prominence in oncology in recent years [bib_ref] A Fractal Fractional Model for Cervical Cancer Due to Human Papillomavirus Infection, Akgül [/bib_ref]. Immunotherapy is often used to complement traditional cancer treatments such as surgery, chemotherapy, and radiation therapy. During clinical practice, it is used as a first-line treatment for some cancers [bib_ref] The Promise of Combining Radiation Therapy With Immunotherapy, Jagodinsky [/bib_ref] and involves the patient's immune system to modify or increase the defense mechanisms against the developing cancer cells [bib_ref] The Promise of Combining Radiation Therapy With Immunotherapy, Jagodinsky [/bib_ref]. The first clinical application of immunotherapy was documented in the 1890s when William Coley first used a bacterial agent called Coley's toxin. Clinical trials showed minimal results. Importantly, this toxin provided the first compelling evidence of the potential to produce an antitumor response using the patient's immune system [bib_ref] The Promise of Combining Radiation Therapy With Immunotherapy, Jagodinsky [/bib_ref]. Immunotherapy became part of standard cancer treatment in the mid-20th century, although it exhibited significant toxicity. Treatment with cell therapy and the development of bone marrow transplantation was initiated by Fritz Bach et al. in the 1960s, as well as the production, testing and approval of high doses of IL-2 (interleukin 2) for the treatment of metastatic kidney cancer and melanoma in clinical trials in the 1990s [bib_ref] High-Dose Recombinant Interleukin 2 Therapy for Patients With Metastatic Melanoma: Analysis of..., Atkins [/bib_ref]. Several types of immunotherapies are currently used to treat cancer, including immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies, therapeutic vaccines, and immune system modulators.
Immunotherapy has an anti-cancer effect because it activates the immune response against cancer cells more specifically and strongly, thus killing them. In tumors, mutated or dysregulated proteins are processed into peptides, then loaded onto major histocompatibility complex I (MHCI) molecules to form immune complexes recognized by CD8+ T cells [bib_ref] The Evolving Landscape of Biomarkers for Checkpoint Inhibitor Immunotherapy, Havel [/bib_ref]. Then cytotoxic T lymphocytes are activated [bib_ref] Toxicities of Immunotherapy for the Practitioner, Weber [/bib_ref] , which not only kill cancer cells and inevitably cause some damage to normal cells, but may eventually attack any of the body's healthy or normal tissues or organs, leading to unpredictable side effects, also known as "immune-related adverse events (irAE)". Organ specificity, incidence, and severity of irAEs vary according to each agent and its dose, but also differ across tumor types [bib_ref] Immune-Related Adverse Events From Immune Checkpoint Inhibitors, Marrone [/bib_ref]. Immune-related adverse events include non-specific symptoms and damage to the skin and mucous membrane system, head and five sense organs, digestive system, cardiovascular system, respiratory system, endocrine system, blood system, neuropsychiatric system, bone and joint system, and immune system [bib_ref] Immune-Related Adverse Events With Immune Checkpoint Blockade: A Comprehensive Review, Michot [/bib_ref]. Immunotherapy has benefited a significant proportion of BC patients and has even been able to cure cancer in some patients in combination with other drugs. This new treatment modality offers hope to cancer patients but emphasizes that the associated toxic side-effects are currently a challenge for effective clinical treatment [fig_ref] TABLE 1 |: List of side effects, indications and serious complications for immunotherapy for bladder... [/fig_ref].
## Immunotherapy drugs and side effects
## Non-targeted immunotherapy drugs
## Bacillus calmette-guerin
It is widely acknowledged that Everolimus (Afinitor) is an attenuated strain of Mycobacterium Bovis. Although it has been discovered for decades, its exact mechanism of action remains unknown [bib_ref] Mechanisms of Immune Evasion in Bladder Cancer, Crispen [/bib_ref]. BCG is used as a vaccine and is now used stably in patients with carcinoma in situ or moderate or high non-muscle invasive BC [bib_ref] Analysis and Dynamical Behavior of Fractional-Order Cancer Model With Vaccine Strategy, Farman [/bib_ref]. It has been shown that BCG can cause a massive release of cytokines and chemokines after attachment to tumor cells by fibronectin and then internalization into tumor cells [bib_ref] Bacillus-Calmette-Gueŕin (BCG) and 3D Tumors: An in Vitro Model for the Study..., Durek [/bib_ref]. BCG also promotes tumor antigen presentation to cells of the immune system [bib_ref] Induction of Urinary Interleukin-1 (IL-1), IL-2, IL-6, and Tumour Necrosis Factor During..., Boer [/bib_ref] [bib_ref] Mycobacterium Bovis Bacillus Calmette-Gueŕin (BCG) Induces Human CC-and CXC-Chemokines In Vitro and..., Luo [/bib_ref] , and induction of long-term adaptive immunity [bib_ref] Mechanisms of Immune Evasion in Bladder Cancer, Crispen [/bib_ref] [bib_ref] Bacillus Calmette-Guerin (BCG) Immunotherapy for Bladder Cancer: Current Understanding and Perspectives on..., Kawai [/bib_ref]. It has been shown that BCG treatment elicits an inflammatory response involving different immune cell subsets, including CD4+ and CD8+ lymphocytes [bib_ref] Intravesical Evans Strain BCG Therapy: Quantitative Immunohistochemical Analysis of the Immune Response..., Prescott [/bib_ref] [bib_ref] T-Cell Subsets Required for Intravesical BCG Immunotherapy for Bladder Cancer, Ratliff [/bib_ref] , natural killer (NK) cells [bib_ref] NK Cells Are Essential for Effective BCG Immunotherapy, Brandau [/bib_ref] , granulocytes [bib_ref] NK Cells Are Essential for Effective BCG Immunotherapy, Brandau [/bib_ref] [bib_ref] Neutrophil Granulocytes are Required for Effective Bacillus Calmette-Gueŕin Immunotherapy of Bladder Cancer..., Suttmann [/bib_ref] and macrophages [bib_ref] Bacillus Calmette-Guerin (BCG) Enhances Monocyte-and Lymphocyte-Mediated Bladder Tumour Cell Killing, Pryor [/bib_ref] [bib_ref] Presence of Activated Lymphocytes in the Urine of Patients With Superficial Bladder..., Boer [/bib_ref] , among other cell subsets. In vitro experiments have shown that integrin cross-linking of BCG leads to cell cycle arrest at the G1/S interface in proliferating cells of human urothelial carcinoma cells, resulting in a direct cytostatic effect on the cancer cell line [bib_ref] BCG Directly Induces Cell Cycle Arrest in Human Transitional Carcinoma Cell Lines..., Chen [/bib_ref].
BCG is currently the most common and important tool in treating and preventing different forms of superficial BC. In this regard, treatment with BCG after transurethral resection of bladder tumor (TUPRBT) reduces the risk of tumor recurrence or high-grade tumor development, and this is now standard practice in the treatment of non-muscle invasive bladder cancer (NMIBC, including carcinoma in situ, high-grade papillary tumors, and invasive plaque intrinsic tumors) [bib_ref] Identification of Key Biomarkers in Bladder Cancer: Evidence From a, Zhang [/bib_ref]. Indeed, BCG treatment is also associated with concomitant side effects. Currently, side effects such as fatigue, fever, mild lower urinary tract symptoms and frank hematuria have been reported in the literature after BCG intravesical infusion therapy for BC [bib_ref] BCG Immunotherapy for Superficial Bladder Cancer, Lockyer [/bib_ref]. Additional side-effects include infections such as granulomatous inflammation of the genitourinary tract (bladder, testes, or prostate), pneumonia, arthritis, and hepatitis. Indeed, tuberculosis may take years to be expressed clinically and often presents as local discomfort, recurrent fever, and night sweats. If the infection worsens, severe systemic manifestations such as high fever, hypotension, organ failure, or septic shock may be observed. Therefore, the BCG vaccine should be used in the prescribed concentration range as much as possible, which will not only increase its effectiveness but also reduce the side effects to some extent [bib_ref] Bacillus Calmette Guerin (BCG) Immunotherapy for Bladder Cancer: A Control and Mathematical..., Akgül [/bib_ref].
## The mtor kinase inhibitors
Studies on the use of the mTOR Kinase Inhibitors for BC are ongoing. An increasing body of evidence shows that these drugs act by binding to the tacrolimus binding protein 12 (FKBP-12) protein, forming a complex that inhibits mTOR activity. This phenomenon leads to cell cycle arrest and inhibition of angiogenesis, proliferation, and glucose delivery to cells [bib_ref] Research on New Drugs in the Therapy of Bladder Cancer (BC) Postepy..., Jurkowska [/bib_ref]. Angiogenesis is inhibited by downregulated expression of hypoxia-inducible factor 1, which reduces the levels of vascular endothelial growth factor [bib_ref] mTOR Inhibitors in Urinary Bladder Cancer, Pinto-Leite [/bib_ref]. In 2016, the Food and Drug Administration (FDA) approved everolimus for adult patients with unresectable, locally advanced or metastatic disease with progressive neuroendocrine tumors of gastrointestinal or pulmonary origin. The most common side effects of this class of drugs include stomatitis, rash, fatigue, hyperglycemia, hyperlipidemia, and myelosuppression; most of these are mild and disappear with drug interruption or dose reduction.
## Cox-2 inhibitors
Cyclooxygenase inhibitors are compounds that have inhibitory effects on cyclooxygenase. Cyclooxygenase inhibitors include two major groups: nonspecific cyclooxygenase inhibitors, which can inhibit both COX-1 and COX-2, such as aspirin and specific COX-2 inhibitors, such as celecoxib. Interestingly, the Cyclooxygenase-2 (COX-2) inhibitor has been shown to exhibit chemopreventive activity against various cancers, including BC, by inhibiting the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of BC cells. However, its mechanism of action is not fully understood [bib_ref] Celecoxib Inhibits the Epithelial-to-Mesenchymal Transition in Bladder Cancer Via the miRNA-145/TGFBR2/Smad3 Axis, Liu [/bib_ref]. Common adverse reactions mainly involve the digestive, cardiovascular, and urinary systems. Other adverse reactions include systemic reactions, which are generally mild.
## Targeted immunotherapy drugs
Immune checkpoints are molecules involved in maintaining immune homeostasis and therefore contribute to maintaining peripheral tolerance to their own molecules. The main immune checkpoint inhibitors include blockade of programmed cell death protein-1/programmed cell death protein ligand 1 (PD-1/PD-L1) and cytotoxic T cell antigen (CTLA4). The use of monoclonal antibodies that block co-inhibitory immune checkpoint molecules helps to increase T cell-specific immune responses and thus harness the immune system against tumors [bib_ref] Cancer Immunoediting: From Immunosurveillance to Tumor Escape, Dunn [/bib_ref]. Responsiveness to checkpoint inhibitors is key to treatment, but this does not necessarily mean that all patients have good outcomes since some can also experience drug side effects. Other immune cells can also play an important role in developing irAEs, including B cells, which can secrete antibodies to conduct toxicity [bib_ref] PD-1 Regulates Germinal Center B Cell Survival and the Formation and Affinity..., Good-Jacobson [/bib_ref] [bib_ref] Pituitary Expression of CTLA-4 Mediates Hypophysitis Secondary to Administration of CTLA-4 Blocking..., Iwama [/bib_ref] , and granulocytes, which secrete inflammatory mediators and cytokines [bib_ref] PD-1 Regulates Germinal Center B Cell Survival and the Formation and Affinity..., Good-Jacobson [/bib_ref] [bib_ref] Targeting PD-1/PD-L1 Interactions for Cancer Immunotherapy, Zitvogel [/bib_ref]. Indeed, it should be borne in mind that the side effects of a drug may not significantly alter its effectiveness; however, the patient's quality of life may be affected during treatment. Overall, side effects associated with anti-PD-1/ PD-L1 are less common and severe than with anti-CTLA-4 antibodies [bib_ref] Toxicities of the Anti-PD-1 and Anti-PD-L1 Immune Checkpoint Antibodies, Naidoo [/bib_ref]. The most typical manifestations involve the skin, gastrointestinal tract, liver, and endocrine system [bib_ref] At the Bedside: CTLA-4-and PD-1-Blocking Antibodies in Cancer Immunotherapy, Callahan [/bib_ref]. Cutaneous toxicity is the most common irAE, although GI involvement is usually more clinically relevant because of its potential morbidity and management, requiring steroids and hospitalization. Other rarely reported irAEs include uveitis, conjunctivitis, neuropathy, myopathy, pancreatitis, pneumonia, hemocytopenia, and nephritis [bib_ref] At the Bedside: CTLA-4-and PD-1-Blocking Antibodies in Cancer Immunotherapy, Callahan [/bib_ref]. Immune-related adverse events associated with a certain immune checkpoint inhibitor is usually consistent across tumor types [fig_ref] FIGURE 1 |: Immune checkpoint inhibitors in BC treatment [/fig_ref].
## Pd-1/pd-l1
PD-1 and PD-L1 are important immune checkpoints that negatively modulate the immune system, impairing its response to antigens. PD-1 is expressed on the surface of activated T and B lymphocytes and macrophages, and PD-L1 on antigen-presenting cells [bib_ref] Advanced Deep Learning Embedded Motion Radiomics Pipeline for Predicting Anti-PD-1/PD-L1 Immunotherapy Response..., Rundo [/bib_ref]. The binding of PD-1 and PD-L1 blocks the activation of T lymphocytes, thereby reducing the production of IL-2 (interleukin 2) and interferon-gamma (59). Anti-PD-1 and PD-L1 drugs can block either of these two molecules, preventing both from binding, thereby increasing the production of both cytokines (60).
## Nivolumab
Nivolumab, a human monoclonal antibody of IgG4 type, was approved by the FDA in 2017 for use in advanced BC. The common complications are elevated lipase and amylase, fatigue, skin rash, dyspnea, neutropenia, and lymphopenia (62-64).
## Pembrolizumab
Pembrolizumab is a humanized IgG4/kappa monoclonal antibody that can be used to treat various types of cancer. Approved by the FDA in 2019 for the treatment of BC, especially for advanced BC cases [bib_ref] Pembrolizumab as Second-Line Therapy for Advanced Urothelial Carcinoma, Bellmunt [/bib_ref] [bib_ref] Cost-Effectiveness of Pembrolizumab for Patients With Advanced, Unresectable, or Metastatic Urothelial Cancer..., Patterson [/bib_ref] , pembrolizumab may be used as a first-line treatment for uroepithelial cancer [bib_ref] Therapeutic Implications of PD-L1 Expression in Bladder Cancer With Squamous Differentiation, Morsch [/bib_ref]. Moreover, it can be used to treat patients with DNA repair defects [bib_ref] Cancer Immunoprevention: A Case Report Raising the Possibility of "Immuno-Interception", Mancuso [/bib_ref] , and the reported overall survival with pembrolizumab is significantly higher than with chemotherapy drugs [bib_ref] Immunotherapy in Urothelial Cancer: Recent Results and Future Perspectives, Farina [/bib_ref]. Importantly, Pembrolizumab has a better safety profile than other drugs, although it may cause immune-related adverse effects such as myocarditis and myasthenia gravis.
## Durvalumab
Durvalumab is an IgG1k monoclonal antibody approved by the FDA to treat BC in 2017. Studies have shown that although Durvalumab has high activity in PD-L1-positive and negative patients, it exhibits relatively higher efficacy in patients with high PD-L1 expression [bib_ref] Analytical Validation and Clinical Utility of an Immunohistochemical Programmed Death Ligand-1 Diagnostic..., Zajac [/bib_ref].
## Atezolizumab
Atezolizumab, a humanized IgG1 isotype monoclonal antibody [bib_ref] Anti-PD-L1) Treatment Leads to Clinical Activity in Metastatic Bladder Cancer, Powles [/bib_ref] , was the first PD-1/PD-L1 checkpoint inhibitor approved by the FDA and is often used in the second-line treatment of patients with advanced BC. Most treatment-related adverse events are mild to moderate, including fatigue, nausea, decreased appetite, pruritus, fever, diarrhea, rash, and arthralgia [bib_ref] Atezolizumab in Patients With Locally Advanced and Metastatic Urothelial Carcinoma Who Have..., Rosenberg [/bib_ref].
## Avelumab
Avelumab is also an IgG1 antibody that primarily targets PD-L1 and was approved by the FDA in 2017 for uroepithelial cancer. When combined with platinum-based drugs, Avelumab produces a sustained antitumor response in patients with advanced or present metastatic uroepithelial carcinoma [bib_ref] Atezolizumab in Patients With Locally Advanced and Metastatic Urothelial Carcinoma Who Have..., Rosenberg [/bib_ref]. Patients may experience side effects such as fatigue, weakness, nausea, and infusion-related reactions [bib_ref] Avelumab, an Anti-Programmed Death-Ligand 1 Antibody, In Patients With Refractory Metastatic Urothelial..., Apolo [/bib_ref].
## Anti-ctla-4 antibodies
CTLA-4 is a surface molecule expressed by activated T cells that binds to B7.1 and B7.2 ligands expressed on B lymphocytes, dendritic cells, and macrophages [bib_ref] Immunotherapy in Urothelial Cancer: Recent Results and Future Perspectives, Farina [/bib_ref]. CTLA-4 is a costimulatory molecule necessary for the activation of T lymphocytes [bib_ref] Immunotherapy in Urothelial Cancer: Recent Results and Future Perspectives, Farina [/bib_ref] [bib_ref] Immunogenic Human Papillomavirus Pseudovirus-Mediated Suicide-Gene Therapy for Bladder Cancer, Hojeij [/bib_ref] [bib_ref] Epigenetics of Urological Cancers, Schulz [/bib_ref]. It has been established to negatively regulate the immune system; nonetheless, the mechanism of its action is not fully understood. Given that CTLA-4 is structurally related to CD28, it has been suggested that CTLA-4 can compete with CD28 in terms of ligand binding. and another also suggested that it can directly signal all the way to the CTLA-4 cytoplasmic tail [bib_ref] Blockade of CTLA-4 on Both Effector and Regulatory T Cell Compartments Contributes..., Peggs [/bib_ref] [bib_ref] Molecular Interactions Mediating T Cell Antigen Recognition, Van Der Merwe [/bib_ref] [bib_ref] CTLA-4 (CD152) can Inhibit T Cell Activation by Two Different Mechanisms Depending..., Carreno [/bib_ref] , and inhibition of CTLA-4 enhances the immune response.
## Ipilimumab
Ipilimumab, originally developed by Bristol-Meyers Squibb as an anti-CTLA-4 monoclonal antibody for the treatment of melanoma, is also used in combination with nivolumab for the treatment of advanced kidney cancer and different types of metastatic colorectal cancer (79), with common side effects including toxicity in the dermal system, gastrointestinal tract, liver, and neurological and endocrine systems [bib_ref] Management of Immune-Related Adverse Events and Kinetics of Response With Ipilimumab, Weber [/bib_ref] [bib_ref] Neurological Immune-Related Adverse Events of Ipilimumab, Bot [/bib_ref]. However, the efficacy of this drug in BC is largely unknown, warranting further studies.
## Tremelimumab
Tremelimumab is a well-recognized humanized monoclonal antibody against CTLA-4, however, it has not been approved by the FDA for cancer treatment.
## Chimeric antigen receptor weight-targeted t cells
CAR-T is a novel precision-targeted therapy for the treatment of tumors. The Chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner, enabling them to recognize a broader range of target antigens than natural T-cell surface receptors (TCRs) [bib_ref] Targeting Tumours With Genetically Enhanced T Lymphocytes, Sadelain [/bib_ref]. It is a highly promising immunotherapy approach that has yielded good results in clinical tumor treatment in recent years through optimization and improvement. There are currently two FDA-approved CAR-T therapies: Kymriah (Tisagenlecleucel) and Yescarta (Axicabtagene ciloleucel). CAR-T is now predominantly used for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) [bib_ref] Trial Watch: Adoptively Transferred Cells for Anticancer Immunotherapy, Fournier [/bib_ref]. Due to the complexity of BC and its location in the body, the treatment of solid tumors with CAR-T cells faces multiple obstacles, such as a harsh tumor microenvironment, on-tumor or off-tumor toxicity, and unpredictable antigen specificity [bib_ref] New Strategies for the Treatment of Solid Tumors With CAR-T Cells, Zhang [/bib_ref]. Notwithstanding that CAR-T is already approved to treat solid tumors such as BC, clinical trials on CAR-T cells for solid tumors are still being conducted on multiple fronts. CAR-T is also associated with serious adverse effects (85), mainly cytokine release syndrome (CRS) [bib_ref] B-Cell Depletion and Remissions of Malignancy Along With Cytokine-Associated Toxicity in a..., Kochenderfer [/bib_ref] , immune effector cell-associated neurotoxic syndrome (ICANS), infection, bone marrow suppression, phagocytic lymphohistiocytosis (HLH) (88), B-cell dysplasia, neurotoxicity [bib_ref] CD19 CAR-T Cells of Defined CD4+:CD8+ Composition in Adult B Cell ALL..., Turtle [/bib_ref] , and disseminated intravascular coagulation (DIC) [bib_ref] Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B..., Davila [/bib_ref] , and toxicity to other organs.
## Prevention and treatment of side effects
The side effects of immune checkpoint inhibitors therapies are usually caused by the immune system attacking normal body parts in the same way it attacks cancer cells. Different types of immunotherapies can cause various side effects, many of which depend on the type of treatment, the tumor type and location, and the patient's general health condition. Immunotherapy side effects can be mild, moderate, or even life-threatening. Some side effects can resolve on their own within a certain time frame while others persist and worsen. In such cases, it should be considered to taper the dosage, discontinue, or change the medication. Indeed, prevention of the occurrence or worsening of side effects is essential for effective treatment of these patients population. At the end of immunotherapy, it is important to observe side effects, some of which may occur months or years later [bib_ref] Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitors, Day [/bib_ref] [bib_ref] Safety Profiles of Anti-CTLA-4 and Anti-PD-1 Antibodies Alone and in Combination, Boutros [/bib_ref]. Side effects of immune checkpoint inhibitors therapies may affect the following parts of the body. [fig_ref] TABLE 2 |: List of serious complications and brief prevention methods for bladder cancer [/fig_ref] When an immunotherapy drug is given to the patients through a vein, it is called an infusion. Patients receiving infusions may experience different reactions, mainly including fever, chills, accompanied by nausea, vomiting, headache and peripheral discomfort. When a mild reaction is observed, the infusion rate can be slowed down, and attention can be paid to keeping the patients warm. In case of a severe reaction, the infusion should be immediately stopped, external cooling should be provided to pyrexial patients, and anti-allergy drugs should be prescribed if necessary [bib_ref] Management and Preparedness for Infusion and Hypersensitivity Reactions, Lenz [/bib_ref] [bib_ref] Brentuximab Vedotin Infusion Reaction Management: A Case Study, Comer [/bib_ref] [bib_ref] Management of Infusion Reactions to Systemic Anticancer Therapy: ESMO Clinical Practice Guidelines, Rosellós [/bib_ref].
Skin problems, like rash, itching and skin photosensitivity, are most common in people with BC. Skin problem caused by immunotherapy are usually not serious but can be significantly uncomfortable for the patients. For rashes, corticosteroid ointments or antibiotic ointments remain the mainstay of treatment, and oral medications may be required for severe cases. For dry skin, it is recommended to use a hypoallergenic, cream-based moisturizer to prevent skin dryness, try bath products that are gentle on the skin and shower with warm water. It is essential for patients complaining of itchy skin to avoid scented skin products and use topical steroids and oral antihistamines. Indeed, such patients should pay attention to hydration daily, avoiding contact with allergens and exposure to sunlight [bib_ref] The Role of Diet in Preventing Photoaging and Treating Common Skin Conditions, Soliman [/bib_ref].
Problems with the gastrointestinal tract are also some of the most common side effects related to immune checkpoint inhibitors therapies. These include colitis, diarrhea, swallowing problems, nausea and vomiting, and pain in the upper abdomen. Regular examination of abdominal signs, abdominal X-rays, abdominal ultrasound, CT, gastroscopy and enteroscopy can be used for diagnosis [bib_ref] Intestinal Obstruction: Evaluation and Management, Jackson [/bib_ref]. The treatment regimen usually includes medications to inhibit hydrochloric acid and protect the digestive tract, such as proton pump inhibitors, and medications such as gastric mucosal protectors and hepatoprotective drugs can also be considered. Daily diet is carefully chosen by avoiding caffeine, alcohol, and spicy foods, eating less and more often, and regular monitoring of electrolyte levels, coupled with proper hydration and electrolyte supplementation to prevent further aggravation of the condition [bib_ref] Mechanisms, Prevention, and Management of Diarrhea in Enteral Nutrition, Whelan [/bib_ref].
Muscle, joint and bone problems can also occur in people who receive immune checkpoint inhibitors therapies. These can result in arthritis-type pain, swelling in joints, and muscle cramping, and even myasthenia gravis, manifest with limited range of motion and stiffness after inactivity or activity, swelling or pressure pain and redness or warmth at the joint. The diagnosis is usually made with X-rays, bone scans, CT, MRI and bone densitometry. Pain can be relieved with medications such as painkillers, corticosteroids, calcium tablets, vitamin D and antibiotics [bib_ref] Management of Toxicities of Immune Checkpoint Inhibitors, Spain [/bib_ref] [bib_ref] Management of Severe Osteoporosis, Miller [/bib_ref]. In addition, some physical therapies such as acupuncture, hot or cold compresses and massage can also be used to relieve pain [bib_ref] Respiratory, Musculoskeletal, Cardiac and Ocular Side-Effects of Anti-PD-1 Therapy, Zimmer [/bib_ref]. It is worth mentioning that myasthenia gravis is a chronic autoimmune disease, the diagnosis is usually made by conducting the Tensilon test or a nerve conduction test. Acetylcholinesterase inhibitors such as neostigmine or pyridostigmine remain the mainstay of treatment of myasthenia gravis; immunosuppressive drugs such as prednisone or azathioprine also can be considered [bib_ref] Myasthenia Gravis: An Emerging Toxicity of Immune Checkpoint Inhibitors, Makarious [/bib_ref]. Treatment with plasmapheresis and high doses of intravenous immunoglobulin may be required for cases presenting with sudden onset of symptoms [bib_ref] Management of Toxicities From Immunotherapy: ESMO Clinical Practice Guidelines for Diagnosis, Treatment..., Haanen [/bib_ref]. Adjunctive use of a ventilator may be required in cases of respiratory muscle weakness. For these problems, proper exercise, weight maintenance, and taking precautions to avoid falls are essential [bib_ref] Management of Toxicities of Immune Checkpoint Inhibitors, Spain [/bib_ref] [bib_ref] Management of Severe Osteoporosis, Miller [/bib_ref].
In the urinary tract, renal inflammation and hematuria is more likely to occur in patients who with immune checkpoint inhibitors therapies compared to kidney damage and kidney failure. These can be diagnosed through complete blood count (CBC), creatinine, blood urea nitrogen, abdominal ultrasound, abdominal CT and ureteroscopy. The treatment mainly focuses on protecting kidney function, ensuring adequate rest, proper nutrition and strict control of blood pressure, blood lipids and blood sugar, coupled with management of major and minor symptoms [bib_ref] Urinary Tract Infection Syndromes: Occurrence, Recurrence, Bacteriology, Risk Factors, and Disease Burden, Foxman [/bib_ref] [bib_ref] Diagnosis and Management of Hematuria, Avellino [/bib_ref] [bib_ref] Evaluation and Management of Chronic Kidney Disease: Synopsis of the Kidney Disease:..., Stevens [/bib_ref] [bib_ref] Diagnosis, Management, and Prevention of Catheter-Associated Urinary Tract Infections, Chenoweth [/bib_ref].
The neurological side effects of immune checkpoint inhibitors therapies are mainly in the central and peripheral nervous system [bib_ref] Neurological Sequelae of Cancer Immunotherapies and Targeted Therapies, Wick [/bib_ref] [bib_ref] Peripheral Nerve Injury, Scarring, and Recovery, Wang [/bib_ref] , affecting your brain, senses, mind, and even movement. These are rare but can be serious side effects. A cranial CT or MRI would be a good choice for the diagnostic workup in patients with dizziness and headache combined with a history of severe illness. The treatment of neurological problems is based on neurotrophy and then, take appropriate treatment measures to deal with corresponding symptoms. For example, analgesics for headaches, anti-dizziness drugs for dizziness, etc. Although nerve damage and neurological symptoms are not preventable, most are manageable if detected early, and early treatment can also prevent symptoms from exacerbating.
Immunotherapy may cause changes in the number of blood cells and blood factors, which can lead to anemia, coagulation disorders and sepsis. It can be diagnosed with a CBC, clotting assays and blood protein tests. Anemia can be treated with blood transfusions or erythropoiesis-stimulating agents (ESAs), and a diet rich in iron, folic acid, and vitamin B12 can play a preventive role. Blood coagulation is a complex process involving a series of reactions involving platelets and clotting factors. Hemorrhage and thrombosis occur when the balance between clotting factors is disrupted [bib_ref] Bleeding and Thrombosis in Patients With Liver Diseases, Lisman [/bib_ref]. In the case of bleeding disorder, prompt supplementation of platelets, vitamin K and other pro-coagulant medications; A blood clot is a serious condition that needs treatment right away, the management of thrombosis consists of anticoagulation with warfarin or rivaroxaban, followed by thrombolytic therapy with urokinase or streptokinase. Besides, extra care should be taken during daily activities to avoid circumstances that may lead to bleeding and thrombosis [bib_ref] Bleeding in Cancer Patients and its Treatment: A Review, Johnstone [/bib_ref] [bib_ref] How I Manage Cancer-Associated Thrombosis, Moik [/bib_ref]. Sepsis, on the other hand, requires the General prevention: reasonable diet, pay attention to environmental hygiene, regular work and rest, avoid straining and staying up late, limit smoking and alcohol, strengthen exercise, and enhance their resistance.
selection of appropriate antibiotics, aggressive anti-infection treatment, increasing resistance, avoiding late nights and exertion, and avoiding the intake of unclean water and food. Immune checkpoint inhibitors therapies may cause pneumonitis, which is inflammation of the lung that can cause a cough or trouble breathing. Pneumonitis is uncommon but may be serious. Inflammatory serum biomarkers, chest X-rays, contrast-enhanced CT, and pulmonary function tests are common diagnostic methods [bib_ref] Pleural Effusion in Adults-Etiology, Diagnosis, and Treatment, Jany [/bib_ref]. The management involves aggressive treatment with anti-inflammatory drugs, coupled with symptomatic management to relieve respiratory spasms and alleviate wheezing [bib_ref] Evaluation of the Patient With Pleural Effusion, Beaudoin [/bib_ref]. In patients with pneumonia and pleural effusion, light exercise is recommended to accelerate resorption of inflammation. It is essential for patients with pulmonary vascular thrombosis to lie down to prevent dislodging of the thrombus that can block other blood vessels. The patient should refrain from smoking and exposure to secondhand smoke. Indeed, lots of fluids are required to keep hydrated, and exposure to irritants should be avoided to avoid exacerbating the cough (120-122).
The endocrine system controls the hormones that help the body regulate many important functions, like blood pressure, blood sugar, energy, and the ability to respond to stresses like infections and injuries [bib_ref] Immune-Related Adverse Events With Immune Checkpoint Blockade: A Comprehensive Review, Michot [/bib_ref] [bib_ref] Endocrine-Related Adverse Events Associated With Immune Checkpoint Blockade and Expert Insights on..., Sznol [/bib_ref]. The thyroid, adrenal, pancreatic, sexual gland is a vital part of the endocrine system, and it may be triggered to become either more or less productive by immune checkpoint inhibitor treatments. The diagnosis focuses on the examination of the corresponding glands and the hormones they secrete. To treat the above endocrine side effects, the patient's hormone levels should be assessed. If a decline is observed, treatment with hormone replacement therapy is indicated. Drugs that inhibit endocrine gland hormone release are prescribed if high levels are found. Given the insidious nature of these autoimmune events, the consequences are often ongoing and even permanent, requiring long-term hormone replacement therapy [bib_ref] Management of Toxicities From Immunotherapy: ESMO Clinical Practice Guidelines for Diagnosis, Treatment..., Haanen [/bib_ref]. Pay attention to exercise and healthy diet in daily life.
Immunotherapy may affect the heart and blood vessels. These side effects are rare but are often very serious and can be lifethreatening. Includes cardiomyopathy, congestive heart failure (CHF), myocarditis, coronary artery disease, arrhythmias, heart valve damage, and pericardial disease. The clinical presentation usually consists of shortness of breath, dizziness, chest pain, edema, fatigue, etc. [bib_ref] Immune Checkpoint Inhibitors and Cardiac Toxicity: An Emerging Issue, Varricchi [/bib_ref]. Regular physical examinations heartbeat sounds, vascular murmurs, laboratory tests, cardiac enzyme profile, BNP, echocardiograms, chest X-rays, electrocardiograms, multi-gate acquisition scans (MUGA), cardiac MRI and angiograms can be used to diagnose heart problems. The treatment plan usually consists of cardioprotective drugs such as dexrazoxane (Zinecard) which help prevent cardiac problems induced by anthracyclines. Inotropes (digitalis), diuretics and hypertensive drugs should also be considered [bib_ref] An Overview, Brieler [/bib_ref] [bib_ref] Congestive Heart Failure: Diagnosis, Pathophysiology, Therapy, and Implications for Respiratory Care, Figueroa [/bib_ref] [bib_ref] Management of Neoplastic Pericardial Disease, Zhang [/bib_ref]. Take care to avoid emotional excitement in daily life and have a light diet is necessary.
# Conclusion
Immunotherapy is regarded as a promising and more effective therapeutic measure in the treatment of various types of cancer. However, the side effects of it are underestimated currently. The unpredictable occurrence of serious side effects not only causes serious health damage to patients, but also increases the medical burden to some extent. The clinical management of side effects in patients today is mainly empirical. Therefore, a uniform and systematic guideline to control the side effects of immunotherapy is necessary. Based on the insufficiency understanding on the side effects of immunotherapy, more follow-up data on the side effect is needed, as well as prospective, multicenter, large-scale trials on the preventive measures. Above all, further research on the molecular mechanisms and clinical efficacy of the side effects of immunotherapy is still urgent.
## Prospect
Although immunotherapy developed and achieved widespread application in clinical cancer therapy, further research is necessary in immunotherapy for bladder cancer, especially in the systemic response, which may exert role in the development of side effects of immunotherapy. Furthermore, the genetic diversity of bladder cancer and epigenetic modification are also relevant to efficacy and side effects.
During recent years, researchers proposed new methods to improve the efficacy of immunotherapy and mitigate side effects, such as activation of thioredoxin, bacterial colony transplantation, and ferroptosis induction. At the same time, alternative therapeutic measures are beneficial in alleviating the symptoms of drug complications, such as rehabilitation therapy, Chinese acupuncture, and physiotherapy. In addition, combination or individualized treatments depending on the diversity of the patient is also a good choice. Hence, develop new methods to alleviate side effect would be an important subject in immunotherapy.
[fig] FIGURE 1 |: Immune checkpoint inhibitors in BC treatment. PD-1/PD-L1 and CTLA-4 blockers interfere with suppression of checkpoint molecules of the immune system, leading to T-cell activation and tumor cell killing. CTLA-4, cytotoxic T-lymphocyte antigen 4; MHC, major histocompatibility complex; PD-1, programmed cell death-1; PD-L1, programmed cell death-1 ligand; TCR, T cell receptor; APC: Antigen-presenting cell. [/fig]
[table] TABLE 1 |: List of side effects, indications and serious complications for immunotherapy for bladder cancer.Patients with locally advanced or metastatic urothelial carcinoma that experience exacerbations during or following platinumcontaining chemotherapy, or within 12 months of receiving platinum-containing chemotherapy, either before (neoadjuvant) or after (adjuvant) surgical treatment. [/table]
[table] TABLE 2 |: List of serious complications and brief prevention methods for bladder cancer. Absorb oxygen, prevent respiratory tract infection, use ventilator if necessary. Endocrine diseases Pay attention to diet, strengthen exercise, use long-term maintenance medication if necessary. [/table]
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Comparison of qPCR and culture methods for group B Streptococcus colonization detection in pregnant women: evaluation of a new qPCR assay
Background: Streptococcus Group B (GBS) colonization in pregnant women is the most important risk factor for newborn disease due to vertical transmission during delivery. GBS colonization during pregnancy has been implicated as a leading cause of perinatal infections. Traditionally, pregnant women are screened for GBS between 35 and 37 weeks of gestation. However, antenatal culture-based screening yields no information on GBS colonization status and offers low predictive value for GBS colonization at delivery. Numerous assays have been evaluated for GBS screening in an attempt to validate a fast and efficient method. The aim of this study was to compare bacteria isolation by culture and two qPCR techniques, targeting sip and cfb genes, respectively, for detecting colonizing GBS. Methods: Culturesthe gold-standard technique, a previous qPCR technique targeting the sip gene, and a new proposed qPCR assay targeting the cfb gene were evaluated as diagnostic tools on 320 samples. Results: Considering cultures as the gold standard, the evaluated qPCR method detected 75 out of 78 samples, representing a sensitivity of 93.58% (95% confidence interval (CI), 90.89-96.27) and specificity of 94.62% (95% CI, 91. 78-97.46). However, an additional analysis was performed for true positives that included not only samples showing positives by culture but samples showing positive for both qPCR assays. The sensitivity and specificity were recalculated including these discrepant samples and a total of 89 samples were considered as positive, giving a prevalence of 27.81%. With this new analysis, the qPCR targeting the cfb gene showed a sensitivity of 95.5% (95% CI, 88.65-98.59) and specificity of 99.13% (95% CI, 96.69-99.97).Conclusions:The new qPCR method is a sensitive and specific assay for detecting GBS colonization and represents a valuable tool for identifying candidates for intrapartum antibiotic prophylaxis. Cultures should be retained as the reference and the routine technique because of its specificity and cost analysis ratio, but it would be convenient to introduce PCR techniques to check negative culture samples or when an urgent detection is required to reduce risk of infection among infants.
# Background
Group B Streptococcus (GBS) is a common commensal bacteria of gastrointestinal and vaginal flora with reported carriage rates ranging from 4 to 40% [bib_ref] Real-time polymerase chain reaction and culture in the diagnosis of invasive group..., Meehan [/bib_ref]. Colonization during pregnancy has been implicated as a leading cause of severe neonatal infections, including sepsis, pneumonia, and meningitis [bib_ref] Strategies for chemoprophylaxis of GBS early-onset infections, Boyer [/bib_ref] [bib_ref] Prevention of perinatal group B streptococcal disease-revised guidelines from CDC, Verani [/bib_ref]. Vertical transmission to the newborn occurs during labor via fetal aspiration of infected amniotic fluid or during passage through the birth canal [bib_ref] Evaluation of a novel real-time PCR test based on the ssrA gene..., Wernecke [/bib_ref]. Due to this vertical transmission, GBS infection is the a important cause of neonatal morbidity and mortality in the United States.
Determination of infection at the time of delivery is essential for neonatal vertical transmission prevention [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref] , because some women are intermittent carriers of GBS and the rate of GBS colonization may vary during pregnancy [bib_ref] Dynamics of Streptococcus agalactiae colonization in women during and after pregnancy and..., Hansen [/bib_ref]. The predictive value of antenatal screening decreases if it is performed more than a few weeks before delivery [bib_ref] The accuracy of late antenatal screening cultures in predicting genital group B..., Yancey [/bib_ref]. Neonatal infections can be prevented in most cases by providing intrapartum antibiotic prophylaxis to the colonized mother [bib_ref] Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC, Schrag [/bib_ref]. However the use of antibiotic prophylaxis on the basis of risk assessment leads to unnecessary treatment in many women [bib_ref] Group B streptococcal disease in the era of intrapartum antibiotic prophylaxis, Schrag [/bib_ref].
Different methods are used to detect GBS, mainly culture and nucleic acids amplification methods [bib_ref] Culture-based method with performance comparable to that of PCR-based methods for detection..., Berg [/bib_ref]. Since 2002, the Center for Disease Control and Prevention (CDC) published guidelines for the prevention of perinatal GBS disease, recommending routine culture for all pregnant women between 35 and 37 weeks of gestation [bib_ref] Prevention of perinatal group B streptococcal disease-revised guidelines from CDC, Verani [/bib_ref] [bib_ref] Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC, Schrag [/bib_ref]. The current CDC gold-standard method for GBS detection is rectovaginal sample incubation in a selective broth medium followed by subculture on a blood agar plate [bib_ref] Prevention of perinatal group B streptococcal disease-revised guidelines from CDC, Verani [/bib_ref]. However, negative culture results have been observed in some women whose infants subsequently develop GBS infection [bib_ref] Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC, Schrag [/bib_ref]. The culture method for GBS detection has many disadvantages: the sensitivity is limited, a large turnaround time is needed, requiring up to 36 to 72 h before results can be reported [bib_ref] Prevention of perinatal group B streptococcal disease-revised guidelines from CDC, Verani [/bib_ref] , the lack of information on the GBS colonization status of some women at delivery, and the low predictive value of antenatal culture findings for GBS colonization at delivery [bib_ref] Multicenter study of a rapid molecular-based assay for the diagnosis of group..., Davies [/bib_ref] [bib_ref] Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal..., El Helali [/bib_ref].
More sensitive and faster methods for detecting GBS colonization would help to obviate the need for prenatal screening to identify GBS, as well as reducing postpartum complications and severe infections in infants and avoiding the unnecessary use of antibiotic prophylaxis in women who are not colonized [bib_ref] Rapid detection of group B streptococci in pregnant women at delivery, Bergeron [/bib_ref]. Nucleic acids amplification assays for detection of GBS colonization in pregnant women at delivery have shown good sensitivity and specificity [bib_ref] Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal..., El Helali [/bib_ref] [bib_ref] Rapid detection of group B streptococci in pregnant women at delivery, Bergeron [/bib_ref]. In the present study, a new nucleic acids amplification method over a cfb gene fragment has been developed and evaluated. The results are compared with a previously described method that uses the sip gene as a target [bib_ref] Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR..., Bergh [/bib_ref] and with culturing as the current gold-standard method.
# Methods
## The study
A prospective study (November 2013-July 2015) was carried out on 320 clinical samples collected from pregnant women at the Andalucía public health system hospital network. The women included in the sample had not received antibiotic treatment the week prior to sample acquisition, and they did not show contraindication to vaginal examination. Paired vaginal/rectal swabs were used simultaneously to obtain rectovaginal GBS samples from consenting women between 35 and 37 weeks of gestation at a routine antenatal screening. Samples were transported in Stuart's medium and were stored at 4°C for a maximum of 48 h until examination. One swab was used for the GBS microbiological culture and the other was used for GBS qPCR testing. Collected swabs for culturing and qPCR testing, respectively, were stored at 2-8°C and − 80°C until the evaluation. The specific objective of this study was to compare culturing and the two qPCR techniques, targeting sip and cfb genes respectively, for detecting GBS colonization.
# Data analysis
In a primary analysis, culturing was considered the gold-standard technique for detecting GBS. Sensitivity and specificity data for cultures and both qPCR techniques are expressed in reference to the culture results. An additional analysis was performed for true positives that included not only samples showing positives by culture but samples showing positive for both qPCR assays. The cut-off for both qPCR techniques was established in 35 cycles.
## Specimen collection
The recommended specimen for either of the techniques considered here is a dual swab collected from the vaginal area followed by the rectal area, as previously described [bib_ref] Prevention of perinatal group B streptococcal disease-revised guidelines from CDC, Verani [/bib_ref]. The swabs were then placed into the transport container (see above).
## Culture and identification of gbs
GBS cultures were performed according to the guidelines of the Spanish Society for Clinical Microbiology and Infectious Diseases. For specimen enrichment, samples were inoculated in Todd Hewitt broth containing colistin and nalidixic acid (BD, Cat 296,266) to inhibit growth of enterobacteria and other microbiota present in the genital and gastrointestinal tract. After 18-24 h of enrichment at 37°C in aerobiosis, the broth-enriched cultures were subcultured in Granada medium (BD, Cat 257,079) in anaerobiosis for up to 48 h. Cultures were examined for the presence of orange to red colonies as a presumptive identification of GBS.
## Dna extraction
The swabs that were previously collected and frozen at − 80°C for PCR testing were thawed to carry out DNA extraction. The Amies transport medium swab (CM0425, Oxoid, Basingstoke, UK) was resuspended in 500 μl of 10 mM Tris-EDTA, pH 7.4. The swabs were manually rotated for 20 s inside the 1.5 ml tube and DNA was extracted using a QIAamp DNA mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. The DNA was eluted in 200 μl of Qiagen kit AE buffer and stored at − 20°C until use. An aliquot of 10 μl of the supernatant was used for PCR testing.
# Quantitative pcr methods
## Quantitative pcr targeting the sip gene (pcr-a)
A fragment (78 bp) of the sip gene was amplified following the method described by Bergh et al. [bib_ref] Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR..., Bergh [/bib_ref] with minor modifications [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref]. Real time PCR was performed in a 7500 Applied Biosystems thermocycler (Applied Biosystems, MA, USA) following recommended protocol [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref] [bib_ref] Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR..., Bergh [/bib_ref]. Positive and negative controls were included in each run; a dilution corresponding to 10 3 bacterial-DNA genome copies of Streptococcus agalactiae strain 2603 V/ R was used as positive control and sterile water was added instead of DNA template as a negative control.
## Quantitative pcr targeting the cfb gene (pcr-b)
A fragment (99 bp) of the cfb gene that codified for a diffusible extracellular protein called cAMP factor was amplified. Different cfb gene sequences from different GBS strains obtained from GenBank (X72754, HF952105. [bib_ref] Unipro UGENE: a unified bioinformatics toolkit, Okonechnikov [/bib_ref] to identify highly conserved regions with the oligonucleotide design. We also used this program to identify homologous cfb gene sequences available in databases from S. pyogenes (AF079502), S. uberis (U34322), S. canis (AF488802), and S. faecalis (29374661) to rule out non-specific amplification. No homology was found with non-GBS species. The oligos selected were: 5'-GAAA CATTGATTGCCCAGC-3′ and 5′.-AGGAAGATT TATCGCACCTG-3′. The Taqman probe was FAM 5'-CCATTTGATAGACGTTCGTGAAGAG-3' BHQ-1. Real-time PCR was performed in a 7500 Applied Biosystem thermocycler using the following final concentrations: 2.5 mM C 2 Mg, 0.5 μM of each primer, and 0.25 μM Taqman probe. Positive and negative controls were included in each run; a dilution corresponding to 10 3 bacterial genomes of S. agalactiae strain 2603 V/R was used as positive control. Sterile water was added instead of DNA template as negative control. The following PCR program was used: 94°C (5 min), 40 cycles of 92°C (10 s), 58°C (10 s), and 72°C (10 s).
## Sequencing of discrepant samples
A discrepant result was defined as a result obtained with both PCR methods that did not correlate with the culture results. The discrepant results were resolved using bidirectional sequence analysis of the GBS sip and cfb genes [bib_ref] Multicenter clinical evaluation of the Xpert GBS LB assay for detection of..., Buchan [/bib_ref]. The same oligonucleotides used for amplification that were described previously were used for fragment sequencing (GENYO, Granada, Spain). Sequences were analyzed using Unipro UGENE 1.24 software and Online Standard Nucleotide Blast.
## Sample size
A total of 320 samples collected from individual patients were included in this study. The optimal sample size was determined using the program "Power and size calculation" V3.1.2. [bib_ref] Rapid bacterial antigen detection is not clinically useful, Perkins [/bib_ref] setting α error probability at 0.05 and power (1 − β error probability) at 0.95%, and based on an estimated SGB prevalence of 12-20% previously reported in Spain [bib_ref] Led By Mo Perez-Moreno M. Group B streptococcal bacteriuria during pregnancy as..., Perez-Moreno [/bib_ref]. Taking into account the expected prevalence aforementioned under the established conditions, the estimated minimum sample size was established between 163 (for a 12% prevalence) and 246 (for a 20% prevalence).
# Statistical analysis
The results from the qPCRs targeting sip or cfb were compared to the culture results. The performance characteristics, including sensitivity and specificity, were calculated using standard methods. The 95% confidence intervals (CI) were calculated using XLSTAT software (Addinsoft).
# Results
A total of 320 dual rectovaginal swabs were evaluated. Bacterial isolation by culture was considered the gold standard for GBS diagnosis (100% specificity is assumed) [bib_ref] Rapid bacterial antigen detection is not clinically useful, Perkins [/bib_ref]. Among the 78 samples indicated positive by culture, 75 and 73 samples were positive by PCR-A and PCR-B, respectively. A detailed diagram of the results is show in In the present study, 11 samples tested positive by both PCR tests but negative by culture. Although culture is the reference technique for GBS detection, we considered samples in this group to be true positives, so positive results by culture (regardless of whether they are positive or not by any of the PCRs) and/or both PCR techniques (although negative by culture) were considered to be true positives (89 samples). Results with respect to this standard are given in. A GBS prevalence of 27.81% (95% CI 22.14-31.86) was obtained, and considering power samples sizes parameters defined previously (α = 0.05 and 1 − β = 0.95%) a minimum size of 309 samples was considered necessary to obtain statistically significant results. The sensitivity and specificity values obtained with culture and both qPCR methods are summarized in [fig_ref] Table 4: Sensitivity and specificity for culture and both qPCRs taking into account the... [/fig_ref]. Although five samples that tested positive by culture were negative by at least one of the PCR tests, 100% specificity was assumed for GBS culture. Some discrepancies were found between the three evaluated techniques. Samples found positive by either of the evaluated PCR techniques were sequenced to discard non-specific amplification. Discrepancies and the possible causes are presented in [fig_ref] Table 5: Resume of discrepancies between the three evaluated techniques [/fig_ref]. Two samples were positive only by culture, and 11 samples were positive by both PCR tests and negative by culture. What's more, two samples were positive by culture and PCR-A but negative by PCR-B, while one sample was positive by culture and PCR-B but negative by PCR-A. Finally, two samples were only positive by PCR-B.
# Discussion
Since the publication of the CDC guidelines for the prevention of perinatal GBS disease in 2002, the incidence of neonatal infections has decreased more than 60% [bib_ref] Group B Streptococcus detection: comparison of PCR assay and culture as a..., De-Paris [/bib_ref].
Several published studies have demonstrated the usefulness of culture-based [bib_ref] Comparative evaluation of Strepto B ID chromogenic medium and Granada media for..., Tazi [/bib_ref] [bib_ref] Use of GBS media for rapid detection of group B streptococci in..., Votava [/bib_ref] and PCR-based methods [bib_ref] Evaluation of a novel real-time PCR test based on the ssrA gene..., Wernecke [/bib_ref] [bib_ref] Multicenter study evaluating performance of the smart group B Streptococcus (GBS) assay..., Jordan [/bib_ref] [bib_ref] Multicenter evaluation of the BD max GBS assay for detection of group..., Riedlinger [/bib_ref] for detecting GBS. Traditionally, the culture method has been used as the common reference method, including in the Andalusian Sanitary System. However, the culture method may not be absolutely effective for GBS identification [bib_ref] Comparison of group B streptococci colonization in vaginal and rectal specimens by..., Bidgani [/bib_ref]. The use of a sensitive and accurate PCR method would provide the best means of GBS detection [bib_ref] A population-based comparison of strategies to prevent early-onset group B streptococcal disease..., Schrag [/bib_ref] [bib_ref] Intrapartum polymerase chain reaction for detection of group B streptococcus colonisation, Abdelazim [/bib_ref]. Numerous qPCR procedures have been developed in recent years, and different genes have been selected as targets for the specific amplification of GBS, including the cfb gene, which encodes for cAMP factor and is the most frequently used [bib_ref] Rapid detection of group B streptococci in pregnant women at delivery, Bergeron [/bib_ref] [bib_ref] Evaluation of culture, antigen detection and polymerase chain reaction for detection of..., Konikkara [/bib_ref] ; cylE gene [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref] ; dltR gene [bib_ref] Real-time polymerase chain reaction and culture in the diagnosis of invasive group..., Meehan [/bib_ref] ; sip gene [bib_ref] Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR..., Bergh [/bib_ref] ; scpB gene [bib_ref] Clinical diagnosis of group B streptococci by scpB gene based PCR, Dmitriev [/bib_ref] ; and C-protein gene [bib_ref] Detection of the C protein gene among group B streptococci using PCR, Mawn [/bib_ref].. Even commercial methods based on amplification techniques such as Xpert GBS (Cepheid, Sunnyvale, CA, USA) have appeared, helping to establish molecular techniques for GBS detection [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref].
In the present study, a total of 320 vaginal/rectal swabs were tested by two qPCR methods. A new oligo selection, not previously used by other authors, was performed for cfb gene amplification. All samples were also tested by selective culture methods. A specificity of 100% was assumed for bacterial cultures as the gold-standard method; the specificity of PCR-A and PCR-B was calculated to be 95.45 and 94.62%, respectively. Sensitivity was 96.15 and 93.58% for PCR-A and PCR-B, respectively. Among samples that were negative by culture methods, 3.44% (11/320) were indicated to be positive by both qPCR methods (targeting sip and cfb). Therefore, non-specific amplification or contamination were unlikely to be the cause of this discrepancy. If we only consider the samples that were indicated positive by culture, we lose positives detected by PCR but not by culture. Traditionally, these results have been considered false PCR positives, but really, they are false culture negatives and must be considered for greater strength [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref]. These results may indicate that culture may not be absolutely effective in the detection of GBS [bib_ref] Group B Streptococcus detection: comparison of PCR assay and culture as a..., De-Paris [/bib_ref] [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref]. The sensitivity and specificity were recalculated including these discrepant samples. A total of 89 samples that tested positive by culture (regardless of whether they are positive or not by any of the PCRs) and/or positive by both PCR techniques (although negative by culture) were considered true positives, giving an overall detection rate of 27.81%. The GBS detection rated reported in this study was slightly higher compared to previous studies in Spain [bib_ref] Led By Mo Perez-Moreno M. Group B streptococcal bacteriuria during pregnancy as..., Perez-Moreno [/bib_ref] , but in agreement with the SGB prevalence reported in different studies performed in Europe [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref] [bib_ref] Accuracy of prenatal culture in predicting intrapartum group B streptococcus colonization status, Florindo [/bib_ref] [bib_ref] Diagnostic accuracy of polymerase chain reaction for intrapartum detection of group B..., Helmig [/bib_ref]. According to previous studies, the detection rate for GBS ranged between 10 and 35% [bib_ref] Evaluation of a novel real-time PCR test based on the ssrA gene..., Wernecke [/bib_ref] [bib_ref] Real-time PCR targeting the sip gene for detection of group B Streptococcus..., Bergseng [/bib_ref] [bib_ref] Group B Streptococcus detection: comparison of PCR assay and culture as a..., De-Paris [/bib_ref] [bib_ref] Risk factors for group B Streptococcus colonisation and disease in Gambian women..., Doare [/bib_ref]. The high variation in detection rates may be explained by differences in prevalence among different populations, differences in culture techniques and protocols, or differences in prevalence over time [bib_ref] Prevalence of colonisation with group B streptococci in pregnant women of a..., Valkenburg-Van Den Berg [/bib_ref]. A diagnostic sensitivity of 96.62 and 95.50% was obtained for PCR-A and PCR-B, respectively, while culture sensitivity was reduced to 87.64%. The diagnostic specificity was estimated as 100% for both culture and PCR-A and as 99.13% for PCR-B. Based on these results, and in accordance with other studies, it can be concluded that qPCR techniques are more sensitive than culture methods [bib_ref] Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR..., Bergh [/bib_ref] [bib_ref] Group B Streptococcus detection: comparison of PCR assay and culture as a..., De-Paris [/bib_ref] [bib_ref] Comparison of group B streptococci colonization in vaginal and rectal specimens by..., Bidgani [/bib_ref] [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref] [bib_ref] Evaluation of culture and PCR methods for diagnosis of group B streptococcus..., Bakhtiari [/bib_ref] [bib_ref] Direct identification of Streptococcus agalactiae and capsular type by real-time PCR in..., Morozumi [/bib_ref]. Different studies have compared PCR detection of the GBS cfb gene to broth culture and have reported sensitivities between 86.7 and 100% and specificities between 95.9 and 100% [bib_ref] Multicenter study of a rapid molecular-based assay for the diagnosis of group..., Davies [/bib_ref] [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref]. Some authors have reported sensitivity values between 94 to 97% for an sip gene end-point PCR [bib_ref] Rapid detection of group B streptococci in pregnant women at delivery, Bergeron [/bib_ref] [bib_ref] Inadequacy of rapid immunoassays for intrapartum detection of group B streptococcal carriers, Baker [/bib_ref]. Real-time PCR assays in pregnant women that targeted the cfb gene or ptsI gene in GBS have shown sensitivities of 45 to 100% compared to culture methods [bib_ref] Multicenter study of a rapid molecular-based assay for the diagnosis of group..., Davies [/bib_ref] [bib_ref] Rapid detection of group B streptococci in pregnant women at delivery, Bergeron [/bib_ref] [bib_ref] Sensitivities of antigen detection and PCR assays greatly increased compared to that..., Rallu [/bib_ref] [bib_ref] Prospective evaluation of a real-time PCR assay for detection of group B..., Reglier-Poupet [/bib_ref] [bib_ref] Evaluation of the sensitivity of a rapid polymerase chain reaction for detection..., Chan [/bib_ref] [bib_ref] Use of the Roche LightCycler strep B assay for detection of group..., Uhl [/bib_ref].
We observed discrepant results in 18 samples among the three evaluated tests. Specifically, two samples tested positive only by culture. As it is assumed that culture has a specificity of 100%, those samples were considered as false negatives by qPCR, although the samples were not checked by another technique to resolve discrepancies. These false negatives obtained by PCR were reported in samples with low colony growth (one colony observed in culture), which is probably below the detection limit of PCR techniques [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref] [bib_ref] Comparison of BD GeneOhm real-time polymerase chain reaction with chromogenic and conventional..., Smith [/bib_ref]. Moreover, the use of a double swab collection (one processed for culture and the other for PCR) may cause variability in sensitivity between paired samples near the detection limit. Finally, another possible reason for these false negative PCR results may be the fact that in the present study the recommendations of the CDC for the optimization of DNA extraction have not been followed, which constitutes a study limitation. A second group of discrepant samples, considered true positives, included three samples that were weak positives by culture (one or two colonies per plate) and weak positives by only one qPCR. A third group of discrepant samples included 11 samples testing positive via both qPCRs methods but negative by culture. As per previous discussion, the two qPCR methods used in this study were based on different target genes, so non-specific amplification was unlikely to occur and the specificity was confirmed by sequencing. Therefore, those 11 samples were considered as false negatives by culture. A possible explanation for those negative culture samples is loss of bacterial viability during specimen collection and/or transport, where the nonviable bacterial DNA remained available for amplification [bib_ref] Evaluation of polymerase chain reaction for group B streptococcus detection using an..., Atkins [/bib_ref]. Also, it must be considered that the selective culture medium used in this study cannot detect non-hemolytic GBS colonies, which are estimated to constitute up to 4% of GBS infections. A third explanation for the false culture negatives might be high density of recto-vaginal flora present in the culture that might cover up or inhibit GBS growth even when using selective broth medium [bib_ref] Group B Streptococcus detection: comparison of PCR assay and culture as a..., De-Paris [/bib_ref] [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref] [bib_ref] Evaluation of culture and PCR methods for diagnosis of group B streptococcus..., Bakhtiari [/bib_ref] [bib_ref] Comparison of NNA agar culture and selective broth culture for detection of..., Dunne [/bib_ref]. Finally two samples were only positive for PCR-B, which was confirmed by sequencing. Both weak positive samples appeared in cycles > 32 and could be due to a contamination event or to a higher sensitivity of PCR-B, although these samples were considered as negative in the statistical analyses performed, according to the standards established in the study.
In conclusion, different methods can be used for GBS detection in rectovaginal swabs from pregnant women. The new qPCR method evaluated in the present study may be a rapid diagnostic alternative that is easy to use as routinely diagnostic method. The method choice Positive results and the negative ones are indicated with P and N respectively depends on technical aspects (lab expertise and lab equipment) or clinical aspects (preterm delivery, maternal fever during labor, or history of GBS disease in previous infants). Although qPCR techniques are more expensive than culture, they have been shown to have higher sensitivity and are faster than culture. Different authors have studied the cost of GBS culture screening versus PCR testing. Taking into account that the lower sensitivity of GBS culture methods results in unnecessary intrapartum antibiotic prophylaxis, an increase in hospital stay expenses, and early-onset GBS disease not detected by culture, they concluded that the final cost for both techniques was similar [bib_ref] Cost and effectiveness of intrapartum group B streptococcus polymerase chain reaction screening..., El Helali [/bib_ref]. Considering the results of the present study, in which 11 samples (3.44% of total) that were positive for GBS were not detected by the culture method, introduction of amplification techniques in the diagnosis routine appears to be supported. Culture methods must continue to be the routine reference technique, but it would be convenient to introduce PCR techniques to at least verify culture negative samples. Commercial Xpert GBS proposes a fast and completely automated but expensive technique (1 to 2 h) with reduced manipulation that may be especially useful in pregnant women with ruptured membranes [bib_ref] Usefulness of a rapid real-time PCR assay in prenatal screening for group..., Park [/bib_ref]. However, a considerable proportion of invalid or erroneous results (10.8 to 19%) have been described due to the presence of mucus or feces that inhibits the PCR and blocks the microfluidic channel in the cartridge [bib_ref] Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal..., El Helali [/bib_ref] [bib_ref] Intrapartum group B Streptococcus screening in the labor ward by Xpert(R) GBS..., Plainvert [/bib_ref]. The test described in this paper, which has a turn-around time of 2 h, is mainly presented as a routine test for pregnant woman in the diagnosis routine, although it could also be used with patients in a relatively critical state, such as ruptured membranes at delivery, as long as there is availability of qPCR thermocycler in the hospital. PCR is easy to perform and easy to automate; however, the future improvement and automatization of DNA extraction procedures, would facilitate the implementation of amplification techniques for diagnosis.
# Conclusions
The determination of GBS infection at the time of delivery is essential for prevention of neonatal vertical transmission. A combined strategy based on bacteria culture technique and nucleic acid amplification techniques would be more effective than a single diagnostic method to avoid the appearance of false negatives, and the consequent increase in GBS infections in neonates. The new PCR technique described in this manuscript allow for a rapid and effective detection of GBS in carriers pregnant women. The introduction of molecular tools does not imply an increase in diagnostic costs, since their greater sensitivity reduces unnecessary intrapartum antibiotic prophylaxis, and reduces both hospital stays and costs. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
## Availability of data and materials
The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.
Authors' contributions JACA, LGGL and JGF carried out experiments and wrote the manuscript; JGF, AIGE and EGT were involved in samples acquisition, data analysis and critical revision of manuscript. All authors read and approved the final manuscript.
## Ethics approval and consent to participate
The study protocol was carried out in accordance with the Helsinki Declaration. The study was explained to the patients who participated voluntarily in the project and gave a written consent. This was a noninterventional study, with no additional investigation into routine procedures. The biological material was used for the standard diagnosis of the presence of GBS, following the prescriptions of the doctors. No additional sampling or modification of the Protocol was performed. Data analyses were performed using an anonymous database. Therefore, the approval was considered unnecessary according to the guidelines of our country (Law on Data Protection -Organic Law 15/1999 of 13 December on the protection of data of a personal nature, https://www.boe.es/buscar/doc.php?id=BOE-A-1999-23750).
## Consent for publication
Not applicable.
## Competing interests
The authors declare that they have no competing interests.
## Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Author details 1
[table] Table 2: Sensitivity and specificity for both qPCR techniques considering culture as gold standard [/table]
[table] Table 4: Sensitivity and specificity for culture and both qPCRs taking into account the new standard defined [/table]
[table] Table 5: Resume of discrepancies between the three evaluated techniques [/table]
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Potato CYCLING DOF FACTOR 1 and its lncRNA counterpart StFLORE link tuber development and drought response
Plants regulate their reproductive cycles under the influence of environmental cues, such as day length, temperature and water availability. In Solanum tuberosum (potato), vegetative reproduction via tuberization is known to be regulated by photoperiod, in a very similar way to flowering. The central clock output transcription factor CYCLING DOF FACTOR 1 (StCDF1) was shown to regulate tuberization. We now show that StCDF1, together with a long non-coding RNA (lncRNA) counterpart, named StFLORE, also regulates water loss through affecting stomatal growth and diurnal opening. Both natural and CRISPR-Cas9 mutations in the StFLORE transcript produce plants with increased sensitivity to water-limiting conditions. Conversely, elevated expression of StFLORE, both by the overexpression of StFLORE or by the downregulation of StCDF1, results in an increased tolerance to drought through reducing water loss. Although StFLORE appears to act as a natural antisense transcript, it is in turn regulated by the StCDF1 transcription factor. We further show that StCDF1 is a non-redundant regulator of tuberization that affects the expression of two other members of the potato StCDF gene family, as well as StCO genes, through binding to a canonical sequence motif. Taken together, we demonstrate that the StCDF1-StFLORE locus is important for vegetative reproduction and water homeostasis, both of which are important traits for potato plant breeding.
# Introduction
Solanum tuberosum L. (potato) is one of the most important non-grain food crops in the world, with increasing importance for the growing economies of India and China. Potato originated in the equatorial Andean region of South America [bib_ref] Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation..., Spooner [/bib_ref] , where it tuberizes in response to a short-day (SD) photoperiod. During domestication, the potato crop has also adapted to the long-day photoperiod in the northern latitudes of North America, Europe and Asia [bib_ref] The origins and adaptation of European potatoes reconstructed from historical genomes, Gutaker [/bib_ref]. The molecular regulation of tuberization is well understood and bears a striking similarity to the regulation of flowering in most angiosperms [bib_ref] Flowering and tuberization: a tale of two nightshades, Abelenda [/bib_ref].
In Arabidopsis thaliana, flowering is regulated by an integrated light-and clock-dependent signalling cascade that includes proteins such as GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), which together bind to the carboxyl terminus of CYCLING DOF FACTORs (CDFs), targeting them for degradation [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref]. Arabidopsis CDFs act redundantly on the CONSTANS (CO) promoter, which in turn is an inducer of the florigen, FLOWERING LOCUS T (FT) [bib_ref] The genetic basis of flowering responses to seasonal cues, Andres [/bib_ref]. CDF proteins belong to a larger group of DNA-BINDING WITH ONE FINGER (DOF) transcription factors that bind to a consensus motif in promoters of their target genes [bib_ref] Diversity and similarity among recognition sequences of Dof transcription factors, Yanagisawa [/bib_ref] [bib_ref] Involvement of TAAAG elements suggests a role for Dof transcription factors in..., Plesch [/bib_ref]. Multiple tandem repeats of this motif are also present in the CO promoter, and AtCDF1 protein can repress CO by binding to these motifs [bib_ref] Photoperiodic control of flowering: not only by coincidence, Imaizumi [/bib_ref].
In potato, allelic variation in the 3 0 of the StCDF1 gene can lead to a truncation of the coding region, thereby eliminating the StFKF1 binding site in the protein. The resulting lack of StFKF1-mediated ubiquitination, and subsequent degradation by the proteosome, allows the StCDF1 protein to evade normal diurnal degradation [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref]. This transposon insertion-mediated truncation results in the indirect induction of tuberization through the constitutive repression of StCO and StSP5G genes, which together repress the transcription of the tuberigen StSP6A [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref] [bib_ref] Potato StCONSTANS-like1 suppresses storage organ formation by directly activating the FT-like StSP5G..., Abelenda [/bib_ref]. Potato plants carrying one or more of these truncated allelic variants (StCDF1.2 and StCDF1.3) become 'early' and long-day adapted, whereas potato genotypes with the full-length wild-type protein (StCDF1.1) are generally 'late', and are therefore SD dependent for tuberization [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref].
Beyond the regulation of flowering, CDF transcription factors have been associated with abiotic stress tolerance. Overexpression of Solanum lycopersicum (tomato) SlCDF1 and SlCDF3 genes in Arabidopsis leads to higher tolerance to drought stress and salt stress [bib_ref] Characterization of tomato Cycling Dof Factors reveals conserved and new functions in..., Corrales [/bib_ref]. Furthermore, mutations in CDF3 of Arabidopsis results in increased sensitivity to both drought and low temperatures, whereas increased expression enhances tolerance to drought, cold and osmotic stress [bib_ref] Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of..., Corrales [/bib_ref]. A further level of complexity is added to this gene family by the finding that some CDF genes also encode a divergently transcribed long non-coding RNA (lncRNA; [bib_ref] Battles and hijacks: noncoding transcription in plants, Ariel [/bib_ref] that appears to act as a natural antisense transcript (NAT), regulating CDF transcription [bib_ref] The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links..., Henriques [/bib_ref].
Here, we further characterize the molecular mechanisms by which the StCDF1 locus regulates tuberization and impacts on drought tolerance in potato. We show that StCDF1 strongly binds to the DOF consensus sequence present in the promoters of StCO1, StCO2 and StCO3 genes. We also demonstrate that although CDFs in Arabidopsis show functional redundancy for the regulation of flowering, StCDF1 in potato has a non-redundant role for tuberization. In addition, we find that StCDF1 has its own natural antisense transcript (StFLORE) with antiphasic gene expression over the circadian cycle. Finally, we show that changing StFLORE expression has powerful effects on water homeostasis in plants, and that this response is due to the regulation of stomatal opening in an ABA-dependent manner.
# Results
## Stcdf1 binds to the promoter of potato constans genes
We have previously demonstrated that StCDF1 regulates the transcription of StCO genes in potato [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref]. To gain more understanding about the sequences that the StCDF1 protein binds to, we used a protein binding microarray (PBM) to identify the consensus binding sequence for this transcription factor [bib_ref] Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors, Godoy [/bib_ref] [bib_ref] DNA-binding specificities of plant transcription factors and their potential to define target..., Franco-Zorrilla [/bib_ref]. For this, the StCDF1 coding region was cloned in a translational fusion to a maltose binding domain and expressed in Escherichia coli, from which protein was extracted and incubated on the PBM. Results from this experiment clearly confirmed the sequence specificity of the StCDF1 protein for the core DOF motif: AAAG [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. We were also able to define a wider sequence consensus represented by a 9-bp sequence YWAAAGRYC motif [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. Individual nucleotide deviations from the consensus dramatically reduce the specificity of the binding [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. Additionally, in a genome-wide scan for the presence of the canonical StCDF1 cognate using the entire upstream region (À1.5 kb) of the annotated genes of the DM1-3 516 R44 (DM) reference genome (Potato Genome Sequencing Consortium, PGSC, 2011), the best-fit curve shows that the most abundant location of the DOF motif is relatively close to the transcriptional start site [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref] [bib_ref] DNA-binding specificities of plant transcription factors and their potential to define target..., Franco-Zorrilla [/bib_ref]. This finding adds biological weight to the determination of the sequence motif [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref].
We have shown previously that StCDF1 transcriptionally targets StCO1 and StCO2 [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref]. Here, we identify an unannotated homolog StCO3 that is also regulated by circadian rhythm, with a peak during the night [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. The three StCO genes are in a tandem repeat at chromosome 2 in potato. Using chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR), we demonstrated the direct binding of StCDF1 to the DOF motifs present in the promoters of StCO genes in vivo [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. These experiments clearly show a strong binding and specificity to the promoter region of the StCO1, weak binding to the StCO2 promoter and high relative levels of enrichment in the StCO3 promoter in the wild-type Solanum tuberosum group Andigenum background. Another amplified control region of the genome used as a negative control (Actin) shows no binding. The StCO3 gene is repressed in 35S:StCDF1.2 overexpression lines [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref] , confirming transcriptional regulation by StCDF1. Similarly, it was previously shown that StCO1 and StCO2 were also repressed in 35S: StCDF1.2 overexpression lines [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref]. Strikingly, we observed differences between 35S:StCDF1.2 and wild-type (WT) backgrounds in the StCO3 promoter, probably indicating some type of negative transcriptional regulation of StCDF1 by itself in the overexpression lines. Taken together, from the ChIP-qPCR and the expression studies in transgenic lines, we conclude that StCDF1 is likely to regulate all of the StCOs, with StCO1 and StCO3 promoters being the targets with the highest affinity.
## Stcdf1 is a non-redundant regulator of tuberization
We have previously shown that the overexpression of StCDF1.2 strongly promotes tuberization in potato and (c) Box plot of enrichment scores (ES) of the elements indicated, determined by PBM; note the low enrichment score when the element is disrupted. (d) Plot showing the YWAAAGRYC motif average distribution and local enrichment probability using all possible promoters of annotated potato genes as the input. The x-axis represents the distance relative to the transcription start site (TSS). The y-axis represents the enrichment score (ES).
(e) Binding interaction of StCDF1 in CO promoter through the AAAG binding motif evaluated by Chip-qPCR in WT Solanum tuberosum group Andigenum (WT) and 35S:CDF1 plants. Values are averages of three biological replicates and regions with significant enrichment above the actin negative control are indicated (*P < 0.05, **P < 0.01; Holm-Sidak multiple comparison test). The relative fold enrichment was calculated using the pre-immunized serum as a background control. A schematic description of the studied genomic region is presented below. Red dots and numbers represent the qPCR primer pairs and green vertical bars represent putative StCDF1 binding sites in the StCO1, StCO2 and StCO3 promoters. All samples were collected at ZT3 under short days.
© 2020 The Authors. we made an StCDF1-specific RNAi construct and transformed it into the diploid potato clone carrying the homozygous wild-type StCDF1.1 allele (CE3027). Ten transgenic potato plants were grown in the glasshouse and periodically checked for tuberization together with untransformed controls in three replicates. The CE3027 control began to tuberize 14 weeks after planting, under long-day conditions. All plants were grown until 18 weeks after planting. Six of the 10 transgenic lines did not produce any tubers (or swelling stolons). The remaining four transgenic plants had between one and three tubers and a mean total tuber fresh weight of 9.4 g per plant, compared with an average of six tubers and a mean total tuber fresh weight of 42.9 g per plant in the control plants . The StCDF1 RNAi plants were otherwise phenotypically normal in their growth habit and flowered at the same time as the non-transformed controls. We analysed the expression of the StCDF genes that are phylogenetically closest to StCDF1: StCDF2 and StCDF3 [fig_ref] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation [/fig_ref]. Interestingly, although StCDF1 expression was significantly downregulated in the StCDF1 RNAi lines, the expression of both StCDF2 and StCDF3 were upregulated, compared with the controls [fig_ref] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation [/fig_ref]. We therefore checked the expression of these two homologues in the 35S:StCDF1.2 overexpression lines and found that StCDF2 was downregulated in a light-stable version whereas StCDF3 was markedly downregulated [fig_ref] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation [/fig_ref]. Overall, these results showed first that StCDF1 non-redundantly promotes tuberization and second that StCDF1 appears to be a master regulator of a potato gene network also comprising other StCDF genes.
The StCDF1 locus also codes for an antisense lncRNA called StFLORE
Recently, a CDF5 lncRNA was identified in Arabidopsis, and molecular analysis showed that FLORE and AtCDF5 exhibit antiphasic expression that reflects a mutual inhibitory regulation of controlling flowering time [bib_ref] The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links..., Henriques [/bib_ref]. Aiming to check whether the StCDF1 locus would also encode an NAT-lncRNA, we analysed the RNA-Seq data from the DM1-3 516 R44 potato reference genome (PGSC 2011; [fig_ref] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation [/fig_ref]. The reannotation of the strand-specific reads indicates an additional gene model in the StCDF1 locus, possibly through the presence of an lncRNA on its antisense strand, similar to the CDF5/FLORE NAT pair in Arabidopsis. To confirm this, we performed strand-specific cDNA synthesis using four different primers to map the 3 0 end of the lncRNA transcript. PCR amplification in these four cDNA templates gave rise to only three products, allowing an approximate 3 0 end mapping of the transcript into the intron of the StCDF1 gene [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. When the transcription levels of StFLORE were tested over a 24-h time course under SDs in the diploid genotype CE3027 control, StFLORE peaked at night and showed an StCDF1-antiphasic expression profile similar to FLORE expression in Arabidopsis [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. To visualize the spatial localization of both StCDF1 and StFLORE transcripts, we fused an StCDF1 or StFLORE upstream promoter region (2.0 and 3.3 kb, respectively) to the b-glucuronidase (GUS) gene for histochemical localization. After incubation and staining with X-Gluc, plants carrying pStCDF1:GUS showed clear macroscopic vascular staining [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. Furthermore, stomatal guard cells staining was detected also in pStCDF1:GUS [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. No staining was detected in vascular tissue and stomata guard cells in untransformed plants CE3027 [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. The staining of pStFLORE:GUS partially overlapped with pStCDF1:GUS, as we detected vascular staining [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref] , e). In comparison with StCDF1 GUS staining, it was not possible to observe expression in stomata guard cells of pStFLORE:GUS. Thus, StCDF1 and StFLORE cellular localization overlap in vascular tissue, where the main activity of StCDF1 is controlling StCO genes and StSP6A gene expression, as expected [bib_ref] CONSTANS acts in the phloem to regulate a systemic signal that induces..., An [/bib_ref] [bib_ref] Targets of the StBEL5 transcription factor include the FT ortholog StSP6A, Sharma [/bib_ref]. Only StCDF1 expression was found to be located in stomatal guard cells, however, indicating a specific regulatory role in this tissue.
## Stcdf1 locus is involved in drought stress responses
The CDF genes have been linked to abiotic stress responses to stresses such as drought, salt and extreme temperature [bib_ref] Characterization of tomato Cycling Dof Factors reveals conserved and new functions in..., Corrales [/bib_ref] [bib_ref] The GI-CDF module of Arabidopsis affects freezing tolerance and growth as well..., Fornara [/bib_ref] [bib_ref] Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of..., Corrales [/bib_ref]. Using heterozygous potato plants carrying either StCDF1.1/StCDF1.2 or StCDF1.1/StCDF1.3, compared with the homozygous StCDF1.1/StCDF1.1 (CE3027) allelic configuration, we tested whether these potato clones show differences in tolerance to drought stress. The results show that the clones carrying a single copy of the StCDF1.3 allele were significantly less tolerant to water-limiting conditions compared with either the heterozygous StCDF1.1/1.2 or the homozygous StCDF1.1 controls [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. To further understand the impact of the different StCDF1 allele combinations, we generated diploid potato genotypes that were StCDF1.3 homozygotes and StCDF1.2/StCDF1.3 heterozygotes. These plants were obtained from a cross between the diploid clones E and RH (see Experimental procedures). The presence of the homozygous StCDF1.3 allele results in very early tuberization and leads to extremely weak plants with a stunted growth habit, compared with the parental controls . We also analysed plants with an StCDF1.2/1.3 allele combination and these plants had fewer branches and a smaller size than the control CE3027 [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. StCDF1.2 carries a 7-bp insertion, whereas StCDF1.3 carries a transposon insertion of 860 bp; however, both insertions are situated in the C-terminal region of StCDF1 and both result in a very similar functional truncated protein (Kloosterman et al., 2013) [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. We hypothesized that the different insertions have a differential effect on StFLORE transcription, which we tested by reverse-transcriptase qPCR (qRT-PCR) using strand-specific cDNA templates of the various allelic variants from StCDF1 at zeitgeber time 9 (ZT9; peak of StFLORE expression) under SD conditions. StFLORE expression could not be detected in the StCDF1.3 homozygotes [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref] , probably as a result of the 860-bp displacement of the StFLORE promoter [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. Furthermore, heterozygotes carrying one copy of the StCDF1.3 allele showed lower StFLORE expression than the control, whereas StCDF1.1/1.2 heterozygotes showed an even higher StFLORE expression than the control [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. These results indicate that the presence of the StCDF1.3 allele disrupts StFLORE expression and results in detrimental growth and reduced fitness in potato.
[formula] (f) (a) (d) (c) (h) (i) (b) (e) (g) [/formula]
To ascertain the abiotic stress susceptibility of the potato StCDF1 locus, we exposed StCDF1 RNAi plants to drought stress. The StCDF1 RNAi showed a dramatic increase in drought tolerance . StCDF1 RNAi had a lower water loss rate under drought stress and a lower number of tubers under both optimal water conditions and drought treatment, compared with the control . In parallel, we grew plants overexpressing StCDF1.2, which showed a weaker phenotype even under optimal water conditions . Sequence analysis of the putative StFLORE promoter reveals the presence of multiple StCDF1 binding motifs . We therefore also checked StFLORE expression in the StCDF1.2 overexpression plants as well as the StCDF1 RNAi plants in a 24-h time course under SD conditions. We found that the highest peak of StFLORE at ZT9 was twofold upregulated in StCDF1 RNAi plants compared with the controls , consistent with the high level of drought tolerance of these plants . In contrast, plants overexpressing StCDF1 showed a 30-fold decrease of StFLORE expression at both ZT6 and ZT12, compared with the control .
Taken together, the expression analysis indicates a repression of StFLORE expression by StCDF1. To check whether this regulation is direct, we performed a ChIP-qPCR assay using CE3027 plants expressing a GFP-tagged StCDF1 under the phloem-specific promoter SUC2. After the ChIP assay, an enrichment of chromatin was visible in the proximal region (P1) of the transcription start site of StFLORE . Enrichment values are close to those of the StCDF3 promoter, used as a positive control, and probably under similar transcriptional regulation by StCDF1. From these results, we can conclude that StCDF1 negatively regulates StFLORE by directly binding to its promoter.
## Increasing stflore expression confers drought tolerance
To gain more insight into StFLORE function we overexpressed and knocked down StFLORE gene expression. We constructed an StFLORE transcript, driven by the cauliflower mosaic virus (CaMV) 35S promoter (35S:StFLORE) and a CRISPR-Cas9 cassette targeting 1 kb of the StFLORE promoter using four RNA guides, including putative DOFbinding motifs, proximal to the lncRNA start of transcription . We found that using guides 1, 2 and 3, produced deletions of 770 and 952 bp, named DpStFLORE#22 and DpStFLORE#53 . Under normal growing conditions, DpStFLORE plants showed a delay in development, with smaller leaves and lower heights compared with the controls, whereas overexpressing StFLORE did not show any difference compared with the controls [fig_ref] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and... [/fig_ref]. Regarding tuberization, DpStFLORE plant lines showed early tuberization and a slightly higher number of tubers compared with the control [fig_ref] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and... [/fig_ref]. 35S:StFLORE showed a decrease in tuber number and also a delay in tuberization compared with the control [fig_ref] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and... [/fig_ref]. 35S:StFLORE and DpStFLORE plants were exposed to 2 weeks of moderate drought stress (see Experimental procedures). 35S:StFLORE plants were more drought tolerant and DpStFLORE plants were more susceptible to drought stress, compared with control CE3027 plants . StCDF1 expression was lower in the 35S:StFLORE plants compared with the controls . In contrast, DpStFLORE plants showed on average a sixfold lower expression of StFLORE at ZT6 and a threefold lower expression at ZT9 and ZT12, compared with controls , and a higher expression of StCDF1 at ZT0 (1.4-fold), ZT6 (13-fold), ZT9 (8-fold), and ZT12 and ZT15 (22-fold), compared with controls .
In 35S:StFLORE plants, we also checked for the expression of StCDF2 and StCDF3 in a 24-h time course under SD conditions. We found that these two other StCDF family members showed slightly higher expression than the controls, which is likely linked to the lower expression of StCDF1. In DpStFLORE plants there was a reversed pattern, where the expression of StCDF2 and StCDF3 was slightly lower than that observed in controls [fig_ref] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and... [/fig_ref]. In addition, our results revealed that StCDF1 and StFLORE also affect . For instance, StCDF1 RNAi and 35S:StFLORE overexpression plants have higher stomatal density but smaller guard cell size, and 35S:StCDF1 overexpression plants have lower stomatal density but larger stomatal guard cells . Moreover, previously we found that StCDF1 RNAi and 35S: StFLORE overexpression plants also have the lowest values of water loss under drought stress . Taken together, these results show that our tolerant transgenic StCDF1 RNAi and 35S:StFLORE overexpression plants are able to decrease their water loss not only by responding to ABA but also through the regulation of stomata size and number.
[formula] (a) (c) (b) (d) (e) [/formula]
# Discussion
Cycling DOF transcription factors are transcriptional repressors that, together with TOPLESS, regulate downstream initiators of reproductive development [bib_ref] Flowering and tuberization: a tale of two nightshades, Abelenda [/bib_ref] [bib_ref] Constitutive expression of OsDof4, encoding a C2-C2 zinc finger transcription factor, confesses..., Wu [/bib_ref]. In potato, this includes the indirect activation of tuber development [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref]. We show here that StCDF1 protein binds to canonical DOF motifs in the promoter regions of a tandem array of three potato CON-STANS genes located on chromosome 2. The repression of these StCO genes, and especially of StCO1, by StCDF1 is likely to prevent the induction of an FT-like repressor (StSP5G) of the tuberigen StSP6A, making StCDF1 an indirect positive regulator of tuberization. This is similar to the situation in Arabidopsis where CDF1 also represses CO [bib_ref] FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering, Song [/bib_ref]. Unlike Arabidopsis, however, where a quadruple CDF mutant is required to exhibit early flowering and be photoperiod insensitive [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] , we show here that StCDF1 is able to affect tuberization by itself. This confirms that a single potato CDF homologue has evolved specifically to regulate tuberization. In addition to its regulation of potato StCO and StSP5G gene expression, StCDF1 represses the expression of StCDF2 and StCDF3. We noted from an analysis of PGSC RNA-Seq data that the StCDF4 locus also has a secondary gene model that is very similar to that of StCDF1, indicating the possible presence of an lncRNA transcript for this locus . Recently, [bib_ref] BEL1-like protein (StBEL5) regulates CYCLING DOF FACTOR1 (StCDF1) through tandem TGAC core..., Kondhare [/bib_ref] showed that StCDF1 gene expression is regulated by a BEL1-like protein (StBEL5). Interestingly, we found that the KNOX-partner of StBEL5 (StPOTH1) is downregulated in 35S:StCDF1 plants, indicating complex upstream regulatory circuitry that will require further analysis.
As shown for Arabidopsis [bib_ref] The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links..., Henriques [/bib_ref] , potato StCDF1 also has an lncRNA natural antisense transcript, StFLORE. In potato, the antiphasic expression pattern of [bib_ref] Evaluation of breeding strategies for drought tolerance in potato by means of..., Spitters [/bib_ref] [bib_ref] Genetic mapping of tuber size distribution and marketable tuber yield under drought..., Aliche [/bib_ref]. Interestingly, a preponderance of DOF binding motifs has been detected in promoters of stomatal guard cell-expressed genes [bib_ref] Involvement of TAAAG elements suggests a role for Dof transcription factors in..., Plesch [/bib_ref] ; however, a direct mechanistic link of StCDF1 and StFLORE with the regulation of stomatal guard cell opening and closing remains unclear on a molecular level. Our results from stomatal density and stomatal guard cell sizes in our tolerant transgenics, 35S:StFLORE and StCDF1 RNAi plants, concur with previous studies where water deficit leads to a decrease in stomatal size [bib_ref] The importance of cell size in the water relations of plants, Cutler [/bib_ref] [bib_ref] Effects of abscisic acid and water stress on development and morphology of..., Quarrie [/bib_ref] [bib_ref] The problem of variability in stomatal responses, particularly aperture variance, Spence [/bib_ref] and an increase in stomatal density [bib_ref] Effect of water stress and temperature on leaf size and on size..., Mccree [/bib_ref] [bib_ref] The importance of cell size in the water relations of plants, Cutler [/bib_ref] [bib_ref] Leaf stomatal densities and distribution in triticum aestivum under drought and CO_..., Yang [/bib_ref] [bib_ref] Stomatal characteristics of different green organs in wheat under different irrigation regimes, Zhang [/bib_ref] , with stomatal density linked to wateruse efficiency [bib_ref] The changes in water-use efficiency and stoma density of Leymus chinensis along..., Yang [/bib_ref]. The co-expression of StCDF1 and StFLORE in the vasculature indicates a primary role for StFLORE regulation of StCDF1 in this tissue. If not a result of technical limitations in the promoter:GUS approach, the lack of StFLORE expression in stomatal guard cells indicates that there may be further intermediates affecting the influence of this transcript, however. Significant differences in the expression of ABA biosynthesis genes in StFLORE overexpression and knockout plants, as well as the transcriptional responsiveness of StFLORE gene expression to ABA, may provide an interesting lead for this signalling pathway. Nevertheless, we present data from natural StFLORE promoter knockdowns, CRISPR-Cas-9 mutants and overexpression transgenics indicating that StFLORE regulates stomatal guard cell dynamics. There is a vast volume of literature that links lncRNAs to abiotic stress, however, only a few of them have been fully characterized [bib_ref] The involvement of long noncoding RNAs in response to plant stress, Matsui [/bib_ref]. Our results show that the expression of the StFLORE lncRNA is regulated by promotor binding by StCDF1 protein, and in turn this influences stomatal aperture and guard cell size. These effects are not linked to the earliness effect of StCDF1 truncation alone, as the StCDF1.2 variant appears not to have adverse effects on StFLORE expression or plant fitness under normal or drought-stress conditions. Earliness of tuberization and life-cycle length are critical traits for plant breeding and agriculture, as they have profound effects on yield and production in various geographic locations. This importance is accentuated in the new true hybrid breeding programmes where life-cycle length is essential when planning to start with true-seed potato material. Potato crosses based solely on the phenotype do not guarantee avoiding early allelic variants of StCDF1 in the progeny. Moreover, knowing that a single StCDF1.3 allele produces a negative effect on fitness under abiotic stress situations, it is indispensable to develop specific molecular markers to distinguish the early alleles in breeding programmes so as to not introduce adverse effects of knocking down the StFLORE transcript.
## Experimental procedures
## Potato material and growth conditions
Potato plants with different allelic combinations of StCDF1 were obtained from C 9 E diploid clones. Clone C (USW5337.3) is a hybrid between Solanum phureja PI225696.1 and S. tuberosum dihaploid USW42. Clone E (VPH4 77.2102.37) is the result of a backcross between clone C and Solanum vernei-S. tuberosum clone VH3 4211. RH (RH89-039-16), a diploid heterozygous potato clone, and E were crossed to obtain the 1.3/1.3 allelic combination of StCDF1. The C 9 E (CE3027, CE605 and CE630) and RH 9 E (RHE25) progeny, together with transgenic plants generated in this study, were vegetatively propagated and grown in vitro on MS medium supplemented with 2% w/v sucrose [bib_ref] A revised medium for rapid growth and bio assays with tobacco tissue..., Murashige [/bib_ref]. Two-week-old plants were planted in soil and grown either in the glasshouse at 23°C under long days (LDs) or in controlled-environment chambers at 2°C under SDs (with 8 h of light and 16 h of dark). The plants used for this study are listed in , including CE3027, CE605, CE630 and RHE25, which possess 1.1/1.1, 1.1/1.2, 1.1/1.3 and 1.3/1.3 allelic compositions, respectively, which were used for molecular analysis.
## Drought exposure
After 2 months of growing in the glasshouse, the C 9 E population and transgenic plants were divided into two treatments: optimal irrigation and drought stress. Optimal irrigation was considered to be, manual watering every 2 days, corresponding to 100% field capacity. Plants grown under drought conditions were also irrigated every 2 days, but with decreasing field capacities of 60% for 5 days and 40% for the following 5 days. In total, these plants were exposed to 10 days of drought stress. After approximately 120 days after sowing, tuber number and tuber fresh weight were measured for all individuals grown under optimal irrigation and drought treatments. To obtain tuberization timing we check the tubers from week 10, when they begin to tuberize, onwards. To measure water loss under drought conditions, fully developed leaves of control CE3027 and transgenic plants were cut after 10 days of drought treatment and exposed at room temperature. The leaves were weighed 4 h after being cut. Water loss (%) was calculated as [(fresh weightdry weight after 4 h)/fresh weight] 9 100 of eight leaves under drought stress [bib_ref] Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought..., Campo [/bib_ref].To measure water loss under drought conditions, fully developed leaves of control CE3027 and transgenic plants were cut after 10 days of drought treatment and exposed at room temperature. The leaves were weighed 4 h after being cut. Water loss (%) was calculated as = (fresh weight-dry weight after 4 h)/fresh weight 9 100) of 8 leaves under drought stress.
## Physiological evaluation
Stomatal aperture was measured as reported previously [bib_ref] Effect of abscisic acid on stomatal opening in isolated epidermal strips of..., Roelfsema [/bib_ref] [bib_ref] A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells, Desikan [/bib_ref]. Briefly, the leaves of 4-week-old plants growing in a glasshouse were cut and first submerged into MOCK solution (a buffer that favours stomata opening). After 3 h, half of the leaves were submerged in ABA (10 µM) and the other half was kept in the MOCK solution as a control [bib_ref] A rapid and simple method for microscopy-based stomata analyses, Eisele [/bib_ref]. Stomatal aperture in leaves treated with ABA and Mock solution was calculated by measuring width over length from the stomata aperture. The reading was performed for a total of 30 stomata in four leaves per genotype under the microscope, with a 409 magnification. Furthermore, we calculated stomata density as stomata number per mm 2 area and stomata size by using the oval area formula (µm 2 ).
# Histological analysis
For the pStFLORE:GUS construct, a 3.3-kb fragment upstream of the 5 0 transcription start site of StFLORE (for further details of the exact position, see was amplified by using 5 0 -CACCCT-CATAAGTGGAGTAAGCCTTACGA-3 0 and 5 0 -TCACTAAT-TATGTTGCTCATCCT-3 0 and cloned into pENTR TOPO vector to generate the ENTRY Gatewayâ clones first, and then transferred to pkGWFS7 following the manufacturer's instructions. For the promoter StCDF1:GUS construct, a 2-kb upstream StCDF1 was amplified by PCR using 5 0 -CACCCAATATGAACTTTGTTTGTATA-TAAAAATATAAA-3 0 and 5 0 -GATGAAGAAGAAAAAGGGTTTTAGA-3 0 and then cloned following the same procedures as described above. Both the pStFLORE:GUS and pStCDF1:GUS constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into diploid potato CE3027 and Solanum tuberosum, respectively. The subcellular localization of transgenic lines were used for GUS staining, as described previously [bib_ref] GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in..., Jefferson [/bib_ref]. As a negative control, untransformed CE3027 was stained with GUS. Photos were taken of 3-week-old transgenic plants under the microscope with a magnification of 409 and 209 for stomata and vasculature cells visualization, respectively.
## Generation of constructs and transformation
RNAi lines (StCDF1#7 RNAi and StCDF1#13 RNAi) were constructed by cloning the StCDF1 cDNA fragment using 5 0 -CACC ATGTCTGAAGTTAGAGATCCTGCT-3 0 and 5 0 -GACACAAGAACCC GCTATGC-3 0 that contained the full coding sequence . The inverted repeat is assembled in the binary vector by a twostep cloning process with specific restriction enzyme sites [bib_ref] GATEWAY((TM)) vectors for Agrobacterium-mediated plant transformation, Karimi [/bib_ref]. The fragment was subsequently recombined into the binary vector pK7GWIWG2 using LR clonase (Invitrogen, now ThermoFisher Scientific, https://www.thermofisher.com) and transformed into TOP10 E. coli cells. The presence of the insert was confirmed by PCR amplification and the direction was verified by digestion and sequencing. Successful constructs were then transformed by electroporation into Agrobacterium tumefaciens GV3101 and confirmed by PCR before genetic transformation.
A plasmid with the specific SUC2 promoter was generated to obtain plants expressing GFP-CDF1.1 in the phloem. pALLIGA-TOR2pSUC2UTR_GW, kindly provided by G. Coupland, was used as a template to amplify the SUC2 promoter with primers including HindIII and XbaI restriction sequences. The PCR product was cloned in pGEMTeasy, sequenced, digested with HindIII and XbaI, and ligated again in pGWB406 after digestion and excision of its 35S promoter with the same enzymes, obtaining the pGWB06SUC2 plasmid. Finally, LR recombination between StCDF1.1 ORF in TOPO pENTRD and pGWB06SUC2 was performed, and the final SUC2:GFP-StCDF1.1 plasmid transferred to CE3027 plants. In parallel, an empty SUC2:GFP-Stop construct was created to transform plants and use them as negative controls.
The CRISPR/Cas9 knockout of the StFLORE promoter was performed by using Golden Gate cloning. We selected four sgRNAs along 1.5 kb of the StFLORE promoter including StCDF1 binding sites (see . The four sgRNA scaffold clones with 20-bp target sequences were previously obtained by PCR using a pair of synthetic specific primers using gRNA_GFP_T1 as a template. They were recombined with pICH47732:NOSp:NPTII-OCST, pICH47742:35Sp:Cas9-NOST, the linker pICH41780 and cloned in pAGM4723 in a single-cut ligation reaction with BbsI and T4-ligase. (ThermoFisher Scientific). The final binary plasmid contained an hCas9 gene under the CaMV 35S promoter and four single-guide RNAs (sgRNAs) under the control of the AtU6 promoter.
To detect mutagenesis, we amplified by PCR using DreamTaq DNA polymerase (ThermoFisher Scientific), with fragments containing guides sg1, sg2, sg3 and sg4 with the forward primer 5 0 -TCCCTTTCTACTTCGATCTACCTC-3 0 and reverse primer 5 0 -CAGAGTCTTCAAGTTTTATAGTTGTGC-3 0 . From 100 regenerated plants we obtained two transgenic plants (DpStFLORE#22 and DpStFLORE#53) from which we amplified the 1.5-kb region upstream of the transcription site of StFLORE to clone into pGEMT Easy vector (Promega, https://www.promega. com), and 10 colonies from each transgenic plant were sent for sequencing with M13 primers. In all the plants tested, at least one colony carries the intact StFLORE promoter. From these transgenics, we checked that the StCDF1 gene was not affected by our guides.
Overexpression plants of StFLORE (35S:StFLORE#207 and 35S: StFLORE#249) were constructed by amplifying the reverse complement from the StCDF1 gene with the forward primer 5 0 -CACCGGAGAGTGTAGTAGGATTT-3 0 and reverse primer 5 0 -CTA-CACTCTTCAGATCCCATTTG-3 0 . The PCR-generated full-length sequence was cloned into pENTR TOPO vector and TOP10F 0 cells were transformed according to the manufacturer's instructions (Invitrogen, now ThermoFisher Scientific). An LR recombination reaction was performed between the entry clone (pENTR TOPO) and the destination vector (pK7WG2), according to the manufacturer's protocols (Invitrogen, now ThermoFisher Scientific). Transgenic plants overexpressing StCDF1.2 (35S: StCDF1#3 and 35S:StCDF1#10) were described previously [bib_ref] Naturally occurring allele diversity allows potato cultivation in northern latitudes, Kloosterman [/bib_ref].
## Rna extraction and qrt-qpcr analysis
The samples were harvested and stored in liquid nitrogen until RNA extraction. The isolation of total RNA was performed with the RNeasy mini kit (Invitrogen, now ThermoFisher Scientific) following the corresponding protocol. DNA digestion was accomplished by DNase I (TaKaRa, https://www.takarabio.com), and the first-strand cDNA was obtained according to the manufacturer's instructions, by either superscript VI reverse transcriptase (Invitrogen, now ThermoFisher Scientific) to quantify StFLORE expression or by iScript cDNA synthesis kit to quantify StCDF1, StCDF2 and StCDF3. The NAC gene was chosen as the housekeeping gene, and qRT-PCR was performed using SYBR green MasterMix (Bio-Rad, https://www.bio-rad.com) on a real-time PCR System (CFX96; Bio-Rad) with the specific primers listed in . The qRT-PCR programme consists of 95°C for 3 min and 42 cycles of 95°C for 5 sec and 60°C for 10 sec. The relative expression level of each examined gene was quantified by a relative quantification method.
# Chip-qpcr analysis
Chromatin immunoprecipitation (ChIP) on the StCOs cluster was performed as described elsewhere, with minor modifications [bib_ref] Potato StCONSTANS-like1 suppresses storage organ formation by directly activating the FT-like StSP5G..., Abelenda [/bib_ref]. We used a specific StCDF1 antibody in a ChIP experiment on nuclear extracts from WT S. andigena plants and transgenic S. andigena overexpressing StCDF1.2 from CaMV 35S promoter (p35S:StCDF1.2). A preliminary step of incubation and chromatin clean-up with the pre-immunized serum was included. After incubation with the antibody, chromatin isolation using G protein coupled to paramagnetic beads (Dynabeads Protein G; Novex Life Technologies, now ThermoFisher Scientific) was performed. After reverse crosslinking, chromatin was recovered by column purification using the QIAquick PCR clean up Kit (Qiagen, https://www.qiagen.com). ChIP-qPCR was assayed with specific primers to quantify StCDF1 affinity for different StCO1-StCO3 DNA binding sites, and the enrichment of eight separate amplified regions of the StCO gene cluster was quantified. Regarding StCDF1 binding detection in the StFLORE promoter by ChIP, SUC2:GFP-CDF1.1 transgenic plants in the CE3027 background were used. Plants were grown under LDs for 3 weeks, and material was collected at ZT4. Rabbit polyclonal anti-GFP antibody (Ab290; Abcam, https://www.abcam.com) was used in combination with Dynabeads Protein G and protein A (50/50 V/V). Control plants expressing GFP alone were used as a control. Four different regions were assayed by qPCR. Primer sequences are listed in . Regarding StCDF1 binding detection in the StFLORE promoter by ChIP, SUC2:GFP-CDF1.1 transgenic plants in the CE3027 background were used. Plants were grown under LDs for 3 weeks, and material was collected at ZT4. Rabbit polyclonal anti-GFP antibody (Ab290; Abcam) was used in combination with Dynabeads Protein G and protein A (50/50 V/V). Control plants expressing GFP alone were used as a control. Four different regions were assayed by qPCR. Primer sequences are listed in .
All ChIP enrichment calculations were performed using the SuperArray ChIP-qPCR Data Analysis Template (Qiagen/SABiosciences), following the manufacturer's instructions.
## Protein binding microarray assay and analysis
Recombinant MBP-StCDF1 protein was obtained in E. coli Rosetta TM strain (Novogen, https://novogen-layers.com) after cloning in pMAL-c2 vector (New England Biolabs, https://www. neb.com) using Gateway technology [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. Proteins were purified according to the manufacturer's instructions. PBM-11 array design, incubation with transcription factors and binding affinity analysis are described elsewhere [bib_ref] Potato StCONSTANS-like1 suppresses storage organ formation by directly activating the FT-like StSP5G..., Abelenda [/bib_ref].
## Analysis of phenotypic data
Data analysis was performed using SPSS 22 (IBM, https://www.ibm. com/analytics/spss-statistics-software). Data obtained from the phenotypic variables evaluated were subjected to analysis of variance homogeneity to determine whether the distribution was normal. As the data presented a non-normal distribution, we applied a non-parametric test using the Kruskal-Wallis test. The differences among the genotypes were found through Fisher's least significant difference (LSD) test. The differences were considered significant for a value of P ≤ 0.05.
## Supporting information
Additional Supporting Information may be found in the online version of this article. [fig_ref] Figure 1: StCDF1 binding specificity assessed by protein binding microarray [/fig_ref]. StCDF1 acts a repressor of StCO3. [fig_ref] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation [/fig_ref]. StCDF1 knockdown non-redundantly delays tuberization. [fig_ref] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE [/fig_ref]. The StCDF1 locus encodes for an antisense lncRNA called StFLORE. . StCDF1 knockdown shows enhanced drought tolerance. . Analysis of CRISPR/Cas9 pStFLORE plants and overexpressing StFLORE lines. [fig_ref] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and... [/fig_ref]. Analysis of CRISPR/Cas9 pStFLORE plants and overexpressing StFLORE lines. . Analysis of CRISPR/Cas9 pStFLORE plants and 35S: StFLORE lines. . Effect of StCDF1 expression on stomatal response to ABA treatment. . Effect of StFLORE expression on stomatal response to ABA treatment. . Summary of the tuberization data of control (CE3027), StCDF1 RNAi and overexpressing StCDF1.2 independent lines. . Summary of average stomatal features from StCDF1 RNAi, 35S:StCDF1, DpStFLORE, 35S:StFLORE and CE3027, as a control. . List of plants from the C 9 E population used in this study. . Oligonucleotides used in this study.
[fig] Figure 1: StCDF1 binding specificity assessed by protein binding microarray (PBM) and chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR). (a) Three different secondary position weight matrices (PWM) representing obtained motifs in the experiment, with Z score indicated. (b) Representative conserved DNA binding site motif (YWAAAGRYC). [/fig]
[fig] Figure 2: StCDF1 knock-down non-redundantly delays tuberisation. Relative gene expression profile of StCDF1 and its close CYCLING DOF FACTOR family members StCDF2 and StCDF3 in two StCDF1 RNAi lines and two 35S: CDF1.2 lines during a 24-h time course under short-day conditions. CE3027 was used as a control for 1.1/1.1 allele combination. Error bars: means AE SEMs, with n = 3 biological replicates. Significant changes of gene expression compared with control CE3027 (1.1/1.1): *P ≤ 0.05. © 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2021), 105, 855-869 [/fig]
[fig] Figure 3: StCDF1 locus encodes for an antisense lncRNA called StFLORE. (a-e) b-Glucuronidase (GUS) staining of pStCDF1:GUS (#12 and #7) and pStFLORE:GUS (#12 and #15) plants, showing StCDF1 localization in young leaf (a-b) and stomata guard cells (c), and StFLORE localization in young leaf (d-e). StCDF1 and StFLORE were both expressed in vascular tissue. Scale bars: (a) 0.5 cm; (b) 209, 20 µm; (c) 409, 20 µm; (d) 0.5 cm; (e) 409, 20 µm. (f) Plants with different allele combinations of StCDF1 under normal (left) and drought conditions (right [/fig]
[fig] Figure 4, Figure 5: StCDF1 knock down shows enhanced drought tolerance and increased StFLORE expression. (a) CE3027 (1.1/1.1) untransformed control and two representative StCDF1 RNAi transgenic lines under fully watered conditions (left) and after 10 days of drought (right). (b) Water loss percentage after drought-stress conditions in StCDF1 RNAi lines. CE3027 (1.1/1.1) untransformed was used as a control. Error bars: means AE SEMs, with n = 3 biological replicates. *P < 0.05, with respect to control. For further details, see Experimental procedures. (c) Average number of tubers under drought and fully watered conditions in StCDF1 RNAi lines. CE3027 (1.1/1.1) untransformed was used as a control. Error bars: means AE SEMs, with n = 3 biological replicates. *P < 0.05, with respect to control. (d) Relative gene expression of StFLORE using two representative lines of StCDF1 RNAi and 35S:StCDF1.2 during a 24-h time course under optimal water conditions in short days. Untransformed CE3027 (1.1/1.1) was used as a control. Error bars: means AE SEMs, with n = 3 biological replicates. Significant changes of gene expression compared with control CE3027 (1.1/1.1): *P ≤ 0.05. (e) StCDF1 physically associates with StFLORE promoter detected by ChIP-qPCR in SUC2:GFP-CDF1 plants. Values are averages of three biological replicates + SDs. Regions with significant enrichment above actin negative control are indicated (**P < 0.01; ***P < 0.001, adjusted P value, Dunnett's multiple comparison test). The relative fold enrichment was calculated using GFP alone with transformed plants as a control. As a positive control a region with a theoretically high number of StCDF1 binding sites in the StCDF3 promoter was used. All samples were collected at ZT4 under long days. Amplicons are representative of consecutive genomic regions in the promoter, from proximal (P1) to distal (P4) StFLORE transcription start site.© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2021), 105, Analysis of CRISPR/Cas9 pStFLORE plants and overexpressing StFLORE lines. (a) CE3027 (1.1/1.1) untransformed control (left) and two representative 35S:StFLORE lines after 10 days of drought (right). Error bars: means AE SEMs, with n = 3 biological replicates. (b) CE3027 (1.1/1.1) untransformed control (left) and two representative DpStFLORE transgenic lines after 10 days of drought (right). Error bars: means AE SEMs, with n = 3 biological replicates. (c) Relative gene expression of DpStFLORE and 35S:StFLORE during a 24-h time course under optimal water conditions in short days. The untransformed CE3027 (1.1/1.1) from Figure 4(b) was used as a control. Error bars: means AE SEMs, with n = 3 biological replicates. Significant changes of gene expression compared with control CE3027 (1.1/1.1): *P ≤ 0.05. (d) Expression of StCDF1 in DpStFLORE and 35S:StFLORE during a 24-h time course under optimal water conditions in short days. Error bars: means AE SEMs, with n = 3 biological replicates. CE3027 (1.1/1.1) untransformed from Figure 2(a) was used as a control. Significant changes of gene expression compared with control CE3027 (1.1/1.1): *P ≤ 0.05. © 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2021), 105, 855-869 stomata density and size (Figures S8 and S9; [/fig]
[fig] Figure 6: Stomata opening is affected in plants with higher or lower StCDF1 and StFLORE expression. (a) Stomatal pore aperture of StCDF1 RNAi and 35S:StCDF1.2 under ABA treatment, compared with untransformed CE3027 (1.1/1.1) as a control. Error bars: means AE SEMs, with n = 3 biological replicates. *P < 0.05, with respect to control. For further details, see Experimental procedures. (b) Stomatal aperture ratio of CRISPR/Cas9 StFLORE promoter (DpStFLORE) and 35S:StFLORE under ABA treatment, with untransformed CE3027 (1.1/1.1) as a control. Error bars: means AE SEMs, with n = 3 biological replicates. *P < 0.05, with respect to control. For further details, see Experimental procedures. [/fig]
[fig] © 2020: The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2021), 105, 855-869 [/fig]
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Dopamine release, diffusion and uptake: A computational model for synaptic and volume transmission
Computational modeling of dopamine transmission is challenged by complex underlying mechanisms. Here we present a new computational model that (I) simultaneously regards release, diffusion and uptake of dopamine, (II) considers multiple terminal release events and (III) comprises both synaptic and volume transmission by incorporating the geometry of the synaptic cleft. We were able to validate our model in that it simulates concentration values comparable to physiological values observed in empirical studies. Further, although synaptic dopamine diffuses into extra-synaptic space, our model reflects a very localized signal occurring on the synaptic level, i.e. synaptic dopamine release is negligibly recognized by neighboring synapses. Moreover, increasing evidence suggests that cognitive performance can be predicted by signal variability of neuroimaging data (e.g. BOLD). Signal variability in target areas of dopaminergic neurons (striatum, cortex) may arise from dopamine concentration variability. On that account we compared spatio-temporal variability in a simulation mimicking normal dopamine transmission in striatum to scenarios of enhanced dopamine release and dopamine uptake inhibition. We found different variability characteristics between the three settings, which may in part account for differences in empirical observations. From a clinical perspective, differences in striatal dopaminergic signaling contribute to differential learning and reward processing, with relevant implications for addictiveand compulsive-like behavior. Specifically, dopaminergic tone is assumed to impact on phasic dopamine and hence on the integration of reward-related signals. However, in humans DA tone is classically assessed using PET, which is an indirect measure of endogenous DA availability and suffers from temporal and spatial resolution issues. We discuss how this can lead to discrepancies with observations from other methods such as microdialysis and show how computational modeling can help to refine our understanding of DA transmission.PLOS COMPUTATIONAL BIOLOGYPLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1008410 November 30, 2020 1 / 26 OPEN ACCESS Citation: Wiencke K, Horstmann A, Mathar D, Villringer A, Neumann J (2020) Dopamine release, diffusion and uptake: A computational model for synaptic and volume transmission. PLoS Comput Biol 16(11): e1008410. https://doi.org/10.The dopaminergic system of the brain is very complex and affects various cognitive domains like memory, learning and motor control. Alterations have been observed e.g. in Parkinson's or Huntington's Disease, ADHD, addiction and compulsive disorders, such as pathological gambling and also in obesity. We present a new computational model that allows to simulate the process of dopamine transmission from dopaminergic neurons originated in source brain regions like the VTA to target areas such as the striatum on a synaptic and on a larger, volume-spanning level. The model can further be used for simulations of dopamine related diseases or pharmacological interventions. In general, computational modeling helps to extend our understanding, gained from empirical research, to situations where in vivo measurements are not feasible.PLOS COMPUTATIONAL BIOLOGYComputational dopamine transmission model PLOS Computational Biology | https://doi.
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# Introduction
The neurotransmitter dopamine (DA) impacts on a variety of cognitive functions (e.g. learning, working memory) and behavioral features (e.g. motivation, affective behavior) that interact in complex ways. While learning demands very precise input-(action-)output-associations, motivation might be thought of as a more general, behavior modulating factor. Two types of dopaminergic signaling, phasic and tonic DA, have been identified in target areas of DA neurons such as striatum or cortex and putatively subserve distinct functions. They are frequently associated with a teaching signal and motivational drive respectively [bib_ref] Tonic dopamine: opportunity costs and the control of response vigor, Niv [/bib_ref] [bib_ref] Forgetting in Reinforcement Learning Links Sustained Dopamine Signals to Motivation, Kato [/bib_ref] and arise from distinct spiking patterns of midbrain dopaminergic neurons [fig_ref] Fig 1: DA spiking patterns and transmission influence cognition and behavior [/fig_ref]. Tonic DA is grounded in irregular, low frequency firing, whereas phasic signals arise from simultaneous activity in a (sub-)population of neurons. The two characteristics of dopaminergic signaling, tonic and phasic, strongly interact. While prolonged phasic activity can increase the slowly varying levels of DA tone [bib_ref] Burst activation of dopamine neurons produces prolonged post-burst availability of actively released..., Sweyta [/bib_ref] , a potentiation of tonic efflux can attenuate phasic signals [bib_ref] The tonic/phasic model of dopamine system regulation and its implications for understanding..., Grace [/bib_ref] [bib_ref] Ventral tegmental ionotropic glutamate receptor stimulation of nucleus accumbens tonic dopamine efflux..., Tye [/bib_ref].
DA transmission refers to the process shaping the DA signal. For transmission of DA from dopaminergic neurons to their target areas, synaptic and volume transmission have to be considered differentially. Synaptic transmission describes the precise signaling between two cells. Volume transmission refers to communication between neurons beyond the synaptic cleft (i.e. spillover of DA into extra-synaptic space) and to non-synaptic transmission. The presence of extra-synaptic DA receptors and non-synaptic terminals corroborates the occurrence of volume transmission [bib_ref] Somatodendritic dopamine release: recent mechanistic insights, Rice [/bib_ref] [bib_ref] Geometry and kinetics of dopaminergic transmission in the rat striatum and in..., Gonon [/bib_ref] , which has been investigated with different computational models [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref] [bib_ref] Dopamine spillover after quantal release: Rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref]. These models commonly rely on a point source model for synaptic terminals, which assumes equal diffusion in all directions, and are thus adequate for non-synaptic, e.g. somatodendritic release . Not considered is the confinement of DA efflux by the geometry of a synapse. However, DA diffusion from synaptic terminals and its impact on DA transmission is still not fully understood and might be crucial to explain apparent discrepancies across empirical findings [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref]. It has been proposed that reuptake by DA transporters (DAT) strongly limits diffusion from the synaptic cleft [bib_ref] Grace Anthony A. Afferent modulation of dopamine neuron firing differentially regulates tonic..., Floresco Stan [/bib_ref]. In turn, DA from the synaptic cleft activates receptors in close proximity to release sites in contrast to volume transmission [bib_ref] Vesicular Dopamine Release Elicits an Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons, Beckstead [/bib_ref]. In that view, synaptic DA transmission targets precise input-output representations while volume transmission produces a more global signal. Thus, similarly to phasic and tonic signaling, synaptic and volume transmission may impact on learning and motivational aspects of behavior, respectively [fig_ref] Fig 1: DA spiking patterns and transmission influence cognition and behavior [/fig_ref].
Besides disregarding the precise geometry of a synapse, the aforementioned models rely on a second simplification: the analytical solution solving the partial differential equation for DA concentration is based on the assumption that DA concentration is much smaller than the dissociation constant of DAT, the concentration at which 50% of DATs are occupied. However, this holds only for relatively small values of DA concentration like normal tonic DA levels, but not in close proximity to terminals after DA release or in scenarios of strong tonic DA increase, for example after amphetamine or cocaine administration. In particular, under this assumption Michaelis-Menten uptake becomes linear and uptake will be overestimated, i.e. DA concentration will be underestimated.
Here we present a new DA transmission model, a simulation tool for multiple terminal DA release, diffusion and uptake that incorporates the specific geometry of synapses . Furthermore, while the model is based on the same partial differential equation as previous models, it does not restrict on certain concentration values as it relies on a full numerical approach with the method of finite elements, in contrast to analytical approaches. DA terminal co-localized with a fronto-striatal connection and the two modes of spike-dependent DA release: tonic and phasic spiking. Synaptic spillover facilitates volume transmission. Tonic spiking resulting in tonic DA concentration is primarily associated with volume transmission relevant for pre-synaptic control and, on a functional level, with a motivational drive. Phasic DA acts also pre-synaptically via volume transmission and might be involved in terminating phasic spiking signals. However, phasic spiking, i.e. bursts and pauses, and phasic DA are functionally more relevant for learning. Increased and decreased prevalence of synaptic transmission between DA neuron terminals and specific target post-synaptic neurons contribute to post-synaptic cell excitability. In addition, synaptic spillover following burst firing can increase extra-synaptic DA concentration and modulate fronto-striatal loop connections. Depending on how localized this signal is, the definition of volume transmission may not apply.
https://doi.org/10.1371/journal.pcbi.1008410.g001
The paper is structured as follows. First, in section 'Synaptic transmission' we focus on our small-scale model encompassing a single synapse and its near-synaptic space. We examine how strongly DAT uptake actually acts near a synaptic cleft. This has been a matter of debate so far [bib_ref] Extrasynaptic dopamine and phasic neuronal activity, Paul [/bib_ref] [bib_ref] Extrasynaptic dopamine and phasic neuronal activity, Floresco Stan [/bib_ref] , whereby Floresco and colleagues urge to consider the synaptic and extra-synaptic compartment of the DA system differentially. However, from previous point source models of DA transmission one can not distinguish between these two compartments. We model the potential effect on DA-type2-receptors (D2R), because this allows to relate our simulations to empirical findings using methods based on DA action at these receptors such as [11C]raclopride positron emission tomography (PET). In earlier models Michaelis-Menten kinetics for D2R have been used to quantify D2R occupancy. However, these models assume steady state concentrations. More recently, a dynamic receptor binding model has been proposed, incorporating on-and off-kinetics of the receptors. In their paper, the authors use parameters from the literature that suggest slow kinetics. However, they also mention the possibility of faster kinetics implied by recent findings using optical methods. Our new computational model enables us to discuss both, slow and fast binding.
Second, in section 'Spatio-temporal variability of tonic DA concentration in extra-cellular space' we examine simulations of our large-scale model, which contains � 13000 synaptic and non-synaptic terminals. We compare simulations of increased DA release and of uptake inhibition to our default simulation and focus on temporal and spatial variability in extra-synaptic space. In addition, we test a hypothesis postulated by Laruelle [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref] that bridges apparent discrepancies between observations using PET versus microdialysis. This and similar discrepancies across studies may be traced back to resolution issues of different empirical methods. In this context, we will discuss the two extra-cellular compartments of DA transmission: intra-synaptic and extra-synaptic DA. A full mathematical account of our model and a complete list of model parameters are presented in Section 'Materials and methods' and in in S1 respectively.
# Results and discussion
## Synaptic transmission
Synaptic DA transmission may be of particular importance for understanding the role of DA where striatal DA synapses occur interrelated with synapses from limbic and cortical inputs.
Here, DA is of crucial importance for long-term modulation of connections [bib_ref] Dopamine-mediated regulation of corticostriatal synaptic plasticity, Calabresi [/bib_ref] [bib_ref] Striatal cholinergic interneurons and cortico-striatal synaptic plasticity in health and disease, Deffains [/bib_ref] [bib_ref] Regulation of firing of dopaminergic neurons and control of goal-directed behaviors, Grace [/bib_ref] and ultimately for cognitive functions like reward processing and learning. Empirical studies suggest that the synaptic micro-environment needs special consideration. Laruelle [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref] discussed the possibility that if a challenge for benzamide-D2R binding primarily occurs within the synaptic space, this would partly explain discrepancies across pharmacological agents (nicotine vs. amphetamine) in their ability to affect DA microdialysis and benzamide binding potential measurements. Concerning also the synaptic level, Beckstead et al. [bib_ref] Vesicular Dopamine Release Elicits an Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons, Beckstead [/bib_ref] suggested a special role for synaptic transmission in directly regulating post-synaptic cell excitability. From cell recordings in mice midbrain slices the authors concluded and summarized that "synaptic DA transmission does not depend on volume transmission" and that "uptake is a critical factor controlling shape and duration of inhibitory post-synaptic currents" [bib_ref] Vesicular Dopamine Release Elicits an Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons, Beckstead [/bib_ref]. Similar results were recently reported in striatal interneurons and medium spiny neurons (MSNs) using optical methods [bib_ref] Dopamine Neurons Control Striatal Cholinergic Neurons via Regionally Heterogeneous Dopamine and Glutamate..., Chuhma [/bib_ref] [bib_ref] Phasic Dopamine Release Drives Rapid Activation of Striatal D2-Receptors, Marcott [/bib_ref].
According to the suggestion that the synaptic compartment has to be distinguished from the extra-synaptic compartment, in our analysis of synaptic transmission we particularly focused on the hypotheses that (1) DA release is highly localized to the synapses and that (2) DA uptake strongly contributes to this tight localization. This has been a matter of special debate [bib_ref] Extrasynaptic dopamine and phasic neuronal activity, Paul [/bib_ref] [bib_ref] Extrasynaptic dopamine and phasic neuronal activity, Floresco Stan [/bib_ref] and links to the models of 'private' vs. 'social' synapses by Sulzer and Pothos. In a 'private' synapse molecules of a single release event are restricted to the peri-synaptic area, in contrast to 'social' synapses facilitating volume transmission.
The hypotheses were in part addressed with previous computational models. Garris and colleagues [bib_ref] Efflux of dopamine from the synaptic cleft in the nucleus accumbens of..., Garris [/bib_ref] , for example, considered the geometry of the synaptic cleft but focused on the process of diffusion. The authors aimed at quantifying the maximum possible efflux from the synaptic cleft, and thus disregarded the process of DA uptake. By comparing the fast relaxation time of the DA concentration gradient after release with the half-life of DA uptake, they conclude that diffusion rather than uptake is the dominating process here. Nicholson [bib_ref] Interaction between diffusion and Michaelis-Menten uptake of dopamine after iontophoresis in striatum, Nicholson [/bib_ref] investigated the dynamics of DA after release from a point source (the tip of a microelectrode) using a model integrating diffusion and uptake simultaneously. He demonstrated solutions for the respective partial differential equation assuming linear uptake and a baseline DA concentration equal to zero. To investigate a single terminal release event Cragg and Rice [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref] [bib_ref] Dopamine spillover after quantal release: Rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] used the same point source model. They conclude that the predominant mode of DA signaling is volume transmission. In contrast, we will argue for a potential role for synaptic terminal release in addition to volume transmission. In the following, we will discuss the influence of the synapse geometry, non-zero baseline concentration, multiple release from a single terminal, as well as homogeneous and non-homogeneous Michaelis-Menten uptake. Finally, we discuss models of receptor occupancy, i.e. equilibrium binding [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref] [bib_ref] Dopamine spillover after quantal release: Rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] as well as slow and fast dynamic binding [bib_ref] A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine..., Sun [/bib_ref] [bib_ref] Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors, Patriarchi [/bib_ref].
A single full content release event. First, we simulated a single terminal release event with and without homogeneous Michaelis-Menten uptake kinetics, where the full vesicle content of 3000 molecules was released from the center of a synaptic terminal. Initial baseline concentration was chosen to be at the extreme ends of the range of empirical measures (4nM and 50nM). DA quickly escapes the synaptic cleft, which can not be prevented by homogeneous uptake as illustrated in [fig_ref] Fig 3: Single DA release event from synaptic terminal [/fig_ref]. White isolines indicate DA concentration of 1000, 100 and 10nM. DA concentration higher than 1000, 100 and 10nM lasts for 2, 8 and 19ms, respectively, in the 4nM baseline scenario with volume uptake and for 2, 9 and 25ms, resp., in the 50nM baseline scenario with volume uptake. These different values between the two baseline scenarios arise because it takes more time in the 50nM scenario to decrease concentration in the entire volume. The clearing of the synapse after DA release does not actually depend on baseline concentration values. The concentration gradient is extremely steep such that in scenarios Moreover, isolines in the plots and hence the DA concentration patterns differ between scenarios of no vs. homogeneous uptake. There is no 10nM isoline in the scenario of 50nM without uptake. However, this isoline appears in the scenario with uptake. Thus, DATs contribute substantially to the overall DA flux and facilitate the balance towards DA release. Since, in striatal regions, DAT is localized on axons and axonal terminals near and distant from synaptic junctions [bib_ref] The dopamine transporter is localized to dendritic and axonal plasma membranes of..., Nirenberg [/bib_ref] [bib_ref] Subcellular localization and molecular topology of the dopamine transporter in the striatum..., Hersch [/bib_ref] , volume averaged uptake is a simplified model, that does not reflect the true localization and dynamics of uptake. The question remains, whether increased DAT activity at the synaptic level can prevent synaptic spillover of DA. Thus, we ran a further simulation of this micro-environment and tested the effect of enhanced uptake on the cell membrane surface of the pre-synaptic terminal. In comparison to volume uptake, this surface uptake acts very localized and requires a different set of model parameters. Values for those parameters were chosen to promote very strong surface uptake (see section 'Materials and methods'). Nonetheless the effect is rather small [fig_ref] Fig 3: Single DA release event from synaptic terminal [/fig_ref]. Even though there is a DA concentration difference up to few μM between homogeneous uptake (volume uptake only) and inhomogeneous uptake (surface and volume uptake), the isolines do not shift substantially. We conclude that even strong DAT activity near the synaptic junction can not prevent DA spillover, hence volume transmission plays an important role in DA signaling. However, in line with our second hypothesis, uptake promotes the 'privacy' of a synapse. Whether or not there exists cross-talk between neighboring synapses will strongly depend on how we define this inter-synaptic communication in the model.
Cragg and Rice strongly emphasize the role of volume transmission in DA signaling, because the average distance to neighboring synapses, that was previously estimated to be � 4μm [bib_ref] Efflux of dopamine from the synaptic cleft in the nucleus accumbens of..., Garris [/bib_ref] , is much smaller than the distance at which concentration equals 10nM in their model (the 'outer' isolines in [fig_ref] Fig 3: Single DA release event from synaptic terminal [/fig_ref]. Note that in their model they released three times the number of molecules from our model. We adhere to this number in line with other previous models and our simulations of multiple terminal release as discussed in the next section 'Spatio-temporal variability of tonic DA concentration in extra-cellular space'. All remaining parameters for diffusion, uptake and release are identical (S1 . 10nM was chosen as the critical value, because it corresponds to the equilibrium constant for D2Rs. In this case, half the D2Rs would be occupied at a steady state concentration of 10nM. However, two aspects may be discussed here. First, the model by Rice and Cragg assumed zero baseline concentration. This is inadequate in light of empirical values for tonic DA levels, which can by far exceed the equilibrium constant (up to 50nM for rodents and up to 72nM for humans, S3 . Second, the DA signal is highly transient, which violates the equilibrium assumption for D2R binding. A different opinion is provided by the group around Williams, Beckstead and Ford [bib_ref] Vesicular Dopamine Release Elicits an Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons, Beckstead [/bib_ref] [bib_ref] The Time Course of Dopamine Transmission in the Ventral Tegmental Area, Ford [/bib_ref] [bib_ref] The Timing of Dopamine-and Noradrenaline-Mediated Transmission Reflects Underlying Differences in the Extent..., Courtney [/bib_ref]. Their reasoning is also based on DA acting at D2Rs, but from an empirical perspective that accounts for the dynamic nature of DA release. The authors argue for a post-of volume and surface uptake (inhomogeneous uptake). White isolines refer to the simulation with volume uptake only. Gray and black isolines refer to the simulation where surface uptake was five and one-hundred times elevated, resp. Neither homogeneous uptake nor uptake strongly pronounced at the pre-synaptic terminal can prevent synaptic spillover. F: Concentration difference between simulations of homogeneous (white) and inhomogeneous uptake with five (gray) and one-hundred (black) times elevated uptake at the pre-synaptic terminal. G: Fast D2 receptor binding at different distances from release site. Comparable receptor binding occurs at distances up to 0.5μm. The blue and yellow graph are equivalent. D2R binding at half the distance of neighboring terminals (2.0μm) is still profound but negligible at 4.0μM. Differences between a synaptic (solid line) and a non-synaptic terminal (dashed line) could not be confirmed. H: Slow D2 receptor binding at different distances from release site for a synaptic (solid line) and a nonsynaptic terminal (dashed line). Elevated receptor binding occurs at a distance of 0.1μm, (i.e. inside the synapse for the synaptic terminal with radius = 0.15μm), but not at distances beyond 0.2μm. D2R binding beyond half the distance of neighboring terminals (2.0μm) is small and negligible at 4.0μm.
https://doi.org/10.1371/journal.pcbi.1008410.g003 synaptic mechanism independent of diffusion, in contrast to the principles of volume transmission. Despite the high affinity of D2Rs for DA, a much higher concentration than 10nM is required to produce the activation of receptors that would mimic an electrically evoked D2R mediated response in midbrain DA neurons. Transferring this idea to striatum, from an experimental point of view the 10nM isoline might not be the best indicator where synaptic spillover of DA affects cell properties. Especially since this value is within the range of baseline concentrations. In our simulations the 'middle' isoline of 100nM DA concentration, which is less sensitive to baseline concentration, remains below the average distance to neighboring synapses in all scenarios even without uptake. Our simulation with 4nM (50nM) baseline concentration and homogeneous Michaelis-Menten kinetics yielded a distance of 2.3μm (2.8μm). The 1000nM isoline appears at a distance of 1.14μm (1.15μm). The next question is how the concentration time course looks like, if a single synapse releases DA successively within a short time interval. After examining multiple release events, we will model and discuss receptor binding in more detail and pick up on this discussion.
Multiple releases event from a single terminal. Next, we modeled a single synaptic terminal releasing DA at a frequency of 30Hz (Poisson process), which could be considered a burst mode. However, the model does not capture DA input from other terminals that would release DA synchronously in such case. Initial baseline concentration was 4nM. An entire vesicle content of 3000 molecules was released each time. Notably, a release probability of approx. 6% or partial content release ranging between 0.1 − 21% has been suggested [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref] [bib_ref] Computational modeling of extracellular dopamine kinetics suggests low probability of neurotransmitter release, Rooney [/bib_ref]. Thus our simulation presumably exaggerates realistic DA release. However, between release events DA concentration falls below 10nM [fig_ref] Fig 4: Multiple DA release events from synaptic terminal [/fig_ref]. The 10nM isoline embraces multiple spike events only if they occur within short intervals (<17ms). Hence without the pooling effect of multiple synapses, in the peri-synaptic area of a single terminal DA concentration �10nM will not endure with release frequencies higher than tonic firing modes. These range between 0.5 and 8Hz [bib_ref] Frequency-dependent modulation of dopamine release by nicotine, Zhang [/bib_ref]. Similarly, considering a fraction of release or partial content release such high concentrations do not endure during burst firing, i.e. �15 − 50Hz in rodents and up to 100Hz in primates [bib_ref] Frequency-dependent modulation of dopamine release by nicotine, Zhang [/bib_ref].
Dynamic receptor binding. As stated earlier, the debate about the privacy of a synapse may be based on the ability of synaptic DA to bind to receptors outside the synaptic junction. Directly translating concentration values to dopamine receptor binding using Michaelis-Menten kinetics [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref] [bib_ref] Dopamine spillover after quantal release: Rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] does not take into account the on-and off-binding kinetics when concentration is dynamic. In this case, concentration is simply transformed onto the [0, 1]-interval, reflecting concentration dynamics, but not receptor dynamics [fig_ref] Fig 4: Multiple DA release events from synaptic terminal [/fig_ref]. For transient DA release on the synaptic level we implemented dynamic receptor kinetics as proposed in. Slow binding is illustrated in [fig_ref] Fig 4: Multiple DA release events from synaptic terminal [/fig_ref] In this plot, we assigned colors to different distances from the synapse center. While binding increases stepwise with each terminal release event at small distances (blue, orange, green), this stepwise increase is almost not visible at distances further than 2μm (red & purple), which is half the distance to neighboring synapses. With this result we again argue for the privacy of a single terminal. In this scenario of slow binding and 0% initial receptor occupancy, after 1000ms 18.5% of the receptors are occupied inside the synapse. Over a long period of time occupancy will reach an asymptote, that will characterize the average DA release-uptake balance of the terminal over time. Changes in firing patterns and consequently in striatal DA release are only detectable over time scales of several seconds to minutes.
Recent studies using genetically encoded sensors [bib_ref] A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine..., Sun [/bib_ref] [bib_ref] Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors, Patriarchi [/bib_ref] suggest faster receptor binding. In contrast to the monotonic increase in case of slow receptor binding, in this scenario receptor occupancy is much more dynamic and fluctuates between 44.7% and 100% in synaptic and peri-synaptic space. Also at a distance further than 2μm these fluctuations are prominently pronounced ranging between 28.8% and 80.7% [fig_ref] Fig 4: Multiple DA release events from synaptic terminal [/fig_ref] , but not so much at the average distance of neighboring synapses, where D2R occupancy is between 10.1% and 31.8%. With fast binding kinetics a single release event is capable to increase D2R binding by maximum 5.7% [fig_ref] Fig 3: Single DA release event from synaptic terminal [/fig_ref].
Synaptic vs. volume transmission. We conclude, DA leaves the synaptic cleft due to high gradients between extra-and intra-synaptic concentration after terminal release, which can not be prevented by DAT even if strongly pronounced at the pre-synaptic terminal. Thus diffusion is the predominant process within in the first milliseconds. However, for the subsequent shaping of the concentration pattern DATs contribute to the privacy of the synapse. In summary, we found with respect to slow binding kinetics that synaptic DA is highly localized to the synapse. The model of fast receptor kinetics shows some effect on D2R binding at the average distance of neighboring synapses. However, with DA contribution from multiple synapses, that are missing here, is remains questionable if synapses actually detect neighboring signals. Thus cross-talk between synapses is limited in the sense that a synapse can not actually eavesdrop on a neighboring synapse. This does not exclude that synapses can detect concerted activity, i.e. the average population DA release in their neighborhood, such as changes in tonic activity as well as changes from tonic to burst modes. Thus both, synaptic and volume transmission may contribute to complex DA signaling.
As we previously touched upon, from an experimental point of view data from midbrain neurons suggest locally induced D2 autoreceptor mediated currents, so-called inhibitory postsynaptic currents (IPSCs). Two scenarios of DA application in midbrain could induce an IPSC comparable to electrical stimulation. Either a DA concentration of 10μM for at least 25ms or an instantaneous DA concentration of 100μM. While this was previously interpreted as a postsynaptic response, Rice and Patel [bib_ref] Somatodendritic dopamine release: recent mechanistic insights, Rice [/bib_ref] argue that these midbrain DA neurons are autoregulated primarily by their own DA release, rather than via synaptic DA release. Importantly, IPSC have been studied in striatal MSNs [bib_ref] Phasic Dopamine Release Drives Rapid Activation of Striatal D2-Receptors, Marcott [/bib_ref] and interneurons [bib_ref] Dopamine Neurons Control Striatal Cholinergic Neurons via Regionally Heterogeneous Dopamine and Glutamate..., Chuhma [/bib_ref]. Both studies conclude a localized and discrete action of DA on these neurons.
Directly arguing with the empirical data derived from midbrain neurons, a prolonged concentration of >10μM that is necessary to mimic the electrically evoked IPSC, does not occur in our simulation of a single striatal synapse. It may be realized via volume transmission through the pooling effect of DA release from multiple terminals (e.g. during burst activity). An instantaneous DA concentration of >100μM occurs in our simulation only inside the synapse. Consequently, we conclude that DA acts highly localized to the terminal. However, this is not necessarily restricted to action inside the synapse, but at least to the peri-synaptic area [fig_ref] Fig 4: Multiple DA release events from synaptic terminal [/fig_ref] , particularly in view of the fact that most receptors are located extra-synaptically. In addition, we designed a non-synaptic terminal and compared DA release between both terminal types [fig_ref] Fig 3: Single DA release event from synaptic terminal [/fig_ref]. Our results show that fast DA spillover from the synapse is not very different from non-synaptic DA release and the discussion about synaptic terminals above apply similarly to non-synaptic terminals. The main difference is the high concentration that occurs briefly within the synapse. The figure illustrates the D2R binding difference between synaptic and non-synaptic terminal DA release (solid versus dotted lines). This difference is apparent only directly next to the release site, i.e. inside the synapse.
## Spatio-temporal variability of tonic da concentration in extra-cellular space
As a physiological measure, DA tone refers to the endogenous DA concentration in the brain in the absence of neuronal bursts, spatio-temporally averaged according to the resolution of the empirical measurement method. Alteration of DA tone has attracted much attention as it may be crucial for cognitive impairments in neurological disease and psychiatric disorders, including Parkinson's and Huntington's Disease [bib_ref] The role of dopamine in positive and negative prediction error utilization during..., Mathar [/bib_ref] , addiction [bib_ref] Imaging dopamine's role in drug abuse and addiction, Volkow [/bib_ref] [bib_ref] Aversive Stimuli Drive Drug Seeking in a State of Low Dopamine Tone, Twining [/bib_ref] , pathological gambling [bib_ref] Increased Striatal Dopamine Synthesis Capacity in Gambling Addiction, Van Holst [/bib_ref] [bib_ref] Dopamine and Opioid Neurotransmission in Behavioral Addictions: A Comparative PET Study in..., Joonas [/bib_ref] and binge eating disorder [bib_ref] Dopamine and Opioid Neurotransmission in Behavioral Addictions: A Comparative PET Study in..., Joonas [/bib_ref]. Different empirical methods aim at quantifying DA tone, either directly, but invasively (e.g. microdialysis [bib_ref] Grace Anthony A. Afferent modulation of dopamine neuron firing differentially regulates tonic..., Floresco Stan [/bib_ref] [bib_ref] An In Vivo Study of Dopamine Release and Metabolism in Rat Brain..., Sharp [/bib_ref] [bib_ref] Extracellular dopamine in rat striatum following uptake inhibition by cocaine, nomifensine and..., Church [/bib_ref] [bib_ref] Extracellular Concentration and In Vivo Recovery of Dopamine in the Nucleus Accumbens..., Parsons [/bib_ref] or indirectly (e.g. PET [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref] [bib_ref] Argument for a non-linear relationship between severity of human obesity and dopaminergic..., Horstmann [/bib_ref] [bib_ref] Imaging D2 Receptor Occupancy by Endogenous Dopamine in Humans, Marc [/bib_ref] [bib_ref] Increased baseline occupancy of D2 receptors by dopamine in schizophrenia, Abi-Dargham [/bib_ref] [bib_ref] A Simple Method to Measure Baseline Occupancy of Neostriatal Dopamine D2 Receptors..., Verhoeff [/bib_ref].
DA transmission describes the complex interplay between DA release, diffusion and uptake. The resulting DA tone is determined by the sensitive balance between DA release and uptake. Inside the cell DA is stored in vesicles which fuse with the membrane when the neuron spikes (exocytosis), thereby releasing DA into the extra-cellular space. DA release relies on multiple factors. These include DA synthesis and recycling of DA molecules into vesicles (both of which impact on the amount of DA in vesicles), fraction and probability of vesicles content release per spike event, and the frequency at which tonic spiking occurs. DA uptake on the other side of the balance is also influenced by several factors such as DAT localization, DAT trafficking or the DAT recycling rate. Mathematically, these factors are expressed by the Michaelis-Menten uptake kinetics, which in turn relies on two parameters, the maximum uptake rate V max and the so-called dissociation constant K m which indicates the DA concentration at which 50% of DATs are occupied.
Extra-synaptic DA concentration is highly heterogeneous throughout space and time. To understand the dynamics of dopaminergic signaling and potential consequences, not only the mean DA concentration but also its variability might be of importance. With our model we can simulate arbitrarily small volumes and high temporal resolution, although at the cost of computational effort, and examine DA distribution throughout the simulation space. This allows to bridge apparent discrepancies from empirical observation across methods, that may arise from resolution problems. In the following, we first examine a default simulation, meant to reflect healthy DA transmission. Second, we focus on temporal variability in extra-synaptic space and show how it differs between a simulation of uptake inhibition, increased DA release and our default simulation. Importantly, the uptake inhibition simulation reaches a concentration level that violates the assumption to be much smaller (<< K m ) or much higher (>> K m ) than the dissociation constant of DAT (K m = 210), thus impeding the application of an analytical solution to the differential equation (see 'Materials and methods'). Third, we discuss why both, temporal and spatial variability, have to be taken into account when assessing receptor occupancy as a function of DA concentration.
Finally, we want to test a hypothesis based on imaging pharmacological interventions using PET, postulated in Laruelle 2000: for agents that enhance DA concentration by inhibiting uptake (e.g. amphetamine), the competition between endogenous DA and radioligand occurs prominently at non-synaptic D2Rs. On the other hand, following the administration of drugs that stimulate DA release (e.g. nicotine) the competition is more pronounced at synaptic D2Rs. Laruelle references studies investigating simultaneous PET and microdialysis measurements, which show similar reductions in raclopride BP between administration of uptake inhibitors and DA release stimulants. However, the associated increases in extracellular DA was much larger following uptake inhibition than following elevated DA release. These results can be expected with a predominant intra-synaptic location of the challenge following increased DA release and microdialysis measuring extra-synaptic DA only, while PET measures both, extra-and intra-synaptic D2R challenges. For the following analysis, we ran simulations in three different parameter settings: (1) default DA, (2) direct DA enhancer and (3) indirect DA enhancer.
Default setting. First, as a proof of concept for our model, we simulated DA transmission under default parameter setting. This default setting mimics healthy DA transmission to the striatum, i.e. where DA tone is determined by an undisturbed balance of all subprocesses. To simulate DA transmission to cortical areas an adjustment to various parameters has to be considered. Cortical areas receive much less DA projections compared to striatal areas resulting in much lower DA concentration levels in prefrontal cortex [bib_ref] Imaging dopamine transmission in the frontal cortex: a simultaneous microdialysis and [11C]FLB..., Narendran [/bib_ref].
For reasons of comparability, we choose our default parameter setting in accordance with a previous model by Dreyer and colleagues [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref]. Additionally we implemented parameters needed for synaptic specificity (S1 . Dreyer's model resulted in a slightly lower DA tone of � 37nM, compared to our model, where the average extra-synaptic concentration is � 40nM [fig_ref] Fig 5: Extra-synaptic DA [/fig_ref]. The discrepancy between our and Dreyer's model results can be explained by the approach to solve the partial differential equation describing diffusion and uptake. Dreyer and colleagues used a mixed strategy. Like in our approach, they solved their largescale model numerically, but in this large-scale model the authors integrated an analytical solution for individual terminal release as it was suggested in [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref]. Importantly, this analytical solution holds only for DA concentrations much smaller than the dissociation constant for uptake [bib_ref] Interaction between diffusion and Michaelis-Menten uptake of dopamine after iontophoresis in striatum, Nicholson [/bib_ref] , which is not the case in direct vicinity of terminals. The analytical solution was achieved by assuming linear uptake, where in fact uptake converges to an upper limit due to saturation (see 'Materials and methods'). Consequently, this assumption overestimates DA uptake for high concentration, i.e. around a terminal after DA release. In contrast, we implemented an overall numerical solution that does not violate the saturation effect of DATs.
DA tone resulting from our default simulation setting is well in the range of empirical measures. Studies with rats report values in striatal regions between 4 − 50nM [bib_ref] An In Vivo Study of Dopamine Release and Metabolism in Rat Brain..., Sharp [/bib_ref] [bib_ref] Extracellular dopamine in rat striatum following uptake inhibition by cocaine, nomifensine and..., Church [/bib_ref] [bib_ref] Extracellular Concentration and In Vivo Recovery of Dopamine in the Nucleus Accumbens..., Parsons [/bib_ref] [bib_ref] Transient changes in mesolimbic dopamine and their association with reward, Wightman [/bib_ref]. PET or SPECT studies derived human in vivo steady-state DA levels ranging between 23 and 72nM (S3 . Using the range of empirical measurements for the tonic firing frequency of 2 − 5Hz [bib_ref] Striatal dopamine neurotransmission: regulation of release and uptake, David [/bib_ref] , we simulated DA tone and obtained basal DA levels between approx. 20 and 50nM.
In our model several specific implementations have to be discussed. First, fixed parameters in our healthy condition model have been taken from previous models and are not consistently from the same species. It is particularly difficult to find values for all required parameters in humans in order to realistically simulate human endogenous DA levels. Moreover, often parameter values can not be precisely determined from the literature. For example, reported empirical values for the two parameters describing Michaelis-Menten uptake kinetics in the striatum differ widely across and within species (S2 . These differences might arise in part from methodological differences in measurements, but could also reflect individual differences and even heterogeneity within an individual's striatum [bib_ref] Passive stabilization' of striatal extracellular dopamine across the lesion spectrum encompassing the..., Bergstrom [/bib_ref] [bib_ref] Dopamine Release and Uptake Dynamics within Nonhuman Primate Striatum In Vitro, Cragg [/bib_ref]. Second, for a single terminal there is evidence that not every spike is associated with an event of exocytosis and for such events the vesicle content might not be released entirely. This was investigated in detail by Rooney and Wallace [bib_ref] Computational modeling of extracellular dopamine kinetics suggests low probability of neurotransmitter release, Rooney [/bib_ref] , but based on their computational model, the authors could not conclude which mechanism is actually functional in the brain. However, assuming partial content release at all terminals ranging between 0.1 − 21% they were able to keep a reasonable tonic DA level constant. With our model we adhere to Dreyer and colleagues and implemented full content release at a fraction (default = 6%) of terminals. Finally, in the living being vesicle content is not released at an instance as in our model [bib_ref] Striatal dopamine neurotransmission: regulation of release and uptake, David [/bib_ref] Stimulation of DA release and uptake inhibition. To simulate the presence of an agent stimulating DA release, we increased the release probability from 6% to 7.8%. This elevates DA tone to 129% of the DSS, similar to a representative example mentioned by Laruelle. Here, rodents were administered a high dose of nicotine (5mg/kg). He compared the results with a study of amphetamine administration (0.4mg/kg) in rhesus monkeys which elicits a 1365% increase over baseline in extra-synaptic DA as measured with microdialysis. For the UIS we set the maximum uptake rate V max to 80% of the default and K m to 24μM, as it has been observed after amphetamine superfusion of mice striatal slices using cyclic voltammetry [bib_ref] Amphetamine Distorts Stimulation-Dependent Dopamine Overflow: Effects on D2 Autoreceptors, Transporters, and Synaptic..., Schmitz [/bib_ref]. In addition, we divided the number of molecules being released during an event of exocytosis by 8.5 to obtain approximately the above mentioned increase in DA tone. A reduced number of molecules is a consequence of less DA recycled into the cell and D2R auto-inhibition of stimulation-dependent DA release. Note, however, that we do not simulate the above mentioned interventions themselves. These pharmacological manipulations are much more complex. For example, amphetamine does not only block but also reverse transporters. Hence for a realistic simulation of amphetamine administration stimulationindependent retrograde release via DAT has to be considered. Rather, we are interested in the DAT blocking effect on extra-and intra-synaptic DA concentration compared to promoted DA release and consider simplified models of uptake inhibition and enhanced DA release.
Temporal variability. We first consider temporal fluctuation of the volume-averaged extra-synaptic DA concentration, by comparing default setting simulation (DSS) with simulations of uptake inhibition (UIS) and stimulated release (SRS). In [fig_ref] Fig 5: Extra-synaptic DA [/fig_ref] -synaptic DA for all three simulations are displayed. DSS, SRS and UIS yielded a tonic DA level of �40nM, �52nM and �511nM, respectively. Importantly, in the UIS, DA concentration values are larger than the dissociation constant K m and DA uptake approximates its maximum (see 'Materials and methods').
With respect to temporal variability, we obtained the following results. Firstly, under default conditions and in the SRS, DA tone leveled very quickly compared to the UIS after initialization with high and low concentration values. This indicates an effective balance between release and uptake, capable of reinstating extra-synaptic concentration for example after the occurrence of a burst or pause of neuronal activity. In contrast, DA tone of the UIS needed considerably more time to settle. Secondly, in the default setting and in the SRS extra-synaptic DA concentration oscillated fast around the mean (i.e. DA tone), while in the UIS slower and larger fluctuations appeared.
We examined these fluctuations by fitting an Ornstein-Uhlenbeck process (OUP) to the simulated time series. OUPs are used to describe mean reverting processes and contain three parameters: (1) the mean reversion level or balance level (in our application DA tone), (2) the mean reversion rate, which measures the strength of attraction towards the balance level (in our application the capability to reinstate DA tone) and (3) the volatility parameter describing the size of random jumps (Poisson-distributed spiking). The process was fit to the simulated time series using maximum likelihood estimation. After the fitting procedure, the balance levels of the OUPs corresponded to the means of the extra-synaptic concentration, i.e. � 39.7nM for default, � 52.2nM for SRS and �510.3nM for UIS. We further obtained best estimates for the rate and volatility parameter, however, depending on initialization of the optimization algorithm we found multiple local maxima. In total we ran the fitting procedure with nine different initializations. All results and a more detailed discussion of the locally best estimates is provided in S4 [fig_ref] Table Table: of model parameters [/fig_ref] In the following we will only report the essential outcomes.
First, we will compare default simulation and stimulated release simulation. We found a difference between the rate parameters, which was larger for the SRS than for the DSS and comparable volatility parameters, being just slightly higher for the SRS. The two simulations differ by an enhanced release frequency in the SRS, while the number of DA molecules is the same for every release event in both simulations. Hence the size of the random jumps, which we associate with the volatility parameter is comparable, while elevated DA release in the SRS requires faster reinstatement (i.e. a higher rate parameter).
For the UIS we found that both, the rate and volatility parameter were lower compared to default and SRS. This is not surprising, given that we reduced the amount of DA molecules being released in the UIS, which should in turn result in smaller random jumps. Moreover, the lower rate parameter for UIS signifies that reinstatement is impaired by lower DAT activity.
In addition, the variance of an OUP is given by the ratio volatility 2 /(2 � rate). From a mathematical point of view, this ratio is the variance of the stationary distribution of the stochastic process, the OUP. Specifically, it constitutes a relationship between random tonic DA release and the capacity of the system to reinstate stationarity, thus it is the variance of the concentration time course, when the concentration level is around the mean (tonic) DA level, i.e. when it is not disturbed by bursts and pauses. This value is approximately in the same range for DSS and SRS and lower in case of the UIS that reflects uptake inhibition and diminished DA release. This points towards strong low frequency fluctuations in this scenario, caused by an impaired reinstatement rather than fast oscillations due to transient DA release.
It becomes obvious that, in case of uptake inhibition, the balance between release and uptake is heavily disturbed. In the default setting and also in the SRS tonic DA appears noisy (larger random jumps) but stable (fast reinstatement). In contrast, DA tone in the UIS exhibits smaller random jumps, but is overlaid by longer fluctuations caused by a low reversion rate. Thus, when uptake is inhibited, the overall heightened DA tone together with a reduced capacity to effectively decrease concentration during pauses or after bursts of DA neuronal firing potentially impact on behavior. Additionally, the long fluctuations could easily be misinterpreted as systemic, i.e. as small phasic signals. In turn, small phasic signals might not be detectable due to the relatively high amplitude of the observed fluctuations. In other words, our results suggest a low signal-to-noise-ratio in scenarios of uptake inhibition, which has to be further investigated with simulations of DA bursts and pauses. Finally, not only DA tone but also extra-synaptic DA variability might contribute to altered behavior during uptake inhibition. In fact, in neuroimaging research, brain signal variability has been linked to cognitive performance and was suggested to be considered as a functional property of the human brain [bib_ref] Moment-to-moment brain signal variability: A next frontier in human brain mapping?, Garrett [/bib_ref] [bib_ref] BOLD Variability is Related to Dopaminergic Neurotransmission and Cognitive Aging, Guitart-Masip [/bib_ref] [bib_ref] Dopaminergic modulation of hemodynamic signal variability and the functional connectome during cognitive..., Alavash [/bib_ref].
Spatial variability. In the previous paragraph we discussed temporal characteristics of the volume-averaged extra-synaptic DA concentration. However, throughout the volume there exist strong concentration gradients, which can be very steep near release sites. In default parameter setting, where DA tone is in the range of 10 1 nM, concentration gradients in the range of 10 4 nM/μm can occur milliseconds after such release according to our model of a single terminal release event (see 'Synaptic transmission'). This wide concentration range indicates a very complex spatio-temporal pattern of DA concentration, with very transient occurrence of high values. On a coarser resolution, using fast-scan voltammetry-a method that averages over terminals within a radius of 75μm from the electrode-transients of 10 to 100-fold increased DA concentration have been observed [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref] [bib_ref] Time-dependent assessment of stimulus-evoked regional dopamine release, Lippert [/bib_ref]. Thus, for our further considerations two aspect will be crucial: First, whether a method measures DA directly or indirectly and second, its temporal and spatial resolution.
In the bottom graphs we illustrate DA concentration within four randomly chosen finite elements of the tonic model, i.e. extra-synaptic DA concentration within a radius of � 0.8μM from the terminal. Large concentration peaks indicate DA release from this synapse, while smaller peaks are caused by DA release from neighboring terminals. As the same amount of DA molecules is released every time, peak concentration in larger volumes (coarser resolutions) are smaller. With our simulations we found that volume averaging strongly simplifies the true dynamics of dopaminergic transmission and can in fact have severe consequences when inferring post-synaptic effects. In particular, if we calculate volume-averaged receptor occupancy from the spatio-temporally varying concentration, the result crucially depends on the order of (a) volume averaging and (b) applying the transform function F D2R (x): restricted to immanent resolution and, particularly, when drawing conclusions about DA concentration from PET measurements. Here, binding of tracers and hence binding of competing endogenous DA to D2Rs is frequently used to infer concentration of DA. Specifically, it is important bridge to observations using PET-a method that can be applied to human research -with findings from other methods that can not be used in human studies. In the following we seek evidence for the hypothesis postulated by Laruelle. For that we will rely on temporal1 as the more realistic value for tonic D2R occupancy since it should be less corrupted by spatial resolution. Note however, for our models of dynamic receptor binding we choose on-and off-kinetic parameters such that the resulting dissociation constant is equal to what has been observed on a spacial scale that is coarse compared to the resolution of our model (see 'Materials and methods'). In this case, tonic DA concentration corresponds to receptor binding given by tem-poral2. Two aspects have to be considered here: first, the above spacial averaging assumes homogeneous distribution of D2Rs across the extrasynaptic space and second, parameters derived on a macro-scale resolutions (temporal and spatial) may not be equivalent on a microscale. Alternatively, parameters for our simulations on micro-scale could be adjusted such that temporal1 equals the empirical observations. However, this goes beyond the scope of this article and is not necessary for the following considerations.
Laruelle's hypothesis. We analys the spatio-temporal DA concentration from the stimulated release (SRS) and uptake inhibition simulation (UIS). The criterion for our simulations, that would support Laruelle's hypothesis, is an excessive synaptic D2R occupation in SRS compared to UIS. DA tone for default setting simulation (DSS), SRS and UIS leveled at 40.1nM, 51.8nM and 511.1nM, respectively. With our models of dynamic slow and fast D2 receptor binding we obtain the following results.
First we discuss fast binding. For the SRS, on average, DA binds to 57.08% of the D2Rs in the entire volume, assuming those receptors are homogeneously distributed. D2 occupancy is only slighly increased in peri-synaptic space (57.6%) and slighly more inside the synapse (58.2%). Thus a non-homogeneous distribution of D2Rs, i.e. denser near a terminal, can result in an overall D2R occupancy <58.2%. In contrast, for the UIS we obtain comparable estimates across extracellular compartments of approx. 95.3% D2R occupancy. Consequently, with the model of fast receptor binding we can not confirm Laruelle's hypothesis.
More interesting appears the model of slow binding: For the SRS, on average and under the assumption of homogeneity, DA binds to 57.08% of the D2Rs in the entire volume. Specifically, in peri-synaptic space 70.11% of the D2Rs are being occupied and 87.4% inside the synapse. This difference across different compartments is not pronounced in the UIS. Here our model yields 95.35% in peri-and 95.81% occupancy in synaptic space. High DA concentration in the UIS saturates D2Rs across the entire extracellular volume. Since synaptic and peri-synaptic D2R occupancy in the SRS is much lower compared to the UIS we can not conclude comparable occupancy between these two settings. However, for the SRS, we found differences across compartments. In turn, this means that for PET tracers challenges with endogenous DA can be different in synaptic, peri-synaptic and extra-synaptic space.
In summary, the discrepancy between PET and microdialysis observations can only in part be explained by different challenges across the extra-cellular space, but not completely. This leads to the conclusion that other methodological issues are relevant here. First the observations, i.e the simultaneous PET-microdialysis-measures are from different studies on either nicotine or amphetamine administration and not within the same species. Second, as Laruelle also discussed in his work, even simultaneous intra-subject measures are not always consistent.
# Conclusion
We have proposed a new computational model for DA transmission integrating release from multiple terminals, diffusion and uptake of DA. To our knowledge it is the only model of multiple release sites, to date, that considers both, synaptic and volume transmission. The importance of synaptic transmission has been a matter of debate. With our synaptic model we simulated DA concentration in and near the synaptic cleft. After vesicle release most DA molecules leave the synaptic cleft within less than a millisecond due to fast diffusion. Here peri-synaptic DATs are saturated at early high concentrations.
Experimental observations have led to the conclusion that DATs limit DA spillover [bib_ref] Vesicular Dopamine Release Elicits an Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons, Beckstead [/bib_ref] [bib_ref] The Timing of Dopamine-and Noradrenaline-Mediated Transmission Reflects Underlying Differences in the Extent..., Courtney [/bib_ref] and to the discussion about volume transmission versus synaptic transmission. This discussion was based on empirical evidence in midbrain. Here, somatodentritic release mainly serves to regulate the rate and pattern of firing of the DA neurons and hence release into striatum. Synaptic release sites are sparse, they exist in VTA, but not SNc, and their role remains to be determined [bib_ref] Somatodendritic dopamine release: recent mechanistic insights, Rice [/bib_ref]. For our purpose we simulated DA release in striatum. Here the majority of terminals is non-synaptic, which is classically an argument for volume transmission. In addition, our simulations suggest that strongly pronounced DAT activity at the presynaptic terminal can not prevent DA from escaping the synaptic cleft. Directly after DA release, diffusion rather than uptake is the dominating process.
However, uptake seems not only critical for a balanced DA tone, but intensively shapes the extra-synaptic DA signal near release sites. Thus, neither supporting an exclusive intra-synaptic nor a population signal (volume transmission), overall, our simulations show that DA from a terminal, synaptic or non-synaptic, is highly localized to the release site. Hence, it is likely that autocrine signals are mediated, by a cell's own DA. This does not exclude a pooling effect of DA release from multiple terminals in line with volume transmission.
With respect to post-synaptic effects on neurons in the projection area, we again emphasize that DA is highly localized to its release site, suggesting direct signaling from cell to cell, rather than from cells forming synapses in neighboring distance. In addition, inside synaptic junctions DA concentration gains levels that do not occur with non-synaptic terminals. Beyond the synapse, with models of slow and fast D2R receptor binding kinetics, the difference between synaptically and non-synaptically released DA does not translate into differences in binding. For both release modes the effect on D2R binding was negligible further than half the distance of neighboring synapses with models of slow and fast binding kinetics. Again, this supports the privacy of terminals within their front yard, but does not exclude a mechanism that is based on a population effect. Volume transmission remains an important aspect of DA signaling characterizing the average population activity, hence detecting changes in the amplitude of tonic activity or switches between tonic and burst modes.
However, so far our conclusion about the specificity of synaptic DA was primarily based on the discussion about D2 receptor binding. It is likely that synaptic and also non-synaptic DA is crucial for modulating co-occurring synapses from limbic and cortical afferents. Thus, in addition to the view that the DA system is driven by volume transmission, essential for pre-synaptic control, we suggest to consider the possibility that synaptic transmission is functionally relevant for post-synaptic modulation and hence learning [fig_ref] Fig 1: DA spiking patterns and transmission influence cognition and behavior [/fig_ref].
Importantly, in his work, Berke [bib_ref] What does dopamine mean?, Joshua [/bib_ref] reconciles the distinct association between motivation and tonic DA versus learning and phasic DA. In this context, (peri-)synaptic vs. volume transmission might be an alternative candidate mechanism that allows to distinguish between motivational and teaching signals of DA.
Comprising small-scale and large-scale simulations, our model is suitable to investigate hypotheses concerning a single synapse and also a population of cells. As a first application of our large scale model we assessed the spatio-temporal variability of DA concentration in a volume of [50μm] 3 containing �13000 terminals. We showed how temporal concentration variability in extra-synaptic space can be described by an Ornstein-Uhlenbeck process. With this approach we identified differences between healthy DA transmission, enhanced DA release and uptake inhibition. Importantly, from an empirical point of view temporal variability in DA concentration depends on the sampling resolution of the measurement method which in turn can have severe impact on the interpretation of results. With their recent findings Berke and colleagues challenge the view that motivation is mediated by slow changes in tonic dopamine cell firing [bib_ref] What does dopamine mean?, Joshua [/bib_ref] [bib_ref] Dissociable dopamine dynamics for learning and motivation, Mohebi [/bib_ref].
The analysis of our simulated DA concentration time series further showed how strongly extra-synaptic DA concentration varies across space and how spatial averaging affects inference on receptor occupancy and subsequent post-synaptic effects. Again, this might have direct implications for the interpretation of neuroimaging measurements such as PET, where competitive binding of radioligands to D2Rs has been inversely related to the availability of endogenous DA.
Physiological measures of DA concentration are either indirect (PET, SPECT, fMRI), invasive (microdialysis, optogenetics) or both and suffer from resolution limitations or poor signal-to-noise-ratio. Computational modeling advances our understanding of dopamine transmission and the complex underlying mechanisms in addition to empirical observations. Furthermore, it can be used to postulate hypotheses before expensive experiments are being conducted or to consolidate apparent discrepancies between results from different empirical observations. Our model may be used to mimic pharmacological manipulations (e.g. the administration of amphetamine or L-DOPA) or DA-related disease and disorders.
In this work, we tested a hypothesis postulated by Laruelle [bib_ref] Imaging Synaptic Neurotransmission with in Vivo Binding Competition Techniques: A Critical Review, Laruelle [/bib_ref]. Based on empirical observations, he proposed that the challenge at D2Rs between endogenous DA and PET tracers is occurring primarily in the synaptic space when DA release is enhanced. In contrast, if DA uptake is inhibited, then this challenge takes place mainly in the extra-synaptic space. This could explain why amphetamine administration (i.e. uptake inhibition) elevates DA concentration levels far beyond elevated levels following nicotine administration as measured with microdialysis, but at the same time reduces D2R binding potential to a comparable amount as measured with PET. We indeed found differences between the synaptic, peri-syaptic and extra-synaptic compartments. Our model suggests the possibility for enhanced challenges in synaptic compared to extra-synaptic space following nicotine administration. However, this could not completely explain the strong difference in concentration occurring in parallel with no difference in D2R binding.
Despite some limitations and discrepancies in empirical research concerning resolution, expense and inference, we want to emphasize the importance of using different empirical methods that have previously established theories for dopamine transmission. These merging theories were necessary to build simulation tools like the one presented here. In turn, model simulations can provide a level of detail not observable with empirical methods or can help to disentangle effects on indirect measures. In that sense, we would like to encourage the combination of empirical and simulation methods, such that they optimize each other. Here we presented such a simulation tool, reported its potentials and limitations and applied it to current research of interest.
# Materials and methods
## Principal processes and model architecture
Our model implements the general process of DA transmission between brain regions, whereby our default parameters account for transmission to striatal regions. The three dominating processes that control DA distribution in extra-cellular space are release, diffusion and uptake. DA is being released (exocytosis) from randomly distributed synaptic and nonsynaptic terminals of neurons that fire in a Poisson-distributed manner. From previous model simulations, propose partial vesicle content release between 0.1 and 21%. In our default model we used full content release with a release probability of 6% per spike as suggested by Dreyer and colleagues [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref]. Diffusion strongly depends on the volume fraction of the extracellular space (α) and its tortuosity (λ). Both vary across species, brain regions and age [bib_ref] Diffusion in Brain Extracellular Space, Sykova [/bib_ref]. Diffusion can thus be described by the following partial differential equation, where D is the free diffusion coefficient and C(x, t) denotes concentration at time t and point x:
[formula] dCðx; tÞ dt ¼D � DCðx; tÞ; whereD ¼ D l 2 :ð1Þ [/formula]
Our model considers isotropic diffusion which is only limited by cellular borders on the synaptic level and by the boundaries of the simulation space. The latter was restricted to zero flux.
[formula] dCðx; tÞ dt ¼ 0; for x 2 @V ;ð2Þ [/formula]
where @V is the boundary of the volume. Note that for evaluations of DA transmission we omitted a small strip from the border of the simulation space (by default: 1μm from each side of the [50μm] 3 cube), in order to minimize artificial boundary effects. DA uptake (endocytosis) is realized via DAT. This process can be described by the following differential equation, the Michaelis-Menten (MM) enzyme kinetics
[formula] dCðx; tÞ dt ¼ À V max � Cðx; tÞ K m þ Cðx; tÞ ;ð3Þ [/formula]
where V max and K m denote the maximum uptake rate and the dissociation constant respectively. Our model was implemented using Fipy, a python-based finite-volume-method to solve the differential equation integrating diffusion (Eq 1) and uptake (Eq 3).
## Resolution
Our model comprises two levels of resolution, the large-scale tonic and the small-scale synaptic level. Separation into these two levels allows simulations to captures release, diffusion and uptake both in extra-synaptic space and inside synaptic clefts with high computational efficiency. The synaptic model accounts for the synaptic and near-synaptic space. It is implemented on a very narrow mesh in the range of 10 −3 μm and simulations are performed at very high temporal resolution of 10 −5 ms. For each synaptic terminal such a synaptic model is then embedded into the tonic model which encompasses the entire simulation space and is coarser in space (10 0 μm) and time (0.25ms) [fig_ref] Fig 7: Model [/fig_ref]. Extending the high resolution of the synaptic model to the entire simulation space would result in infeasible computational effort. Thus, in our tonic model, the synaptic model is used for the finite elements where a releasing terminal is located and only as long as additional DA molecules enter the extra-synaptic space. During every iteration of the tonic model this amount of DA molecules from the synaptic model is transferred into the respective finite element of the tonic model.
## Tonic model and volume transmission
In our approach, extra-synaptic DA is simulated within the tonic model. For non-synaptic terminals, the full content of a DA vesicle is released into the respective finite element instantaneously. For synaptic release, on the other hand, a fraction of the vesicle content is gradually released according to the calculations from the finer grained synaptic model as described below. Running the synaptic model with baseline concentrations between 0.001 and 500nM revealed that within this range DA spillover does not depend on the baseline concentration and that more than 97% of the released molecules have left the synaptic cleft within the first 0.25ms. For both synaptic and non-synaptic terminals, similarly to earlier point source models [bib_ref] DAncing past the DAT at a DA synapse, Cragg [/bib_ref] [bib_ref] Dopamine spillover after quantal release: Rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref] , DA then diffuses in all directions isotropically. In accordance with these models we further assume volumetric Michaelis-Menten uptake on the tonic level. Volume transmission is thus described by the following equation, combining diffusion and uptake: Under this assumption there exists an analytical solution of (Eq 4), which has been demonstrated by Nicholson [bib_ref] Interaction between diffusion and Michaelis-Menten uptake of dopamine after iontophoresis in striatum, Nicholson [/bib_ref]. Under normal conditions, tonic DA levels of approximately 4 − 50nM would satisfy this assumption, but large concentration variability exists within the extra-synaptic volume. High concentration values occur in the vicinity of release sites that would violate the assumption K m >>C. Instead, if K m <<C uptake approximates its maximum, i.e. V max .
## Synaptic model and synaptic transmission
Very high concentrations also appear inside the synaptic cleft after vesicle content release, although these high concentrations are transient. Importantly, with our numerical method of finite elements we can apply our model to any concentration values in the absence of an analytical solution.
DA can only escape the synaptic cleft where it is not constrained by cellular membranes. A point source model may not be appropriate here. Since in striatum approximately 35% of the nerve terminals are synaptic [bib_ref] Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat..., Descarries [/bib_ref] a more realistic model should incorporate this specific geometry. In our model the synapses appear as discs with a diameter of 150nm and a distance of 15nm between pre-and post-synaptic neuron [bib_ref] Efflux of dopamine from the synaptic cleft in the nucleus accumbens of..., Garris [/bib_ref]. DA release occurs centrally from the membrane of the pre-synaptic cell. [fig_ref] Fig 7: Model [/fig_ref] into the synaptic model towards the synaptic cleft. The disk-like synapse appears in red, as DA has just been released. The 'empty space' above and below the disk resemble the pre-and post-synapse.
The diffusion coefficient D � on the synaptic level might differ from volume transmission due the different physical and chemical conditions. However, in the absence of empirical measurements on the synaptic level, we used the same coefficient in both models.
To compare diffusion from non-synaptic terminals to synapses, we also designed a single half shell and released DA from its center, comparable to the synaptic case of two half shells and used identical parameters.
In addition, we wanted to test, if strongly pronounced DAT uptake at the pre-synaptic terminal can prevent DA from escaping the synaptic cleft. In this context, we implemented surface uptake, that is two-dimensional. We derived respective values for the maximum uptake capacity V sur max and the dissociation constant K sur m as follows: For a single axon, we assume a cylinder with an average length l = 467000μm [bib_ref] Single Nigrostriatal Dopaminergic Neurons Form Widely Spread and Highly Dense Axonal Arborizations..., Matsuda [/bib_ref] and diameter d = 2 � r = 0.25μm. The ratio between axonal volume (V A ) and axonal surface (S A ) is then 1LU 3 : 15LU 2 (LU = length unit) and the volume of a single axon is determined by:
[formula] V A ¼ p � r � l ¼ 22923:8089mm 3 [/formula]
Given that there are on average 370,000 terminals per axon and 0.104 terminals per μm 3 striatum (see references in [bib_ref] Influence of Phasic and Tonic Dopamine Release on Receptor Activation, Dreyer [/bib_ref] , we can conclude that per striatal volume unit there exist approximately 0.00644 axonal volume. Since, in striatum, the extrasynaptic volume fraction has been estimated to be 0.21 [bib_ref] Interaction between diffusion and Michaelis-Menten uptake of dopamine after iontophoresis in striatum, Nicholson [/bib_ref] , the ratio of extra-synaptic volume (V E ) to axonal cell volume is approximately 1: 0.03. According to the ratio V A : S A , it follows that 1LU 2 axonal membrane surface should have the same dopamine uptake capacity as approximately 2.174LU 3 extra-synaptic volume and therefore we assume:
[formula] V [/formula]
## Receptor occupancy
Similar to uptake via DAT, receptor occupancy can be described by the Michaelis-Menten kinetics Eq 3. Using V max = 1 for full occupancy and EC50 instead of K m we get:
Occupancyðx; tÞ ¼ Cðx; tÞ EC50 þ Cðx; tÞ EC50 is the effective concentration in equilibrium with 50% occupancy. Values for D1R and D2R are listed in S1 [fig_ref] Table Table: of model parameters [/fig_ref] This is a very simplistic model for the dynamics of receptor occupancy and has been recently advanced in a computational model. This model implements dynamic on-and off kinetics. We solved this equation numerically. Using the Eulermethod we obtained the following recursive equation for D2 occupancy (Occ):
Occðx; t þ 1Þ ¼ Occðx; tÞ þ dt � ðD2 on � Concðx; tÞ � ð1 À Occðx; tÞÞ À D2 off � Occðx; tÞÞ
Where D2 on and D2 off reflect the on-and off binding rate. For slow and fast binding kinetics these parameters were taken fromand [bib_ref] Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors, Patriarchi [/bib_ref] , respectively.
## Ornstein-uhlenbeck-process
The Ornstein-Uhlenbeck Process is a stochastic process used to model mean reverting behavior. X t is an Ornstein-Uhlenbeck process (OUP) if
[formula] dX t ¼ lðm À X t Þdt þ sdW t ; [/formula]
where μ is the mean reversion level to which the process tends to revert, λ is the mean reversion rate, σ measures the volatility of the process and W t is a Wiener process. The process has a stationary distribution X stat that is a normal distribution:
[formula] X stat � N ðm; s 2 =2yÞ [/formula]
We used an OUP to describe the course of spatially averaged extra-synaptic concentration, which fluctuates around a mean level, i.e. DA tone, according to the balance between release and uptake. The OUP was fitted in R using the mle() function.
# Limitations
Our model could be extended to some more aspects of the DA system. First, metabolism of DA that would regulate the impact of DA synthesis has not yet been considered. We expect this mechanism to have very little impact as it occurs at a sufficiently slow rate, not affecting transient changes in concentration [bib_ref] Real-time characterization of dopamine overflow and uptake in the rat striatum, Wightman [/bib_ref] and our simulations of up to 100 seconds. Second, the model could be advanced by a continuous firing frequency depending on D2 auto-receptor activation. This was for example demonstrated in [bib_ref] Mathematical model of dopamine autoreceptors and uptake inhibitors and their influence on..., Dreyer [/bib_ref]. Third, in our model, DA molecules bound to receptor sites have not been subtracted from the extra-cellular concentration. This should not affect steady-state analysis, but there is a potential effect during phasic DA release. Fourth, vesicle content should not be released at an instance but over the time of 10 −1 s [bib_ref] Striatal dopamine neurotransmission: regulation of release and uptake, David [/bib_ref].
Supporting information S1 [fig_ref] Table Table: of model parameters [/fig_ref] Ornstein-Uhlenbeck process. Maximum likelihood fits to default simulation (D1-D3) revealed three local maxima depending on initialization and one global maximum for the uptake inhibition simulation (UI). (PDF)
[fig] Fig 1: DA spiking patterns and transmission influence cognition and behavior. [/fig]
[fig] Fig 3: Single DA release event from synaptic terminal. For all plots the initial baseline concentration was 4nM except in B & D where the inital baseline concentration was 50nM. A-D: Release of 3000 DA molecules from synaptic terminal. White isolines indicate the concentration of 10, 100 and 1000nM. All color scales represent concentration in μM. DA quickly escapes the synaptic cleft in scenarios without uptake (A & B) and also with volume (homogeneous) uptake (C & D). E: Detail of plot C with additional isolines of 10, 100 and 1000nM for simulations with a combination of baseline concentrations between 0.001 and 500nM more than 97% of the released molecules have left the synaptic cleft within 0.25ms. [/fig]
[fig] Fig 4: Multiple DA release events from synaptic terminal. A: Simulated concentration [μM] with 3000 molecules released per event. B: Associated equilibrium receptor binding (Michaelis-Menten kinetics). C: Associated slow receptor binding. Percentage of binding increases with each release event inside and close to release site. At 2μm distance from the synapse center increments become insignificantly small. D: Associated fast receptor binding. Again, at 2μm distance from the synapse center receptor binding becomes insignificantly low. https://doi.org/10.1371/journal.pcbi.1008410.g004 [/fig]
[fig] Fig 5: Extra-synaptic DA. Volume-averaged DA concentration for the three different simulation settings. Fast oscillations occur in the DSS and SRS, while low frequency oscillations occur in the UIS. DSS: default setting simulation, SRS: stimulated release simulation, UIS: uptake inhibition simulation. https://doi.org/10.1371/journal.pcbi.1008410.g005 [/fig]
[fig] Fig 7: Model. A) Tonic (large-scale) and synaptic (small-scale) model. The tonic model contains more than 13000 finite elements that are associated with synaptic and non-synaptic terminals. For non-synaptic terminals the whole vesicle content is released into the respective finite element, while synaptic terminals refer to the synaptic model. B) DA diffusion from a synaptic terminal. Concentration 0.1 ms after release. https://doi.org/10.1371/journal.pcbi.1008410.g007 [/fig]
[table] 1 N X: e2fFEgF D2R ðCðeÞÞ |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } where C(e) is the concentration in the finite element e within the set of all finite elements FE and N is the number of finite elements. This order effect is caused by the non-linearity of F D2R (x), being either the Michaelis-Menten kinetics or dynamic slow or fast receptor binding. As an example, for the model of fast receptor binding, temporal1 = 57.1% while tempo-ral2 = 67.8%. This might have practical implication when interpreting imaging data that isFig 6. Extra-synaptic DA. DA concentration and D2R binding characteristics for four randomly chosen finite elements containing a synaptic terminal. Depicted at the bottom is DA concentration within the finite element of the tonic model, i.e. extra-synaptic DA concentration within a radius of~0.8μM from the terminal. Large concentration peaks indicate DA release from this synapse, while smaller peaks are caused by DA release from neighboring terminals.As the same amount of DA molecules is released every time, peak concentration in larger volumes (coarser resolutions) are smaller. The upper (dotted lines) and middle graphs (solid lines) illustrate slow D2R binding in a short time window for the SRS in synaptic (dotted) and peri-synaptic (solid) space. [/table]
[table] Table Table: of model parameters. Table splits into parameters taken from previous computational modeling studies and additional parameters for synaptic specifity. (PDF) S2 Table. Different Michaelis-Menten uptake parameters reported in the literature. Values differ across species and within a species striatum. (PDF) S3 Table. Physiological measurements of endogenous DA in humans. Mean values range between 23 and 72nM. (PDF) [/table]
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Cerebrospinal fluid leakage after cranial surgery in the pediatric population—a systematic review and meta-analysis
Background Cerebrospinal fluid (CSF) leakage is a common complication after neurosurgical intervention. It is associated with substantial morbidity and increased healthcare costs. The current systematic review and meta-analysis aim to quantify the incidence of cerebrospinal fluid leakage in the pediatric population and identify its risk factors. Methods The authors followed the PRISMA guidelines. The Embase, PubMed, and Cochrane database were searched for studies reporting CSF leakage after intradural cranial surgery in patients up to 18 years old. Meta-analysis of incidences was performed using a generalized linear mixed model. Results Twenty-six articles were included in this systematic review. Data were retrieved of 2929 patients who underwent a total of 3034 intradural cranial surgeries. Surprisingly, only four of the included articles reported their definition of CSF leakage. The overall CSF leakage rate was 4.4% (95% CI 2.6 to 7.3%). The odds of CSF leakage were significantly greater for craniectomy as opposed to craniotomy (OR 4.7, 95% CI 1.7 to 13.4) and infratentorial as opposed to supratentorial surgery (OR 5.9, 95% CI 1.7 to 20.6). The odds of CSF leakage were significantly lower for duraplasty use versus no duraplasty (OR 0.41 95% CI 0.2 to 0.9). Conclusion The overall CSF leakage rate after intradural cranial surgery in the pediatric population is 4.4%. Risk factors are craniectomy and infratentorial surgery. Duraplasty use is negatively associated with CSF leak. We suggest defining a CSF leak as "leakage of CSF through the skin," as an unambiguous definition is fundamental for future research.
# Introduction
Cerebrospinal fluid (CSF) leakage is one of the most common complications after neurosurgical intervention. CSF leakage is associated with substantial morbidity and increased healthcare costs [bib_ref] Costs of postoperative cerebrospinal fluid leakage: 1-Year, retrospective analysis of 412 consecutive..., Grotenhuis [/bib_ref]. One study found an average cost difference of €17.412 for patients with postoperative CSF leakage compared to patients without CSF leakage [bib_ref] Costs of postoperative cerebrospinal fluid leakage: 1-Year, retrospective analysis of 412 consecutive..., Grotenhuis [/bib_ref]. CSF leakage may lead to the development of a pseudomeningocele (PMC), wound healing problems requiring surgical re-closure, surgical site infection, meningitis, and pneumocephalus. CSF leakage rates reported in pediatric studies range between 0 and 38% [bib_ref] The supraorbital eyebrow approach in children: clinical outcomes, cosmetic results, and complications, Dlouhy [/bib_ref] [bib_ref] Time to resolution of symptoms after suboccipital decompression with duraplasty in children..., Hidalgo [/bib_ref] [bib_ref] Comparison of clinical and radiographic outcomes for posterior fossa decompression with and..., Jiang [/bib_ref] [bib_ref] Crescent posterior fossa durotomy for occipito-marginal venous sinus preservation: a pilot study, Panigrahi [/bib_ref] [bib_ref] The interhemispheric approach in children: our experience and review of the literature, Soleman [/bib_ref]. Definitions of CSF leakage vary in the existing body of literature.
The exact magnitude of the problem in children, however, is still unknown and may be larger than in adults for several reasons. First, almost half of all pediatric brain tumors resides in the posterior fossa, and posterior fossa surgeries are thought to be more prone to CSF leakage [bib_ref] Costs of postoperative cerebrospinal fluid leakage: 1-Year, retrospective analysis of 412 consecutive..., Grotenhuis [/bib_ref] [bib_ref] Risk factors for postoperative CSF leakage after elective craniotomy and the efficacy..., Hutter [/bib_ref] [bib_ref] Epidemiology of central nervous system tumors in childhood and adolescence based on..., Rickert [/bib_ref]. Second, intraventricular tumors are more common in the pediatric population [bib_ref] Epidemiology of central nervous system tumors in childhood and adolescence based on..., Rickert [/bib_ref]. Surgical opening of the ventricle may result in higher chance of postoperative CSF leakage [bib_ref] The prognostic impact of ventricular opening in glioblastoma surgery: a retrospective single..., Behling [/bib_ref]. A clear understanding of the incidence and risk factors of CSF leakage in the pediatric population is essential in the prevention of CSF leakage in children. The current systematic review and meta-analysis aim to address these issues.
# Methods
The authors followed the PRISMA guidelines [bib_ref] Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement, Moher [/bib_ref] for this systematic review and meta-analysis.
## Search strategy and selection criteria
Embase, PubMed, and Cochrane databases were searched until August 31, 2020 for studies reporting CSF leakage and related complications after intradural cranial surgery in patients up to 18 years old. The following search terms were used: ""children" OR "child" OR "pediatric" OR "paediatric" OR newborn OR "adolescent" OR "infant"" AND "neurosurgery" OR "craniotomy" OR "craniectomy" OR "cranial surgery" OR "tumor resection" AND ""cerebrospinal fluid leakage" OR "CSF leakage"" OR ""pseudomeningocele" OR "incisional leakage" OR "wound leakage" OR "surgical site infection" OR "surgical wound infection" OR "meningitis"" and relevant Mesh/Emtree terms. A modified version of the filter used to search pediatric studies in PubMed is used [bib_ref] Validation of search filters for identifying pediatric studies in PubMed, Leclercq [/bib_ref] (see Appendix A-C for the full search strings). Studies written in other languages than English, Dutch, German, French, Italian, or Spanish were excluded. Studies written before 1966 were excluded, as those are not included in the PubMed database. Laboratory studies, animal studies, cadaveric studies, case reports, small case series (N < 10), and literature reviews were excluded. Furthermore, studies on transsphenoidal surgery, skull base reconstructions, burr hole surgery (i.e., drainage of chronic subdural hematoma, needle biopsy), and primary CSF diversion surgeries were excluded. Two authors (EMHS and KMvB) independently screened all records from the database search on title and abstract to identify relevant articles. All remaining full text articles were screened on their eligibility for inclusion. A consensus meeting was held to reach agreement on the included articles.
## Data extraction
The following patient specific data items were extracted as proportion or mean per study: age, gender, compromised immune status, previous chemotherapy or radiotherapy, presence of hydrocephalus preoperatively, and CSF diversion surgery (endoscopic third ventriculostomy (ETV)/external ventricle drain (EVD)/ventriculoperitoneal (VP) shunt). The following surgical items were collected as proportion per study: site of durotomy (infratentorial/supratentorial), craniotomy versus craniectomy, indication for surgery (i.e., tumor resection or Chiari decompression), ventricular opening (yes/ no), use of sealant (yes/no), use of duraplasty (yes/no), and whether a "watertight" closure of the dura was attempted or not. The following outcome parameter was collected: proportion of patients with CSF leakage (based on the individual study's definition).
Study quality was assessed according to the National Heart, Lung and Blood Institute of National Institutes of Health (NIH) quality assessment tool for case series studies. Studies with more than 2 items with high risk for bias or unclear risk for bias were classified as poor quality. Studies with a maximum of 2 items with high risk for bias or unclear risk for bias were judged to be of fair quality. Studies with no items with high risk of bias and a maximum of 1 item with unclear risk of bias were deemed of good quality.
# Statistical analysis
A meta-analysis of the incidence of CSF leakage was performed using a generalized linear mixed model. Heterogeneity of the data across studies was determined using Higgins I 2 [bib_ref] Quantifying heterogeneity in a meta-analysis, Higgins [/bib_ref].
The primary outcome measure in this study is the incidence of CSF leakage with 95% confidence interval (CI). Subgroup analyses were performed for the separate surgical indications Chiari decompression (with dural opening) and posterior fossa tumor surgery. Secondary outcome measures are the odds ratio (OR) for CSF leakage for craniotomy versus craniectomy, supratentorial versus infratentorial surgery, cases in which a duraplasty was used or not, and studies in which watertight closure was attempted in all cases or not. Finally, three sensitivity analyses were performed (1) for studies of high quality only, [bib_ref] Analysis of CSF shunting procedure requirement in children with posterior fossa tumors, Bognár [/bib_ref] for studies of > 50 patients only, and (3) including the study of Jiang et al (see Results section) [bib_ref] Comparison of clinical and radiographic outcomes for posterior fossa decompression with and..., Jiang [/bib_ref].
All analyses were performed using SAS version 9.4 (SAS Institute Inc).
# Results
The database search yielded 2123 articles of which 26 were included in this systematic review [fig_ref] Figure 1: Flowchart of study selection [/fig_ref]. Twenty-one articles were included in the meta-analysis, as four articles had to be excluded because of overlapping study populations (the article discussing the largest sample size was included) [bib_ref] The surgical and natural morbidity of aggressive resection for posterior fossa tumors..., Cochrane [/bib_ref] [bib_ref] MRI study of the natural history and risk factors for pseudomeningocoele formation..., Gananalignham [/bib_ref] [bib_ref] Surgical mortality and selected complications in 273 consecutive craniotomies for intracranial tumors..., Lassen [/bib_ref] [bib_ref] The interhemispheric approach in children: our experience and review of the literature, Soleman [/bib_ref]. Additionally, the study of Jiang et al. [bib_ref] Comparison of clinical and radiographic outcomes for posterior fossa decompression with and..., Jiang [/bib_ref] was excluded from the meta-analysis, because they unconventionally diagnosed CSF leak when "drainage from the drainage catheter was clear and transparent" in their patient population in which placement of a low-vacuum suction wound drain was part of the surgical protocol.
A total of 2929 patients were included, who underwent a total of 3034 intradural cranial surgeries, as some patients had more than one surgery. [fig_ref] Table 1: Overview of included studies [/fig_ref] provides an overview of study characteristics.
Most included articles report retrospective consecutive case series. One study was a randomized controlled trial, in which patients were randomized for a crescent incision versus a Yshaped incision of the dura [bib_ref] Crescent posterior fossa durotomy for occipito-marginal venous sinus preservation: a pilot study, Panigrahi [/bib_ref]. Ten studies were of poor quality, based on unclear description of the surgical procedure and poor definition of the outcome measure CSF leakage and either insufficient reporting of the follow up duration or lack of description of statistical methods. Twelve studies were of fair quality, again largely based on a lack of adequate definition of the outcome measures and inadequate reporting of statistical methods. Four studies were of good quality; these studies all provide a clear definition of the outcome measure CSF leakage. A detailed description of the quality assessment is presented in Supplementary Information 1.
## Primary outcome measure
The overall incidence of CSF leakage was 4.4% (95% CI 2.6 to 7.3%) [fig_ref] Figure 2: Forest plot incidence of CSF leakage [/fig_ref].
Subgroup analyses for type of surgery could only be performed for Chiari decompression (with dural opening) and posterior fossa tumor surgery, as only these indications were investigated in sufficient studies. CSF leakage rates in these subgroups were 3.4% (95% CI 1.3 to 8.7%) after Chiari decompression, and 8.0% (95% CI 5.2-12.0%) after posterior fossa tumor surgery. All analyses showed substantial heterogeneity. An overview of outcomes for the primary outcome measure and subgroup analyses can be found in [fig_ref] Table 2: Incidence of CSF leakage based on generalized linear mixed model [/fig_ref].
## Secondary outcome measures
The highest percentage of CSF leakage was found in patients undergoing craniectomy (10.3%, 95% CI 4.3% to 22.7%), with an OR of 4.7 (95% CI 1.7 to 13.4) compared to craniotomy (2.4%, 95% CI 1.0% to 5.4%). A CSF leakage rate of 6.4% (95% CI 4.1 to 10.0%) was found for infratentorial surgery in contrast to 1.2% (95% CI 0.4 to 3.7%) for supratentorial surgery (OR 5.9, 95% CI 1.7 to 20.6).
In patients with a duraplasty for dural closure, the incidence of CSF leakage was 5.3% whereas patients without a duraplasty had a significantly higher incidence of 11.8% (OR 0.4, 95% CI 0.2 to 0.9).
In studies in which watertight closure was attempted in all cases, the CSF leakage rate was 2.3% as compared to 6.4% patients in studies in which watertight closure was not attempted in all cases (OR 0.3 95% CI 0.1 to 2.3). An overview of the secondary outcome measures is presented in [fig_ref] Table 3: Overview secondary outcome measures [/fig_ref].
# Sensitivity analysis
Separate analyses were performed: (1) for studies of high quality only, (2) for studies of > 50 patients only, and (3)
# Discussion
This meta-analysis shows that the overall incidence of CSF leakage after intradural cranial surgery in the pediatric population is 4.4%. Infratentorial as opposed to supratentorial surgery, and craniectomy as opposed to craniotomy are significant risk factors for CSF leakage (OR 5.9 and 4.7, respectively). These results underline the relevance of CSF leakage in clinical practice. In the pediatric population, specifically, the burden of additional treatment that may be required for CSF leakage or related complications is substantial. In studies reporting data on treatment of CSF leakage, a total of 37 out of 114 patients with a CSF leak were treated with a ventriculoperitoneal shunt [bib_ref] Analysis of CSF shunting procedure requirement in children with posterior fossa tumors, Bognár [/bib_ref] [bib_ref] An analysis of factors determining the need for ventriculoperitoneal shunts after posterior..., Culley [/bib_ref] [bib_ref] Surgical procedures for posterior fossa tumors in children: does craniotomy lead to..., Gananalignham [/bib_ref] [bib_ref] Factors predicting the need for cerebrospinal fluid diversion following posterior fossa tumor..., Gopalakrishnan [/bib_ref] [bib_ref] Cerebrospinal fluid disturbances after 381 consecutive craniotomies for intracranial tumors in pediatric..., Hosainey [/bib_ref] [bib_ref] Toward a simpler surgical management of Chiari I malformation in a pediatric..., Krieger [/bib_ref] [bib_ref] A single-center retrospective descriptive cohort study of 211 pediatric patients: cerebrospinal fluid..., Kushel [/bib_ref] [bib_ref] Microsurgical keyhole approach for Mmiddle fossa arachnoid cyst fenestration, Levy [/bib_ref] [bib_ref] The bifrontal olfactory nerve-sparing approach to lesions of the suprasellar region in..., Srinivasan [/bib_ref] [bib_ref] Cerebrospinal fluid (CSF) leak and pseudomeningocele formation after posterior fossa tumor resection..., Steinbok [/bib_ref].
There is a wide range of reported CSF leakage rates (between 0.0 and 38.0%) [bib_ref] The supraorbital eyebrow approach in children: clinical outcomes, cosmetic results, and complications, Dlouhy [/bib_ref] [bib_ref] Time to resolution of symptoms after suboccipital decompression with duraplasty in children..., Hidalgo [/bib_ref] [bib_ref] Comparison of clinical and radiographic outcomes for posterior fossa decompression with and..., Jiang [/bib_ref] [bib_ref] Crescent posterior fossa durotomy for occipito-marginal venous sinus preservation: a pilot study, Panigrahi [/bib_ref]. This may have several reasons. First, there is a large variability in the definition of CSF leakage. Moreover, only four out of 26 studies actually described their definition of CSF leakage. Secondly, the wide incidence range may be due to the different types of surgery included across studies (i.e., supra orbital eyebrow craniotomy, epilepsy surgery, posterior fossa tumor surgery).
No separate analyses could be performed per type of surgery for all these categories, nor for the risk factors like age, immune status, previous chemotherapy or previous radiotherapy, CSF diversion surgery, preoperative hydrocephalus, ventricular opening, and sealant use as Our meta-analysis shows that the proportion of CSF leakage is the highest in the subgroup of patients undergoing craniectomy (10.3%). This difference may be explained by the lack of extra counter pressure that is otherwise provided by the replaced bone flap [bib_ref] Surgical procedures for posterior fossa tumors in children: does craniotomy lead to..., Gananalignham [/bib_ref]. Replacement of the bone flap decreases the continuous short increase and decrease in dural stress caused by the triphasic pulsations of cerebrospinal fluid [bib_ref] Analysis of the cerebrospinal fluid pulse wave in intracranial pressure, Cardoso [/bib_ref]. Furthermore, the bone flap may reduce the dead space which is created after detachment of the muscles in the suboccipital region and support their reattachment to the replaced bone flap, so that collection of CSF in this space is limited and pseudomeningocele is prevented [bib_ref] Surgical procedures for posterior fossa tumors in children: does craniotomy lead to..., Gananalignham [/bib_ref].
This meta-analysis finds a CSF leakage rate of 3.4% after Chiari decompression surgery. The relatively low leakage rate in this population is surprising considering the abovementioned surgical risk factors (infratentorial surgery and craniectomy) as this population essentially represents a combination of these two items.
On the contrary, a high leakage rate in posterior fossa tumor surgery (8.0%) is found. This type of surgery may be prone for leakage because pediatric brain tumors frequently reside in the fourth ventricle, requiring opening of the telovelar membrane and leaving a wide-open ventricle. Furthermore, postoperative hydrocephalus may contribute to the increased incidence of CSF leakage in this population [bib_ref] Graft dural closure is associated with a reduction in CSF leak and..., Hale [/bib_ref].
The effect of watertight closure was not significant in this study. However the effect in this analysis may be limited because it was only possible to compare studies in which all cases were closed in watertight fashion to those in which not all cases were closed with this aim (the dura was left open in all cases in one study [bib_ref] Toward a simpler surgical management of Chiari I malformation in a pediatric..., Krieger [/bib_ref] , in other studies 10-89% [bib_ref] Effects of ventricular drainage and dural closure on cerebrospinal fluid leaks after..., Muszynski [/bib_ref] [bib_ref] Posterior cranial fossa surgery in 454 children: comparison of results obtained in..., Parizek [/bib_ref] of cases were not closed in a watertight manner).
CSF leakage was significantly less frequent in patients in whom a duraplasty was performed (OR 0.4). This may reflect that when careful attention is paid to optimal closure of the dura with or without augmentation such as duraplasty or sealants, the risk of CSF leakage is reduced. No distinction has been made in this study between autologous or synthetic material. A study by [bib_ref] Graft dural closure is associated with a reduction in CSF leak and..., Hale [/bib_ref] indicates that graft dural closure may furthermore be protective against hydrocephalus and wound infection in patients undergoing posterior fossa tumor surgery [bib_ref] Graft dural closure is associated with a reduction in CSF leak and..., Hale [/bib_ref].
Compared to adults, the incidence of CSF leakage found in children is considerably lower, which is contrary to our expectations considering the high number of craniectomies and infratentorial surgeries included. A recent meta-analysis has found that the rate of CSF leakage in adults is 8% [bib_ref] Effectiveness of dural sealants in prevention of cerebrospinal fluid leakage after craniotomy:..., Kinaci [/bib_ref]. As is the case in pediatric literature, the definition of CSF leakage reported in studies on adults is not uniform either. This may explain the discrepancy between the incidence of CSF leakage in both populations. Another factor may be that the metaanalysis on adults includes studies in which sealants use was compared, this patient population may, therefore, be one which is more prone to CSF leakage, considering a substantial number of studies selected patients based on intraoperative CSF leakage. Moreover, this may be a result of increased flexibility of the tissues in children compared to adults allowing for better surgical closure of the dura and skin layers.
This meta-analysis is subject to several limitations. Most importantly, the studies included are heterogenous in their definitions of the outcome measure, population, and followup duration. The majority of studies included in this metaanalysis do not clearly define the outcome measure CSF leakage. Those that do, use a variety of definitions, for example, being "CSF leak through the skin" [bib_ref] Cerebrospinal fluid (CSF) leak and pseudomeningocele formation after posterior fossa tumor resection..., Steinbok [/bib_ref] and "all CSF leaks requiring surgical intervention" [bib_ref] Cerebrospinal fluid disturbances after 381 consecutive craniotomies for intracranial tumors in pediatric..., Hosainey [/bib_ref]. This obviously results in differences in outcome, as is reflected by the I 2 -values found in the meta-analyses. It was not possible to adopt a specific definition of CSF leakage for this meta-analysis, as too few publications mention this. One study has been excluded because it included clear fluid in a low-vacuum suctioning wound drainage system as CSF leakage, resulting in an outstandingly high CSF leakage rate of 38.0% [bib_ref] Comparison of clinical and radiographic outcomes for posterior fossa decompression with and..., Jiang [/bib_ref]. In a sensitivity analysis including this publication, we found an overall CSF leakage rate of 4.8% (4.4% without), indicating this study has no clinically meaningful influence on the overall outcome. Secondly, the risk factor analyses for duraplasty use and watertight closure were based on a limited number of studies. Therefore, caution should be applied in generalizing these results.
Thirdly, we did not exclude patients with subdural-toextracranial implants, such as subdural grid electrodes, which may influence CSF leakage, but the total influence of this population on the overall results is expected to be minimal.
Fourth, the results of the risk factor analysis are potentially influenced by confounding. This is inherent to the design of the included publications and the fact that obtained data do not allow correction for potential bias. Future research should further investigate potential risk factors in a multivariate analysis.
Lastly, quality assessment identified only 3 "good quality" studies out of the 26 included in the meta-analysis, compromising quality for the reported outcome measure. The sensitivity analysis shows a higher incidence of CSF leakage in studies of good quality, 7.4% vs. 4.4% found in all studies which may indicate that the CSF leakage rate in this study may be an underestimation of the true CSF leakage rate.
Despite these limitations, this meta-analysis provides a representative overview of the CSF leakage rate and associated risk factors reported in the current body of literature. Moreover, it emphasizes the need for a uniform definition and future studies evaluating CSF leakage and preventative strategies in the pediatric population. CSF leakage may include both incisional leakage and pseudomeningocele (PMC). Incisional CSF leakage is defined as leakage of CSF through the skin, whereas a PMC is an extradural collection of CSF under the skin [bib_ref] Management of pseudomeningocele following neurotologic procedures, Mehendale [/bib_ref]. Although PMC in the absence of incisional CSF leakage can cause symptoms such as, intracranial hypotension, aseptic meningitis, pain, and psychological distress, the condition is often self-limiting [bib_ref] Management of pseudomeningocele following neurotologic procedures, Mehendale [/bib_ref] [bib_ref] Management of postoperative pseudomeningoceles: an international survey study, Tu [/bib_ref]. Describing and quantifying symptomatic PMC can be difficult because the diagnosis is subjective in contrast to incisional CSF leakage. Therefore, it should be considered a separate entity.
# Conclusions
The overall CSF leakage rate after intradural cranial surgery in the pediatric population is 4.4%. The highest leakage rate is found in patients undergoing a craniectomy. Infratentorial surgery is also associated with higher incidence of CSF leakage, whereas the use of a duraplasty is negatively associated with CSF leak. We emphasize the need for a uniform and clinically meaningful definition of CSF leakage, suggesting "leakage of CSF through the skin."
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00381-021-05036-8.
Availability of data and material The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
[fig] Figure 1: Flowchart of study selection [/fig]
[fig] Figure 2: Forest plot incidence of CSF leakage [/fig]
[table] Table 1: Overview of included studies [/table]
[table] Table 2: Incidence of CSF leakage based on generalized linear mixed model [/table]
[table] Table 3: Overview secondary outcome measures [/table]
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Current material engineering strategies to prevent catheter encrustation in urinary tracts
A B S T R A C TCatheters and ureteric stents have played a vital role in relieving urinary obstruction in many urological conditions. With the increasing use of urinary catheters/stents, catheter/stent-related complications such as infection and encrustation are also increasing because of their design defects. Long-term use of antibiotics and frequent replacement of catheters not only increase the economic burden on patients but also bring the pain of catheter replacement. This is unfavorable for patients with long indwelling catheters or stents but inconvenient to replace. In recent years, some promising technologies and mechanisms have been used to prevent infection and encrustation, mainly drug loading coatings, functional coatings, biodegradable polymers and metallic materials for urinary devices. Obvious effects in anti-encrustation and anti-infection experiments of the above strategies in vivo or in vitro have been conducted, which is very helpful for further clinical trials. This review mainly introduces catheter/stent technology and mechanisms in the past ten years to address the potential impact of antiencrustation coating of catheter/stent materials for the prevention of encrustation and to analyze the progress made in this field.
# Introduction
Urological devices such as catheters and stents are commonly utilized by urologists to treat obstructions through temporary or permanent drainage. The most commonly used catheter today is the Foley catheter, which was introduced by Dr Frederick B Foley in the mid-1930s and is used in hospitals and the community. In 1949, Herdman first described ureteric stents. Thomas Hepperlen and Roy Finney proposed single-J and double-J ureteral stents in the 1970s, which have been in use ever since. The current usage amount is huge and increasing year by year. The indwelling devices mentioned above often lead to complications mainly about pain, discomfort, urinary tract infection (UTI) and encrustation. These complications are not only associated with increasing health care costs but also lead to local or systemic symptoms, sepsis, renal failure, and even death.
Biofilm, made up of bacteria, reducing the antibiotic sensitivity, make UTI easily to occur and difficult to treat. Gram-positive bacteria such as Staphylococcus aureus (S. aureus) and Gram-negative bacteria include Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa), which are the most prevalent bacteria. Candida species also grow and reproduce well in the urinary tract microenvironment. Biofilms can also lead to catheter and stent encrustation, especially infections by P. mirabilis, K. pneumoniae, and P. aeruginosa, which can generate urease. Once urea decomposes into ammonia by urease, the pH in urine increases, which causes cations such as Ca 2þ and Mg 2þ to deposit on the biofilm to form crystals(Scheme 1). Crystals were deposited inside the lumen, which would reduce adequate urine flow and even completely block the lumen, leading to impaired renal function. Moreover, it would deposit on the stent/catheter surface, making stent/catheter difficult to be removed. The suggestions for preventing UTI and encrustation include shorting catheter/stent use, sterile operation, and drinking plenty of water and antibiotics. However, these could be cumbersome, ineffective, and short acting.
In this review, an overview of anti-encrustation and anti-infection strategies was presented by focusing on drug loading coatings, functional coatings, biodegradable urinary devices and metallic materials for urinary devices. Current studies on this topic are mainly based on antimicrobial drug loading on urinary devices. Based on the development of biomaterials and catheter preparation technology, strategies of functional coating on urinary devices, biodegradable urinary devices and metallic materials for urinary devices have attracted increasing attention,
which are exactly what we need to focus on after introducing drug loading on urinary devices. Finally, we provide a forecast on this subject based on existing issues and future developments in biofunctionalized applications. We expect this review to not only offer researchers with a clear background of catheter/stent-related complications and research status but also inspire the development of new strategies for inhibiting the occurrence of encrustation and infection and to reduce the pain of patients and the economic burden on families and society. Antibiotics are effective against bacteria that cause urinary tract infections, and long-term oral or intravenous antibiotics can lead to bacterial drug resistance. Local rather than the systemic of drug use can avoid a certain degree of the development of drug resistance. Triclosan has been widely used for several decades. It can target the enoyl-acyl carrier protein reductase (EACPR), which is a highly conserved enzyme for membrane maintenance involved in fatty acid synthesis in bacteria. Therefore, triclosan can kill bacteria protected by biofilms. Some studies also found that triclosan can decrease the expression and activity of various inflammatory factors. Not only is there no evidence that over decades of extensive use of triclosan can develop antibiotic-resistant organisms, but there is also proof that triclosan can reduce the usage of antibiotics and decrease bacterial drug resistance. Tian and his colleagues patented a synthesis method of nontoxic waterborne biodegradable polyurethanes (WBPU). They produced triclosan-loaded WBPU and tested the bacteria by loading WBPU with different concentrations of triclosan in vitro. The results show that it can inhibit Proteus mirabilis. Proteus mirabilis can form biofilms and increase urinary pH, leading to crystal deposits on the catheter. In an in vitro bladder model, triclosan-loaded WBPU prolonged catheter blockage time and slowed biofilm formation. In this research, no studies have been carried out on other bacteria that cause biofilms and calculi, although there is no evidence that triclosan produces bacterial resistance. Thus, we should also be aware of its resistance.
Belfield and his colleagues produced an antimicrobial urinary catheter (AUC) impregnated with rifampicin, triclosan, and sparfloxacin [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref]. The appearance of the AUC segments without soaking in artificial urine (AU) was different from that of silicone catheters over a total of 2 weeks [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref]. In the static model of encrustation with P. mirabilis, obviously less phosphate was observed on the surface of the AUC segments compared to the control catheter segments at 48, 72 and 96 h [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref]. This suggests that AUC can reduce encrustation produced by P. mirabilis. In the 26-day flow model, SEM showed fewer crystal deposits on AUC lumens compared to the control group when inoculated with P. mirabilis [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref]. They use multiple antimicrobials of different classes to prevent drug resistance, since the bacteria are very unlikely to be resistant to two or more antibiotics at the same time. In the 28-day test period, compared with the control group, none of the AUCs were blocked. As showed in [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref] , during the 12 consecutive weeks of antibacterial experiment, the bacterial (methicillin-resistant Staphylococcus aureus and E. coli) colony of AUC group was significantly lower than control group. Which indicated that coating also prevented methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, carbapenemase-producing E. coli, and extended-spectrum beta-lactamase-producing E. coli adhesion in 12 consecutive weeks [fig_ref] Scheme 1: Schematic of catheter/stent-related infection and encrustation [/fig_ref]. All impregnated antimicrobials have been widely used in the clinic for decades, and their safety has been confirmed. What inspires us is the data from a clinical trial of tolerability and acceptability, which shows safety and no evidence of inflammation and toxicity in 30 patients with AUCs.
## Silver ion incorporation
Silver is an effective antimicrobial agent approved by the Food and Drug Administration (FDA) to prevent UTI. Because of its oligodynamic activity, silver can destroy bacterial membranes, cause cell protein denaturation, and inhibit bacterial and fungal reproduction. The form of silver polymer coating is mainly about silver alloy (with palladium or gold), silver-containing polymers, and silver nanoparticles (AgNPs). However, the great mass of silver-coated catheters showed ineffective anti-infection and encrustation effects. The reason may lie in the low capability of silver release and the formation of biofilms, which further limits silver release.
Polytetrafluoroethylene (PTFE) has excellent nonstick ability and reduces the silver release limitation caused by biofilms. A new coating has been developed based on this property: silver-PTFE nanocomposite coating. In the in vitro bladder model, the Ag-PTFE-coated catheter significantly prolonged the time of bacteriuria from an average of 6 days (control) up to 41 days (P < 0.005). A significantly longer duration of anti-encrustation was found in coated catheters with initial concentrations of 10 6 and 10 3 cells/mL in the bladder. Images and SEM showed that the eye hole and the lumen of uncoated catheters were heavily blocked, and little encrustation was visible on the silver-PTFE nanocomposite-coated catheter. The MTT assay showed no significant cytotoxicity compared to silicone catheters (P < 0.1). In the cytotoxicity assay, there were no dramatic differences in cell growth among the blank wells, wells with uncoated catheter segments and wells with coated catheter segments. The MTT assays showed that, comparable to the control silicone samples, cell viability in silver-PTFE nanocomposite-coated catheter segments were higher than 90% (P > 0.1). It has the advantage of anti-encrustation properties, but further animal studies should be performed to verify its further performance. PDPA, which is a surface anchor, can be coated onto almost any material surface, including silicone. Poly(sulfobetaine methacrylate-co-acrylamide)[poly(SBMA-co-AAm)] can inhibit the formation of biofilms and adhesion of bacteria. selected as the final layer of the coating. AgNP-PDPA, bilayers with grafted poly (SBMA-co-AAm) (P3) as an anti-biofilm and anti-encrustation coating, was successfully developed by Wang and his colleagues. This coating can effectively reduce bacterial adhesion and biofilm formation. In the in vitro model, compared with the Dover™ silver-coated catheter (time of resistant encrustation 7 days), the P3-coated catheter can resist crystal deposits for 45 days. In a mouse model, E. coli biofilm was detected on only one out of four P3-coated catheters; however, biofilm formation was shown in five out of six uncoated catheters. Compared to the Dover™ catheter, the deposition of calcium and magnesium in the bladder of pigs with a P3-coated catheter was reduced by 1.8 times. No cytotoxicity was observed in pigs or mice.
Hydrophilic poly (p-xylylene) PPX-N, a biocompatible polymer, increases the silver ion release rate and improves the wettability of the catheter. Thus, the silver/hydrophilic PPX-N coating can inhibit bacterial adhesion, prevent the biofilm formation of E. coli and S. cohnii, and reduce encrustation compared with the bacteria-only control in vitro model. Although it showed excellent resistance to biofilm and crystal deposits, its cytotoxicity and effectiveness in an in vivo model need further investigation.
## Silver and antibiotic hybrid coating
Silver combined antibiotics can offer a synergistic antibiotic effect, and the coating combined silver and antibiotics not only improve the ability of antibiotic effect but also help address antibiotic resistance. Norfloxacin, a fluoroquinolone with a hydrophobic nature that has broad-spectrum antimicrobial properties, is used to treat UTI caused by gram-positive and gram-negative bacteria. This kind of antibiotic can inhibit the DNA-gyrase enzyme, which is essential in bacterial DNA synthesis. The ionized form of silver is also a broad-spectrum antibacterial agent. It rarely develops into bacterial resistance since it can attack broad sites within the bacterial cell. To avoid silver nanoparticle aggregation, they were coated with tetraether lipids (TEL). TEL-coated silver nanoparticles were distributed in a hydrophobic film of poly (lactic-co-glycolic acid) (PLGA) loaded with norfloxacin. This kind of coating is called tetraetherlipid-Ag-norfloxacin-polylactid (TANP). The burst release of norfloxacin from the PLGA film was observed in the first few days. The burst release rate in the first few days was approximately 60%, and the slow-release rate in the next 50 days was approximately 40%. In vitro bacterial and encrustation tests showed that, compared with the uncoated catheter, the coating could inhibit the adhesion of bacteria effectively and decrease the viability of bacteria. It can also efficiently reduce the encrustation on the surfaceand E). Of note, they established an in vitro encrustation model to evaluate the multifunctional biomaterial effect on biofilm formation and crystallization. They grafted the coating onto a silicone (SIK) catheter and polyurethane (PUR) ureteral stent and used this model to carry out in vitro crusting experiments over 14 days. The concentration of dead bacteria observed on the TANP-coated surfaces was much higher than that on the control surface. On average, the concentration increased from~12% to~41% for PUR and from 27% to~67% for SIK samples. The volume of biofilm is reduced by 75%. The study shows that the time of norfloxacin release from PLGA is up to 52 days, which is very beneficial for patients with long-term indwelling catheters.
Silver sulfadiazine is a broad-spectrum bactericidal antimicrobial that can effectively treat gram-positive and gram-negative bacteria by destroying DNA replication, directly modifying the lipid cell membrane, and forming free radicals. This antibiotic was previously used to treat burns and can inhibit bacterial colonization. In 2017, a clinical trial comparing silver sulfadiazine-coated ureteral stents with bare stents for 4 weeks showed that silver sulfadiazine-coated stents decreased bacterial colonization more than control stents on the stent surface, and the difference in stent culture between the control group and antibacterial group was statistically significant (p ¼ 0.054). Due to the short duration of stenting and the relatively small sample size, it is impossible to prove its effect on biofilm formation, encrustation or infection development in patients with long-term stents. The silver and antibiotic mixed coating has been proven to have the effect of antibacterial adhesion both in vitro and in vivo. Our purpose is not only to study its anti-adhesion effect but also to study its anti-encrustation ability. There is no related research in the above clinical trials, which may be a focus that we need to study in the future.
## Antibacterial peptide coating
Antimicrobial peptides (AMPs) are a set of natural host defense peptidesthat have been revealed to have various functions, including controlling immune responses, stimulating the accumulation of immune cells at the site of infection, anti-inflammatory and neutralizing endotoxin properties, stimulating angiogenesis and accelerating wound repair. Due to their unique structure of cationic and amphipathic residues, AMPs can target the lipopolysaccharide layer of the cell membrane to disrupt the cell wall and then kill bacteria. In addition, AMPs have the abilities to increase the susceptibility to the antimicrobial drug by entering into the bacteria and interact with the lipid bilayer to form a transmembrane pore and to inhibit the formation of biofilms that are resistant to most antibiotics. Other killing bacterial mechanisms of AMPs include inhibiting intracellular functions, extracellular polymeric organisms, intracellular translocation, and the synthesis of DNA/RNA/protein. As one of the most promising alternatives to antibiotics, AMPs have shown promise to improve the outcome of medical devices with a high risk of infections.
Covalent immobilization of AMPs on a surface as an antimicrobial coating has been used as a strategy to prevent the need for further and extra systemic treatment. One covalent immobilization strategy for surface modification was a bioinspired chemistry based on the oxidative self-polymerization of dopamine (DPA) developed by Lee et al. in 2007. This was inspired by the coexistence of catechol and amine in mussel foot proteins and by synergistic salt displacement of catechol and amine at the solid-liquid interface to form a synergistic interface adhesion. Through catechol-metal coordination, π-π interactions, electrostatic interactions, covalent reactions and hydrogen bonding, the deposition of polydopamine (PDPA) can occur on almost all types of materials. This mussel-inspired chemistry has the property of allowing functionalization of biomolecules on a variety of biomaterials through amino-or thiol-mediated Michael addition. Metal-catechol-assisted mussel chemistry was used for a typical AMP (RWRWRWC-NH 2 ) with thiol group functionalization on commercially available stents. Both AFM characterization and roughness compared with uncoated stents showed that the coating was successfully grafted onto the surface of stents. In this work, Yao et al. evaluated its bactericidal and anti-crusting ability both in vitro and in vivo. In the in vitro antibacterial experiment, the stents with AMP coating inhibited E. coli, S. aureus and P. mirabilis growth and biofilm formation in situ; even after 2 weeks, the coating also showed good antibacterial effects. In addition, the coating not only had good biocompatibility but also reduced the deposition of struvite and hydroxyapatite crystals in 2 weeks of in vivo experiments. This study developed a safe, stable, and effective antibacterial coating on urinary tract medical devices for long-term bacterial inhibition and encrustation prevention.
Another covalent immobilization strategy was (1-mercapto-11undecyl)-(tetra(ethylene glycol) (EG4), 1,1 0 -carbonyldiimidazole (CDI), and self-assembled monolayers (SAMs) immobilized Chain201D AMP (AMP-CDI-EG4-SAMs). In an in vitro antibacterial experiment, the AMP-CDI-EG4-SAM coating was highly effective against strains relevant to urinary catheter-associated infections (including clinical bacterial strains such as E. coli, K. pneumoniae, Enterobacter cloacae (E. cloacae), P. aeruginosa, A. baumannii and S. aureus and clinical strains of yeasts such as Candida albicans (C. albicans), Candida glabrata (C. glabrata) and Candida parapsilosis (C. parapsilosis)) and was stable over a wide range of temperatures (up to 45 C), pH values (4-9) and salt concentrations (50, 100 and 200 mM NaCl). This antibacterial property has potential anti-crusting ability, and high stability is beneficial to the long-term use of indwelling catheters or stents.
Except for AMP-CDI-EG4-SAMs, the AMP modification strategy on the surface of PU stents attached by novel polymer-based tethering was also an effective surface modification strategy, which not only had nonfouling characteristics but also provided specific flexible binding sites for peptide conjugation. In vitro, the use of AMP (E6) conferred excellent antimicrobial activity toward P. aeruginosa, S. aureus and S. saprophyticus while providing strong biocompatibility. Importantly, as observed by imaging luminescent-tagged bacteria using the Interactive Video Information System (IVIS), the polymer brush-AMP coating showed a marked reduction in bacterial adhesion in a mouse CAUTI model compared with the control group at 1 and 7 days. This study provides a method of AMP immobilization to prevent and limit the occurrence of CAUTIs.
Three strategies of AMP immobilization as coatings above for medical devices, including ureteric stents or catheters, provide excellent candidates for further development owing to their excellent anti-adhesive, antimicrobial and biocompatible properties against bacterial adhesion, colonization and infection. Owing to the natural properties of biofilm formation inhibition and the excellent antibacterial and anti-encrustation properties of coatings, antibacterial peptide coatings are expected to become a potential advantage of anti-encrustation urinary catheters or stents for clinical use, which needs further investigation for verification.
## Drug-loaded polymer modification
Poloxamers, a series of copolymers, contain a central hydrophobic poly segment and two polyethylene oxide (PEO) chains. The central hydrophobic poly segment is used to load and release the fluoroquinolone antibiotic ofloxacin, and PEO chains keep the bacteria away from the surface. Poloxamer 188 molecules were functionalized However, 10% p(DMP-co-HEMA) has a higher ultimate tensile strength and Young's elastic modulus. After suspended in artificial urine for a week, the content of calcium ions on the 10% p(MMP-co-HEMA) coating decreased by 87% and magnesium by 92% compared with the poly (hydroxyethyl methacrylate) (p(HEMA)) control. The 10% p(MMP/DMP-co-HEMA) coating reduced the surface adherence of E. coli by more than 90% relative to the control p(HEMA). However, in the drug release experiment, they released earlier and more than the control group, so it was difficult to be of long-term antibacterial.
## Bacteriophage coating
Bacteriophages are natural predatory viruses that replicate inside bacteria. In particular, lytic phages can cause the bacterium to lyse by replicating. Bacteriophages have the advantages of high strain specificity, self-replication and easy genome editing, which can protect normal flora, low-dose treatment of diseases and specific treatment for specific infections. The phage was prepared on the surface of the catheter with PVA hydrogel. The bacteriophage release is controlled by the pH-sensitive trigger (self-quenching dye 5 (6)-carboxyfluorescein), which is on the outermost surface of the coating. The pH-sensitive trigger will degrade when the urine pH increases, and the bacteriophage in the lower layer will be released. The in vitro bladder showed that the blockage time of the phage-coated catheter was twice as long as that of the control group (P ¼ 0.0199). It is inferred that bacteriophages can only slow down the time of encrustation but cannot completely prevent it. This phenomenon may be due to a decrease in the amount of bacteriophage released at a later stage. At the same time, although phage cocktail treatment can guard against resistance, the drug resistance of bacteriophages is also a reason for failure to prevent encrustation that cannot be ignored.
## Functional coating on urinary devices
## Polyether-based coating
Pellethane, a kind of aromatic polyether, features flexibility, high strength, and resistance to highly caustic solvents. In the in vitro bladder model, encrustation experiments of pelthane thermoplastic polyurethane (TPU), poly 2-hydroxyethyl methacrylate (HEMA)-coated TPU, tetraethylene glycol dimethyl ether (TETRA)-coated TPU and radio opaque hydrogel-coated ureteral stents (Cook Medical, Inc.) were carried out for 90 days and evaluated for surface crusts. HEMA, a hydrophilic polymer, has the ability to stabilize and resist cell adhesion. Therefore, it is often used as a contact lens material to reduce or prevent the negative effects of biofilms on the surface of the eyes. TETRA is a precursor molecule that may form a hydrophilic linear polymer, whose structure resembles poly (ethylene glycol). TETRA can resist protein adsorption and platelet adhesion under flow conditions. The result show that after 90-day trial, all the average mass of magnesium, calcium, and phosphorus were as follows: stent 0.067 mg, 0.312 mg, 1.149 mg, pellethane TPU 0.022 mg 0.164 mg, 0.348 mg, HEMA coated pellethane TPU 0.006 mg, 0.052 mg, 0.360 mg and TETRA-coated pellethane TPU 0.027 mg, 0.183 mg, 0.530 mg. Compared with traditional urinary device material, HEMA-coated pellethane TPU has better anti-encrustation performance. The ant-encrustation effect of HEMA-coated pellethane TPU in vitro experiments for up to 90 days also shows that it is very suitable for patients with long-term indwelling catheters/stents. At present, there are only data from in vitro experiments; more experiments in vivo or clinical trials are needed to obtain more comprehensive data in the future.
Polyethyleneimine (PEI)is a cationic polymer that can inhibit bacterial adhesionand improve antibacterial properties by disrupting the membrane when in contact with bacteria. This antibacterial activity has also been proven to be effective against common urinary bacteria such as Pneumococcus, E. coli and P. mirabilis. Urea was decomposed by P. mirabilis, and the pH increased. The increase in pH promoted encrustation. PEIs with different molecular weights (Mn: 1800 and 60,000) are grafted onto the surface of the PU catheter. This positive charge density on the surface of PEI can be made, and the antibacterial property can be further improved when PEI is alkylated. The in vitro model showed that high molecular weight (Mn 60,000) and alkylated PEI could effectively inhibit biofilm formation, prevent the deposition of surface crystals, reduce calcium salt and magnesium salt by 81% and 93.4%, respectively, and inhibit bacterial adhesion by 2 orders of magnitude compared with the control group. In the 28-day-old mouse model, there was no obvious crystal deposit on the surface of the high molecular alkylated PEI brush coating, and the tissue sections of the mice also showed that its inflammatory response to the host was much weaker than that of the uncoated PU. Polyethyleneimine brushes show excellent performance and a low degree of inflammation, but further clinical trials are required to verify their effectiveness and safety in vivo.
## Polyvinylpyrrolidone-iodine coating
Several studies show that the hydrophilicity of the material surface, such as hydrogel coating, inhibits the catheter surface crust to some extent. Polyvinylpyrrolidone-iodine (PVP-I), a hydrophilic macromolecular material, was used for bladder irrigation to prevent urinary tract infection. This antibacterial material is not an antibiotic and may greatly reduce the production of drug-resistant bacteria. PVP-I was embedded on the surface of PU, and AFM showed that the surface of PVP-I was rougher than that of PU and that both sides of the PU/PVP-I film were uniformly covered with PVP-I molecules. The contact angle results showed that the hydrophilicity on the PVP-I coating was significantly improved and that the coating was stable in ultrasonication methods. After a 4-h bacterial adhesion test, the PU/PVP-I surface significantly reduced the adhesion of both Pseudomonas aeruginosa (P < 0.05) and Staphylococcus aureus (P < 0.01). SEM images of P. aeruginosa and S. aureus adhesion also showed that fewer bacteria adhered to the surface of the PU/PVP-I film than to PU. In a 2-week in vitro encrustation model, the surface of the PVP-I coating was only partially covered by crystal deposits, far less than the PU surface. The major encrustation components calcium, magnesium and phosphorous on the PU surface were much higher than those of PU/PVP-I according to energy dispersive X-ray mapping. In summary, PU/PVP-I has good antiadhesion and anti-encrustation properties and has good durability. Until now, few in vivo investigations have been performed. Thus, it is urgent to study its effect on animal models before we can make conclusions.
## Papain immobilized polyurethane
Papain, a food-grade enzyme found in many natural products, was the first cysteine protease isolated from papaya. It is covalently cross-linked on polyurethane using glutaraldehyde. This method makes papain more stable and cytocompatible, which means 90% of papain in the solution is immobilized on PU, 88% of which still retains their natural activity. Even after storage at 48 C for 30 days, the enzyme activity decreased by 15%. In addition, it has better hydrophilicity. MTT showed that when grafted onto PU, the cytocompatibility was significantly higher than that of bare PU (P < 0.05). In vitro antibiofilm, bacterial attachment and anti-encrustation experiments indicated that papain-immobilized PU exhibited better antibiofilm ability and less bacterial (E. coli and S. aureus) attachment than bare PU (P < 0.01). In a week of the encrustation test, the amount of calcium deposited on bare PU was approximately 51 mg and on papain immobilized was approximately 33 mg, and the amount of calcium plus magnesium deposited on bare PU was 50 mg, more than 40 mg on the modified polymer, which showed better anti-encrustation performance. However, few studies on the long-term benefit of anti-encrustation and its mechanical properties have been found. In vivo anti-encrustation properties should also be further investigated.
## Natural cyanobacterial coating
A kind of marine cyanobacterium, named Natural Cyanobacterial Polymer-Based Coating (CyanoCoating), was used to develop a coating. This coating has been proven to have good antiadhesion effects against E. coli, S. aureus and P. aeruginosa. Amazing biocompatibility also leads to potential applications in the future. In this study, the authors grafted the CyanoCoating on the surface of a urinary catheter to evaluate its performance on anti UTI and encrustation in the urine environment. The results showed that CyanoCoating had great antiadhesion ability against E. coli (P < 0.05), S. aureus (P < 0.001), and Candida albicans (P < 0.001). Moreover, it has great anti-bioform ability in E. coli (P < 0.05), P. mirabilis (P < 0.01), and Candida albicans (P < 0.01). Large crystals were found on the uncoated surface but not on the CyanoCoating based on the data from scanning electron microscopy (SEM). On the other hand, salt deposition was found on both surfaces. However, it is still unknown how long it is effective in anti-adhesion and anti-encrustation.
## Functional nanoparticle coating
Fullerene-like MoS 2 (IF-MoS 2 ), a kind of inorganic layered nanoparticle compound, is effective in forming a seam-less closed-cage fullerene-like structure. Every nanoparticle wrapped in a fully saturated van der Waals surface has a low affinity with the environment, making it easy for them to roll. When doped with rhenium to form electron rich Re-doped nanoparticles (Re: IF-MoS 2 ), they are self-assembled into a mosaic-like order, and the lubrication performance of this material is further increased. The lubrication performance is effective in decreasing the development of encrustation on the catheter surface. A custom-built model of a catheterized urinary tract was used to conduct the in vitro encrustation process. The energy dispersive spectrometer (EDS) analysis also suggested that the Ca and P contents were 1.2% and 1.9% in the Re: IF-MoS 2 -coated catheter, respectively, when compared with 10.1% and 8.6% in the uncoated specimen. This result was consistent with SEM (BSE and SE modes) imaging. The exact mechanism of Re: IF-MoS2-coated catheter encrustation suppression is not fully understood. Its self-assembling mosaic-like order might be the key to reducing deposits. Although some reports show that these nanoparticles have no toxic risk, their biocompatibility still needs further study in the future.
Chitosan (CS), produced by removing the acetate part of chitin through hydration in concentrated alkali, is a copolymer containing a β-(1,4)-2-acetamido-D-glucose and β-(1,4)-2-amino-D-glucose unit. Recent studies have shown that chitosan is known to have antibacterial potential thanks to its cationic polyelectrolyte nature to bind efficiently with negatively charged molecules and that chitosan-immobilized polyurethane materials have efficient antibacterial activity. Chitosan nanoparticles (CSNPs), owing to their higher surface charge density, can more fully contact bacteria than chitosan. CSNPs were immobilized on the PU ureter stent surface, and their ability to prevent bacterial adhesion was investigated. The results showed that in an in vitro antibacterial experiment, CSNP-coated stents showed good anti-E. coli and P. mirabilis properties by disrupting bacterial membranes through electrostatic interactions upon contact compared with the control group. Because of its excellent killing ability against urease-producing bacteria, it can effectively inhibit bacterial adhesion and biofilm formation and has potential anti-encrustation properties, which need to be further investigated.
## Biodegradable polymers for urinary devices
Polymer biodegradable materials have made remarkable progress in biomedicine. These biomaterials mainly include natural origin polymers, synthetic polymers and metals, which are characterized by biocompatibility, certain mechanical strength and biodegradability. Biodegradable ureteral stents are also attracting increasing attention. The preparation methods of BUS mainly include winding, braiding, injection molding, and immersion technology. They can provide sufficient mechanical support within a certain period of time to drain urine and then degrade into small particles excreted with the urine. They are mainly used in patients with short-and medium-term ureteral stent implantation, and those advantages are to avoid secondary operation to be removed and reduce medical costs. Continuous degradation and shedding of surface materials are also beneficial to resist the adhesion of bacteria and the formation of crystals. Long-term use is not suitable for patients, as the degradation time is not long enough. In addition, catheter degradation and broken fragments left in the urethra will produce strong lower urinary tract symptoms, making the patients extremely uncomfortable. This kind of degradable material is not suitable for use as a Foley catheter. Furthermore, the fragments in the degradation of the stents may lead to ureteral obstruction, hydronephrosis and nephritic damage. Despite this, it still brings us an effective way to prevent encrustation.
Alginate is a biodegradable natural polymer. In 2002, Auge et al. made a ureteral stent with alginate, which was found to be degraded Material engineering strategies to prevent catheter encrustation.
## Category
Anti-encrustation strategies
## Anti-encrustation mechanism
References Drug loading on urinary devices
Antibiotic drug coating AntibacterialSilver ion incorporation Antibacterial, inhibit bacterial adhesionSilver and antibiotic hybrid coating Enhanced antibacterial, antiadhesion abilityAntibacterial peptide coating Antibacterial, inhibit biofilm formationDrug-loaded polymer modification Antibacterial and hydrophobicBacteriophage completely after 7 days in pigs. Pathological examination showed that the material had no obvious toxicity to tissues in pigs. In 2014, Barros et al. added gellan gum on the basis of alginate. Alginate, gellan gum, and their blends are gelated by physical cross-linking at low temperature, which obtains a stable structure by ion exchange, dehydration, solvent exchange and drying treatment. The stents made with different ratios increased the degradation time to 14-60 days in the degradation experiment in vitro. The encrustation test showed that no crystal formation was detected by SEM and EDS. Compared with other commercial stents, there was no significant change in cell viability between cytotoxicity and cell adhesion studies. However, it fails to meet the mechanical strength in a certain period of time, and the degradation rate in vivo is faster than that in vitro, which is only approximately 10 days and makes it difficult to meet the needs of long-term placement of stents. Biodegradable polymers ureteral stents have the advantage of not needing a second surgical removal due to the self-degradation property. At the same time, they are difficult to form encrustation because of the property. However, the potential stent obstruction caused by degradation substances and the rapid degradation cannot meet the needs of long-term indwelling catheters. The material surface peeling of biodegradable catheter/stent caused by degradation makes it difficult to form encrustation, and slow degradation is suitable for patients with long-term indwelling catheter/stent. So, how to long-term degradation or slow degradation is the problem need to be focused in the future.
## Metallic materials for urinary devices
Two commercial metallic ureteral stents are currently available: one is the Silhouette stent produced by Applied Medical, Rancho Santa Margarita, and the other is the Resonance stent produced by Cook Medical, USA. A silhouette stent is constructed with nitinol wire reinforcing the walls of the stent and covered with a polymer. Resonance stents are made up of nickel-cobalt-chromium-molybdenum alloy and have been used in the clinic for several decades. They have higher strength than polymer stents and are suitable for many patients whose tumors oppress the ureter. Many studies show that it can be approved for a maximum dwell time of at least 12 months. However, after stents were removed, 22% of stents showed signs of encrustation [bib_ref] Kyriazis Iason, Constantinos, Evangelos [/bib_ref]. Furthermore, its price in the United States is eight times higher than that of polymer stents. The total medical cost of replacing the stent also includes the cost of drugs and surgery. Despite the high price, long-term use can indeed reduce the financial burden of patients and the pain of surgery.
In recent years, researchers have found that copper (Cu) ions are a kind of antibacterial agent that has the long-term effect of inhibiting bacteria (including some drug-resistant bacteria). Other findings, such as that of metal nanoparticles (MNPs), including Cu or Cu oxide NPs, can kill bacteria by their ion mental interactions with nucleic acids, proteins and peptidoglycan of the cell wall. A kind of hybrid system including copper ion modifying molybdenum disulfide (MoS 2 ) can release MoS 2 and copper ions to oxidize glutathione (GSH), which accelerates bacterial death and does little harm to the human body. Other materials include Cu-TiO 2 nanofibers, Ag-Cu and Cu-Ag nanocomposites (NCs). In addition to their antibacterial properties, they are also effective in decreasing ureteral stent encrustation in vitro and in vivo. In 2017, researchers fabricated Cu-bearing stainless steel ureteral stentsand fewer crystal deposits on Cu-bearing stainless steel ureteral stents than uncoated stainless steel ureteral stents in an in vitro encrustation experiment. After the in vitro encrustation experiment, Cu-bearing stainless steel ureteral stents were implanted into the bladder of New Zealand rabbits for 20, 40 and 80 days, after which they killed the rabbits and removed the stents. They found that the material mentioned above can not only inhibit the adherence of microorganisms but also decrease encrustation formation in an in vivo model. This anti-infection and encrustation mechanism is mainly because copper ions can inhibit the production of urease by bacteria, reduce the decomposition of urea into ammonia, and stabilize the urine pH. Copper ions are known to have certain cytotoxic properties, and there is currently no evidence that the release of copper ions from this material will cause damage to urothelial cells. If it can be proven to be safe in terms of biocompatibility, this coating is ideal for long-term indwelling patients.
Metal has excellent mechanical properties, and magnesium alloys possess attractive biodegradability and biocompatibility properties and can be used as materials for biodegradable ureteral stents. Some studies have shown that this magnesium alloy stenthas a bactericidal effect in the process of degradation, and the mechanism may be that magnesium nanoparticles penetrate the cell membrane of bacteria to kill it. Jin et al. found that they had good antibacterial, biocompatibility and degradation times (4 weeks) on their magnesium and polymer mixed stents in vivo and in vitro. Their anti-encrustation ability was not tested in the above study. By their degradation performance and degradation mechanism, we can infer that this material has similar anti-encrustation performance as alginate, which needs to be further verified in future research.
The current metal material for urinary devices include alloy, nanometal and biodegradable metal. Although alloy, nano-metal stent has obvious advantages in prevention of stent-related encrustation, but the alloy material is very expensive and nano metal stent lack of study of long-term biocompatibility. It is hard to form encrustation in biodegradable metal stent thanks to the biodegradable characteristics, however, there is no relevant research on anti-encrustation at present. Which we need to further investigate in the future.
## New oral composition drug
A prolonged indwelling time of stents or catheters will result in urinary infection and encrustation of ureteral stents, which will lead to the use of extracorporeal lithotripsy, endourological or open surgery to resolve these conditions. One reason for encrustation is the increase in pH, which will result in calcium and magnesium ions depositing on the biofilm. Thus, if urine composition is altered by increasing urine acidification and the urine excretion of crystallization inhibitors, encrustation from the stent could be minimized. A new oral composition, which was first reported as a potential oral treatment to prevent ureteral stent-associated encrustation by changing the urine composition of the patients, contains both crystallization inhibitors (phytate) and a urine acidifier (L-methionine). Phytate, a sort of phytate salt that conforms to the inhibitory property of calcium salt deposits, was used to decrease the risk of encrustation formation. L-Methionine is an essential amino acid that can directly reduce urine pH. A randomized, double-blinded, and placebo-controlled trial containing a total of 105 patients was divided into two groups: the oral composition group and the placebo control group. The time of the clinical trial ranged from 3 to 8 weeks depending on the time lapses between the baseline visit and stent removal, and the average time was 37.54 AE 13.9 days. The results showed that the overall crust of the oral composition group was reduced by 8 times, and the pH reduction of the urine from baseline 1 to day 21 was also 0.47 lower than that of the placebo group. The results revealed a significant decrease in stent encrustation in the intervention group. This confirmed that a higher urinary pH decrease was a protective factor against encrustation. If the effectiveness of crystal inhibition and low side effects in the course of long-term use can be verified, then the application of a new oral composition drug in the field of anti-catheter-associated encrustation is very promising.
## Concluding remarks and future perspectives
Indwelling catheters can indeed solve many clinical problems, benefit many patients, slow down disease progression, reduce the incidence of complications, and reduce the average length of hospital stay. Due to the limitations of the catheter design flaws and materials, there is no perfect solution, leading to catheter-related complications such as infection, encrustation, obstruction, pain, and hematuria. These complications cost the patients extra medical costs.
In recent years, promising technologies mainly include drug loading on urinary devices, functional coating on urinary devices, biodegradable polymers for urinary devices, metallic materials for urinary devices and new oral composition drug . At present, a large amount of most popular research is the antibiotic release coating. One of the reasons is that antibiotics are the natural enemies of bacteria and can effectively kill bacteria. Moreover, close to interdisciplinary cooperation is not necessary in antibiotic coating research. Although the use of antibiotics is becoming increasingly standardized, drug resistance has always been a problem that has not been well solved. Drugs such as bacteriophages and enzymes are used as antibacterial materials; however, phage resistance and enzyme instability have not been well solved. Moreover, pharmacokinetics, coating release rate and other knowledge must be clearly understood when we develop the antibiotic release coating, which is difficult to accurately control without close multidisciplinary cooperation. Therefore, a non-release coating may be more suitable for patients with long-term indwelling catheters. At present, there are two main methods of non-release coating: 1, contact sterilization, in which the catheter surface is connected with antibacterial substances, such as antimicrobial peptides and other popular antimicrobial agents, with low drug resistance and broad-spectrum antibacterial activity. 2, the physical resistance to the adhesion of bacteria and biofilms and the anti-adhesion physical properties of the surface of polymers can resist the extracellular polysaccharides produced by bacteria so that bacteria cannot adhere or form biofilms on the surface of this material. It prevents the formation of crystals on the surface of the catheter, although it cannot kill bacteria, but it can prevent the occurrence of catheter stones. Of course, other methods, such as monitoring catheter obstruction and increasing water consumption, also play a role in preventing catheter stones.
It is difficult for clinicians to understand and apply relevant biomaterials, and experts who specialize in materials do not have a comprehensive understanding of clinical needs and problems. As the boundaries between biomaterials and medicine become increasingly blurred, the scope of their overlap becomes increasingly wider, which requires us to rethink the relationship between biomaterials and medicine. They must be closely related in some aspects. Therefore, multidisciplinary cooperation is necessary to make use of their respective advantages to combine different new technologies, new materials and new products. Although antibacterial coatings will still occupy most of the research on the development of anti-encrustation materials in the future, new materials, especially biomaterials with new characteristics, such as antimicrobial peptide coatings and brush antifouling coatings, will be further investigated in the future. Finally, it will be beneficial for patients who need long-term indwelling catheters.
# Credit author statement
## Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[fig] Scheme 1: Schematic of catheter/stent-related infection and encrustation. [/fig]
[fig] Figure 1: (A) Scheme of the setup of the in vitro flow model. (B) Atomic force microscopy (AFM 3D height sensor) results of silicone and antimicrobial-impregnated urinary catheters (AUCs) soaked in artificial urine (AU) for 1 h, 1 week, or 2 weeks. (C) Phosphate (mg/L) attached to silicone and antimicrobial-impregnated catheter (AUC) segments incubated statically with P. mirabilis and E. coli in artificial urine. (D) Scanning electron microscopy (SEM) images of the lumens of silicone (control) and antimicrobial-impregnated urinary catheters (AUCs) perfused with artificial urine and inoculated with P. mirabilis. (E) Examples of colonization of silicone controls and antimicrobial urinary catheters (AUCs) by NDM-1-producing E. coli and MRSA. Reproduced with permission from ref 24, Copyright 2019, Elsevier BV. [/fig]
[fig] Figure 2: (A) Schematic diagram illustrating in vitro bladder models to study. (B) Time taken to develop bacteriuria in the bladder model for evaluation of the antibacterial performances of whole silicone (control) and Ag-PTFE nanocomposite-coated catheters. (C) The time to blockage of silicone (control, blue bar) and Ag-PTFE nanocomposite-coated (red bar) catheters in the bladder model. (D) Optical images of eye-hole sections and scanning electron microscope images of lumen sections. Scale bar: 1 mm. (E) Growth curves and optical images of cells on well plates under direct incubation with catheter segments. Scale bar: 100 mm. (F) MTT assays of extracts from catheter segments. Reproduced with permission from ref 31, Copyright 2017, Elsevier. (For interpretation of the references to colour/colour in this figure legend, the reader is referred to the Web version of this article.) [/fig]
[fig] Figure 3: (A) Schematic representation of PLGA//TEL-Ag/NF construction. (B) Sustained (a) and cumulative (b) release profiles of norfloxacin in PBS at 37 C. (C) Measurement of bacterial adhesion and viability on unmodified and modified PUR and silicon sheets. (D) Scanning electron microscopic images, 1000x on unmodified PUR and silicon (a and c, respectively) and film-coated PUR and silicon (b and d, respectively). (E) Mass of Ca, Mg, phosphate and oxalate in the crystalline deposits formed on both modified and unmodified PUR and silicon sheets. (F) CLSM imaging of adhered live and dead bacteria on unmodified PUR and silicon (a and c, respectively) and film-coated PUR and silicon (b and d, respectively). Reproduced with permission from ref 42, Copyright 2017, Elsevier. (For interpretation of the references to colour/colour in this figure legend, the reader is referred to the Web version of this article.) [/fig]
[fig] Figure 4: (A) The molecular structures of DOPA and AMPs and the synthesis process of the bioinspired antibacterial coating. (B) AFM and roughness of the bare PU sheet, PDPA@Cu-75 coating and PDPA@Cu-75-AMP coating on PU sheets. Statistically significant differences are indicated by *p < 0.05 or **p < 0.005 compared with the control group. (C) Antibacterial effect of bare stents (control), PDPA@Cu-75 and PDPA@Cu-75-AMP coating against E. coli, S. aureus and P. mirabilis. (D) SEM of rat intravesical stent lumens and deposit surfaces in the control, PDPA@Cu-75 and PDPA@Cu-75-AMP coating groups. Reproduced with permission from ref 76, Copyright 2021, Royal Society of Chemistry. [/fig]
[fig] Figure 5: (A) Schematic representation of AMP (Chain201D) immobilization on EG4-SAMs. (B) Functionalization of polyurethane catheter with AMP-brush coating. (C) Broad spectrum activity of the AMP-conjugated PU catheter against P. aeruginosa (a), S. aureus (b) and S. saprophyticus (c) in vitro. *Indicates P 0.05 and ***indicates P 0.001. (D) Antibacterial activity of the AMP-coated PU catheter in a urinary infection model in vivo. (a) IVIS images of mice bearing either untreated or AMP-coated PU catheters at day 1 and day 7 post-instillation with luminescent-tagged P. aeruginosa into the bladder. (b) Bioluminescence readings (total photon flux (photons/s)) measured for untreated and AMP coating-treated mice using IVIS Lumina at day 1, 4 and 7 days post-bacterial instillation into the mouse bladder. ** indicates P < 0.01. Reproduced with permission from ref 48, Copyright 2019, Nature Publication Group and ref 77 Copyright 2016, Elsevier BV.with methacryloyl moieties (synthesis of mono-and dimethacrylated poloxamer 188 (MMP and DMP)) and then subsequently copolymerized with hydrogel monomer 2-HEMA (p(MMP/DMP-co-HEMA)) to further promote the function of poloxamer derivatives as cross-linking agents. After testing, when the weight of MMP/DMP is 10% of HEMA, their comprehensive performance is the best. Compared with the control group (contact angle, mean AE S.D.: advancing 91.3 AE 1.65, receding 92.5 AE 1.35), the contact angle shows that 10%p(MMP-co-HEMA) (advancing 98.0 AE 3.75, receding 98.1 AE 4.75) has higher hydrophobicity than 10% p(DMP-co-HEMA) (advancing 92.7 AE 1.25, receding 94.1 AE 1.15. [/fig]
[fig] Figure 6: (A) The reaction steps of PEI brush grafting on the PU stent surface and N-alkylation of PEI brushes. (B) The change in pH during the in vitro encrustation experiment and step-by-step encrustation in the bioreactor system at 0 h, 6 h and 24 h. (C) 2, 4 and 6 h of bacterial adherence and 24 h of biofilm formation test against P. mirabilis in biofilm reactor. (D) Encrustation on the ureteral stent samples removed from the rat bladder after 7, 14 and 28 days of implantation. (E) Hematoxylin eosin (H&E) staining micrographs of the rat bladder following urethral stent implantation for different time periods and the histopathological scoring system in terms of inflammation degree in the rat bladder. (Histology score system; 1 ¼ Normal, 2 ¼ Mild inflammation, 3 ¼ Mild-Moderate, 4 ¼ Moderate, 5 ¼ Moderate-Severe, 6 ¼ Severe). * Indicates the significant decrease in PU-g-HPEI-R samples in comparison to PU after 7, 14 and 28 days (p < 0.05). Reproduced with permission from ref 92, Copyright 2017, John Wiley and Sons. [/fig]
[fig] Figure 7: (A) Mechanism of 316 L-Cu SS inhibiting infectious encrustation: (a) 316 L SS, (b) 316 L-Cu SS. The release of Cu 2þ ions from 316 L-Cu SS killed the majority of bacteria that produced urease. The hydrolysis of urea was inhibited, resulting in restricting the elevation of Ph. Fewer crystals were deposited on the surface of 316 L-Cu SS. (B) Crystalline deposits on the surface of samples: (a) 316 L-Cu SS, (b) 316 L SS. (C) Ca (a) and Mg (b) contents in the encrustation on different materials by S. aureus. Models infected with S. aureus (5 Â 10 5 cfu/ml) were supplied with mixed HU and AU solutions. Each value is the mean calculation from three replicated experiments. *, # and ◆ indicate significant differences from the control values at p < 0.05. Reproduced with permission from ref 131, Copyright 2017, Elsevier BV. [/fig]
[fig] Figure 8: (A) Methodology used to generate the different stents. (B) Degradation of the stents in vivo. (a). One week after stent implantation, there were no obvious changes. (b). Two weeks after stent implantation, the distal end had degraded. (c). Three weeks after stent implantation, the proximal end remained. (d). SEM image of the stent. (e). SEM image of the stent implanted in the animals for 1 week. (f). Mass loss ratio of the stent in vivo. Reproduced with permission from ref 107, Copyright 2014, John Wiley and Sons and ref 111, Copyright 2020, John Wiley and Sons. [/fig]
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Family physicians’ professional identity formation: a study protocol to explore impression management processes in institutional academic contexts
Background: Despite significant differences in terms of medical training and health care context, the phenomenon of medical students' declining interest in family medicine has been well documented in North America and in many other developed countries as well. As part of a research program on family physicians' professional identity formation initiated in 2007, the purpose of the present investigation is to examine in-depth how family physicians construct their professional image in academic contexts; in other words, this study will allow us to identify and understand the processes whereby family physicians with an academic appointment seek to control the ideas others form about them as a professional group, i.e. impression management. Methods/Design: The methodology consists of a multiple case study embedded in the perspective of institutional theory. Four international cases from Canada, France, Ireland and Spain will be conducted; the "case" is the medical school. Four levels of analysis will be considered: individual family physicians, interpersonal relationships, family physician professional group, and organization (medical school). Individual interviews and focus groups with academic family physicians will constitute the main technique for data generation, which will be complemented with a variety of documentary sources. Discourse techniques, more particularly rhetorical analysis, will be used to analyze the data gathered. Within-and cross-case analysis will then be performed.Discussion: This empirical study is strongly grounded in theory and will contribute to the scant body of literature on family physicians' professional identity formation processes in medical schools. Findings will potentially have important implications for the practice of family medicine, medical education and health and educational policies.
# Background
The purpose of the present investigation, granted by the Canadian Institutes of Health Research (CIHR MOP-125906) is to examine in-depth how family physicians construct their professional image in academic medical contexts; in other words, the processes whereby family physicians seek to control the ideas others form of them as a professional group, i.e. impression management [bib_ref] Impression management: a literature review and two-component model, Leary [/bib_ref]. The project makes up part of an international research program on family physicians' professional identity initiated with a 2007 CIHR-funded investigation (MOP-85044) in which we studied processes of medical student identification with family medicine in medical schools [bib_ref] The Influence of Academic Discourses on Medical students' Identification With the Discipline..., Rodríguez [/bib_ref] [bib_ref] Exploring professional identification and reputation of family medicine among medical students: a..., Rodríguez [/bib_ref] [bib_ref] Understanding reputation of and professional identification with the practice of family medicine..., López-Roig [/bib_ref]. The trend that called for us to spearhead such research program was medical students' declining interest in family medicine as a career choice.
This disturbing trend is also demonstrated by the health comparative statistics from the 30 countries of the Organization for Economic Co-operation and Development (OECD). The number of medical specialists rose by 60% between 1990 and 2007, while the number of general practitioners increased only by 23%. So far, Canada has been able to maintain a balance, i.e. about 50% each. However, the equilibrium between specialists and generalist in the country has been seriously threatened since 1992, when the proportion of Canadian graduates who chose family medicine as 1 st choice in the residency match started decreasing to attain a minimum of 24.8% in 2003 [bib_ref] Family medicine crisis? Fiend attracts smallest-ever share of residency applicants, Sullivan [/bib_ref]. More recently this negative trend has reversed, reaching 31.8% in 2010and 34.0% in 2011 (1 st iteration). Yet, while encouraging, these percentages are far from the national goal of 50% [bib_ref] Higher percentages of medical school graduates seek family medicine residencies, Collier [/bib_ref]. Furthermore, the number of vacant positions in family medicine residency programs still remains very high: in 2013, 40% of vacancies at the 1 st iteration were in family medicine.
As noted above, and despite significant differences in terms of medical training and health care context, the phenomenon of medical students' declining interest in family medicine has also been documented in many other developed countries such as the United States [bib_ref] Results of the 2002 national resident matching program: family practice, Pugno [/bib_ref] [bib_ref] Results of the 2009 national resident matching program: family medicine, Pugno [/bib_ref] , Australia, the United Kingdom [bib_ref] Experiences and career intentions of general practice registrars in Thames deaneries: postal..., Bowler [/bib_ref] , France [bib_ref] Une disparition programmée de la médecine générale, Rabany [/bib_ref] , Switzerland [bib_ref] The new generation of family physicians: career motivation, life goals and work-life..., Buddeberg-Fischer [/bib_ref] , and Spain. A gap between the income of specialists and family physicians, advances in specialized medical technology, and a loss of professional prestige [bib_ref] The decision to enter general practice, Pearce [/bib_ref] [bib_ref] Primary care at a crossroads, Schroeder [/bib_ref] [bib_ref] Factors related to the choice of family medicine: a reassessment and literature..., Senf [/bib_ref] [bib_ref] The future of primary care medicine, Whitcomb [/bib_ref] [bib_ref] Primary care is dying in The United States: mutatis mutandis, Walker [/bib_ref] have been documented as factors than can help explain this trend.
However, the influence of institutional discourses on the construction and reconstruction of family physicians' professional identity in academic centres during undergraduate medical training and its influence on career choice has been largely ignored. Our international research program was conceived to fulfil this research gap. In the first aforementioned investigation, we provided sound evidence about the relationship between professional reputation, discursively constructed, and students' identification with family medicine practice. More specifically, the study highlighted the clear polarization existing between the medical school in which family medicine was a valued academic discipline, to which students were exposed from the very beginning of their studies (the British case), and those in which students had little or no exposure to this practice and where family medicine was disregarded as a valid career option (Canadian, French and Spanish cases). In the former, the reputation of the profession was very high, the features of this professional practice as well as the knowledge and skills necessary to perform as a GP appearing to be held in high esteem by both students and educators. In this institutional context, the majority of students identified with general practice and chose this medical field as career choice. It was the opposite in the other three medical schools, where family medicine was lessened, either overtly or through a double academic discourse that stressed the importance of the practice for the health care system while denigrating family medicine because of its lack of a hard medical skill set [bib_ref] The Influence of Academic Discourses on Medical students' Identification With the Discipline..., Rodríguez [/bib_ref].
Nevertheless "[r]eputations rise and fall" [bib_ref] How should reputations be managed in good times and bad times? Two-way..., Fiol [/bib_ref]. Put differently, as identity, reputation and image are intimately related processes [bib_ref] Organizational identity, image, and adaptive instability, Gioia [/bib_ref] [bib_ref] Identity, intended image, construed image, and reputation: an interdisciplinary framework and suggested..., Brown [/bib_ref] , family physicians may be extremely motivated to project on others an enhanced perception of what they are as a medical professional group (image) with the aim to improve how the ideas the others have about them as a professional group (reputation) as well as their own sense of self (identity) and the future identification of physicians-to-be with the profession; those are the processes that, within institutional academic contexts, our international research team proposes to focus on in the present investigation.
## Literature review identity and image
The Oxford Dictionary defines 'identity' as the "condition of being a specified person or thing". As a topic of investigation, identity has been very popular among organizational scholars. In this field of inquiry, identity has effectively been treated from many different perspectives, e.g. social identity theory; embedded inter-group theory; race, ethnicity and gender research; organizational demography; ethnology [bib_ref] Diverse identities in organizations, Nkomo [/bib_ref]. In recent times, there is a renewed concern by identity issues, largely justified by current broader changes in society (globalization, the expansion of information technology, and greater diversity), the decline of bureaucratic organizational forms, and a special interest in power and meaning.
In 1985 Albert and Whetten published a landmark article that has strongly influenced the study of identity. Being among the first to define the term as a "clear, distinctive, important, useful and measurable" construct, the authors provoked such a noteworthy impact that most later treatments of the topic were built on their work [bib_ref] Organizational identity, image, and adaptive instability, Gioia [/bib_ref]. In effect, an extensive stream of work on identity has viewed it as a stable and well-defined element. Although this traditional view of identity is still espoused, recent perspectives tend to see identity as a social process in constant reconstruction [bib_ref] Diverse identities in organizations, Nkomo [/bib_ref]. Identity would therefore not be stable or fixed but socially and historically constructed and subject to contradictions, revisions, and change. This new approach to identity also implies that the focus of interest is more on the social aspects of the self than on the individuated self-concept of identity (see the Oxford Dictionary's definition above), the latter being adopted by most social psychological theories of the self in the past [bib_ref] Who is this 'we'? Levels of collective identity and self-representations, Brewer [/bib_ref]. Identity is therefore conceptualized as an ongoing process that encompasses the 'sense of self' created through social interactions. In other words, identity concerns how social actors understand and explain themselves through dynamic interactional processes [bib_ref] Diverse identities in organizations, Nkomo [/bib_ref] [bib_ref] The social construction of gender and race in organizations: a social identity..., Wharton [/bib_ref] [bib_ref] Constructing professional identity: "doing power" in policy units, Holmes [/bib_ref]. In addition, the accomplishment of these interactional processes is mainly made through language-in-use. In other words, actors' discourses are constitutive of their own identity and of the space and role they occupy in the social world. As Ainsworth notes [bib_ref] Discourse analysis as social construction: towards greater integration of approaches and methods, Ainsworth [/bib_ref] : "Discourse constructs social identity by defining groups, groups' interests, their position within society, and their relationship to other groups" (p. 31).
If one accepts the assumption that identity is constructed through social interactions, then two other levels of representation of the self besides the individual level can be identified, namely interpersonal and group levels [bib_ref] Who is this 'we'? Levels of collective identity and self-representations, Brewer [/bib_ref]. At the interpersonal level, the relational self is typically constructed through dyadic relationships with significant others; at the group level, the collective self, which does not require personal relationships among the group members, involves the sense of belonging to a particular social category. As noted by Brewer and Gardner [bib_ref] Who is this 'we'? Levels of collective identity and self-representations, Brewer [/bib_ref] , some social identities can be constructed either through interpersonal relationships or collective identities. This is the case for the medical professional identity, which can be built through the doctor-patient relationship, but also in terms of membership in the social category of the medical profession.
The relational nature of identity intimately connects this concept with the notion of 'image'. For Dutton and Dukerich [bib_ref] Keeping an eye on the mirror: image and identity in organizational adaptation, Dutton [/bib_ref] image is what organizational members believe other think about their organization. According to Hatch and Schultz [bib_ref] Relations between organizational culture, identity and image, Hatch [/bib_ref] , marketing literature has put forward a more external definition of image, considering it as the views of the organization held by various external constituencies such as the customers, suppliers and regulators. Indeed, a comprehensive definition of image that encompasses both perspectives is provided by Alvesson [bib_ref] Organization: from substance to image?, Alvesson [/bib_ref] , for him image is the bright impression held by an actor towards an organization that has been nourished by the organization's projected portrait of itself (see also Hatch and Schultz [bib_ref] Relations between organizational culture, identity and image, Hatch [/bib_ref]. One could therefore assert that identity and image are different but interpenetrated organizational dimensions, which emerge from the conversational process between internal and external organizational stakeholders [bib_ref] Organizational identity, image, and adaptive instability, Gioia [/bib_ref] [bib_ref] Relations between organizational culture, identity and image, Hatch [/bib_ref].
## Professional identity and professional image
Sociologists and organization scholars seem to be more and more interested in exploring the crossroad of professions and organizations (see for example the recent monograph of Current Sociology on this topic, introduced by its editors, Muzio and Kirkpatrick [bib_ref] Introduction: Professions and organizations: a conceptual framework, Muzzio [/bib_ref] entitled 'Professions and organizationsa conceptual framework'). A 'new professionalism' is even advocated as professionals are working increasingly in large-scale workplaces, yet international firms [bib_ref] A new professionalism? Challenges and opportunities, Evett [/bib_ref]. Still professionals are for some "the preeminent institutional agents of our time" [bib_ref] Lords of the dance: professionals as institutional agents, Scott [/bib_ref]. A 'profession' is an occupation characterized by both the possession of a specialized body of knowledge and a commitment to service [bib_ref] The dynamics of interactional and institutional orders in work-related settings, Sarangi [/bib_ref] [bib_ref] Professionalism: an ideal to be sustained, Cruess [/bib_ref] [bib_ref] Medicine under threat: professionalism and professional identity, Sullivan [/bib_ref]. As any other collective self, the medical profession: "reflects internalizations of the norms and characteristics of important reference groups and consists of cognitions about the self that are consistent with that group identification" [bib_ref] Who is this 'we'? Levels of collective identity and self-representations, Brewer [/bib_ref]. This process of differentiation, which comes into being through language-in-use, implies attaching value to a particular group membership [bib_ref] Communicating professional identity in medical socialization: considering the ideological discourse of morning..., Apker [/bib_ref] and simultaneously a separation from other social groups that are not only "different" but usually also "less valuable". A professional identity is therefore created and recreated through professional discourse. According to Sarangi and Roberts [bib_ref] The dynamics of interactional and institutional orders in work-related settings, Sarangi [/bib_ref] , professional discourse would be constituted by everything professionals do in the day-to-day accomplishment of their responsibilities and tasks. Such a discourse would be "not only durable, but also legitimate and authoritative" (p. 15). It is also important to note that professional discourses do not emerge in a vacuum. On the contrary, the fleshing out of professional identity through discursive activity can only be understood and explained within the context in which social interactions are performed. In this regard, we agree with Sarangi and Roberts [bib_ref] The dynamics of interactional and institutional orders in work-related settings, Sarangi [/bib_ref] when they point out: "What counts as legitimate professional discourse will depend on the range of discourses available within an institutional order" (p. 15). Context is thus crucial for both understanding discourses and for creating identity [bib_ref] Constructing professional identity: "doing power" in policy units, Holmes [/bib_ref].
It is important to note that professional identity construction starts in educational institutions such as medical schools where trainees internalize the norms, values and power relations that characterize the collective identity of the profession to which they aspire to be part of. There, a professional trainee learns and assumes the particular discourse of the profession of his/her choice in order to become a legitimated and credible member of that profession. Scholars have conceptually and empirically examined the ideological socialization of medical students in academic centers for more than 40 years [bib_ref] Even if I don't know what I'm doing I can make it..., Beagan [/bib_ref] [bib_ref] Exploring the emergent identity of future physicians: toward an understanding of the..., Harter [/bib_ref] [bib_ref] A certain art of uncertainty: case presentation and the development of professional..., Lingard [/bib_ref] [bib_ref] Professional socialization revised: medical students' own conceptions related to adoption of future..., Pitkala [/bib_ref]. However, as recently noted by Cooke and collaboratorswith regard to medical training in the US, professional identity formation is one of the most important, yet most neglected strategy for reforming medical school and residency.
Professional identity and professional image are also two interrelated constructs. In effect, there is no doubt that what professionals identify about their professional group is influenced by the way others see them. Concomitantly, professionals can project a particular perception of what they are as a professional group. Grounded in self-reflecting appraisals, professional image refers to "an externally oriented, public persona" and is defined as "the aggregate of key constituents […] and perceptions of one's competence and character" [bib_ref] Changing faces: professional image construction in diverse organizational settings, Roberts [/bib_ref]. Indeed, professional image has important consequences in terms of social reward and career success [bib_ref] Impression management: a literature review and two-component model, Leary [/bib_ref] [bib_ref] Provisional selves: experimenting with image and identity in professional adaptation, Ibarra [/bib_ref] [bib_ref] Identity as narrative: prevalence, effectiveness, and consequences of narrative identity work in..., Ibarra [/bib_ref] since, as noted by Roberts [bib_ref] Changing faces: professional image construction in diverse organizational settings, Roberts [/bib_ref] , "people who construct viable professional images are perceived as being capable of meeting the technological and social demands of their jobs" (p. 687). Processes of professional image construction therefore appear crucial to reach and preserve social reputation and legitimacy.
## Impression management
Impression management refers to the processes by which social actors try to create, maintain or modify the ideas others have about them . The roots of this concept can be found in Goffman's notion of selfpresentation. According to this sociologist, people usually seek information about others in their social space. The sources of this information can be different, one of them being the individual him/herself (self-presentation). That is, the individual will wittingly or unwittingly express him/herself to others, and the others will have to be impressed somehow by him/her. Using the metaphor of the play, for Goffman self-presentation involves an actor and its audience interacting in a particular context and jointly defining a particular situation, with the actor selecting the behavior that s/he expects will generate the best impression in the audience.
In the 1960's, social psychologists were also attracted by the conceptualization and empirical research on impression management [bib_ref] When actions reflect attitudes: the politics of impression management, Snyder [/bib_ref] [bib_ref] Toward a general theory of strategic selfpresentation, Jones [/bib_ref] [bib_ref] A self-presentational view of social phenomena, Baumeister [/bib_ref]. In this corpus of texts, the frequently-cited work by Leary and Kowalski [bib_ref] Impression management: a literature review and two-component model, Leary [/bib_ref] deserves to be examined in more detail. Being interested in identifying factors that affect self-presentations to others, these authors recognize two discrete processes involved in what has been called impression management, namely impression motivation and impression construction. Among primary self-presentational motives, they consider that individuals may be motivated to control how others perceive them when they want to maximize their reward-cost ratio in social interactions, when they highly value the goals to achieve, and when there is a discrepancy between desired and current image. Then, once individuals are motivated to act, five factors might have an influence on the way they will behave in order to impress others: (1) their sense of self (i.e. identity),what they desire to be or not to be, (3) social constraints with regard to the role to be played, (4) value attached to the model to be emulated, and (5) current reputation and image. This conceptual framework of impression management, which concerns cognitive/behavioral intra/interpersonal levels, emphasizes that identity, image and reputation are intertwined.
The interest in impression management among organizational scholars was scant until the 1980's [bib_ref] Impression management in organizations, Gardner [/bib_ref]. The willingness to examine this topic has increased since then, but remains mostly conceptual [bib_ref] Impression management in the feedback-seeking process: a literature review and research agenda, Morrison [/bib_ref] [bib_ref] A taxonomy of organizational impression management tactics, Mohamed [/bib_ref] , as empirical research [bib_ref] Political influence behavior and career success, Judge [/bib_ref] [bib_ref] Effects of impression management on performance ratings: a longitudinal study, Wayne [/bib_ref] [bib_ref] Enacting 'team' and 'teamwork': using Goffman's theory of impression management to illuminate..., Lewin [/bib_ref] is still scarce. Likewise, most works have focused on a micro-individual level of analysis [bib_ref] The management of information and impressions: when employees behave opportunistically, Fandt [/bib_ref] [bib_ref] Motivation and opportunity: the role of remote work, demographic dissimilarity, and social..., Barsness [/bib_ref] [bib_ref] Acting on the collegiate stage: managing impressions in the workplace, Lester [/bib_ref] , the macro-organizational level [bib_ref] Acquiring organizational legitimacy through illegitimate actions: a marriage of institutional and impression..., Elsbach [/bib_ref] [bib_ref] Responding to organizational identity threats: exploring the role of organizational culture, Ravasi [/bib_ref] has been considered to a lesser extent [bib_ref] A multi-level review of impression management motives and behaviors, Bolino [/bib_ref].
The taxonomy of organizational impression management tactics proposed by Mohamed and collaborators [bib_ref] A taxonomy of organizational impression management tactics, Mohamed [/bib_ref] has made a significant theoretical contribution in this field. Presented in a 2×2 matrice, and inspired by prior work on impression management tactics at the micro-individual level, these scholars argue that organizations may use direct/indirect and assertive/defensive tactics to construct/preserve desirable images among target audiences. What is more, organizations can use more than one tactic at a time as they are not mutually exclusive. Direct and assertive tactics (e.g., ingratiation, intimidation) are adopted when the organization actively works to create and enhance a desirable organizational image, while direct and defensive tactics (e.g. disclaimers, apologies) aim at protecting the organizational image. On the other hand, indirect impression management tactics, either assertive (e.g. boasting) or defensive (e.g. blurring), seek to manage information about the people and things the organization is associated with.
More recently, Robert [bib_ref] Changing faces: professional image construction in diverse organizational settings, Roberts [/bib_ref] has interestingly theorized about impression management for constructing professional image in organizational settings. Drawing on social identity, impression management and organizational behavior theories, this author proposes a comprehensive model that encompasses both individual and group levels and includes three main components: (1) impression monitoring, i.e. the awareness of how one is perceived in a given situation is generated; (2) impression motivation, i.e. how identity threats and negative image discrepancies trigger actors' desire to adopt impression management; and (3) impression construction, i.e. how actors effectively enact their personal and social identities in order to create their desired professional images. Furthermore, this framework also highlights intended and unintended consequences of impression management behaviors at the individual, interpersonal, group and organizational performance levels.
## Summary of the literature review
In our contemporary globalized world, individual, group and organizational identity are constructs that have attracted increasing attention among scholars from different disciplines. In organizational and management literature, current trends conceive identity and related concepts, i.e. image and reputation, as constructed through social interactions, mostly of a discursive nature. Professionals are leading social actors embedded in organizational fields; therefore the construction of professional identities appears of particular interest not only for themselves as cohesive groups but also for the institutions in which they operate. Two important issues are at stake here. First, professional identity formation are processes that begin in educational institutions (such as medical schools), where trainees espouse the norms, values and power relations akin the profession they aim to integrate. Second, the way professionals understand themselves comes basically into being through the discursive activity developed among group members and between them and external stakeholders in their situated contexts. Processes of enhanced professional image construction, i.e. impression management, do therefore allow the constant reconstruction of professional identities as well as the attainment and preservation of social reputation and legitimacy. Despite its unquestionable interest, our literature review has revealed that empirical works in organizational image construction are still scant; and there is even less research on professional image construction. To our knowledge, no study about processes of family physician professional image construction in medical schools has hitherto been conducted.
## Research questions
As noted above, the present study builds on our previous investigation in which we studied processes of medical student identification with and the reputation of family medicine in medical schools. Findings unveiled both low interest in a family medicine career pathway among medical students trained in medical schools where this profession was devaluated, and high interest among those students trained in the medical school in which general medical practice had a greater legitimacy. As part of an on-going research program on family physicians' professional identity, we are at the present interested in examining how family physicians construct their professional image in the same academic contexts. This being said, it is important to point out that this study will be concomitant with important curricular and fieldlevel institutional changes in all the cases involved in the investigation. Three interrelated research questions will guide the study: (1) Why are academic family physicians motivated to control/improve their professional image in academic contexts? Here, we are interested in describing and understanding the motivations underlying processes of family physicians professional image construction.What are the impression management strategies they have undertaken/will undertake in order to improve the image of the family medicine discipline? The answer of this question will imply the description and comprehension of the different ways academic family physicians behave in order to enhance their professional image. (3) How are these strategies being implemented, and what have been their intended and unintended consequences to date? Indeed, we want to understand and explain processes of impression management adoption, as well as the consequences (projected as well as unintentional) of their adoption at the individual, interpersonal group and organizational levels.
## Theoretical framework
Taking into account identity and impression management organizational literature, institutional theory will be the overarching theoretical perspective we will adopt in this investigation to better describe, understand and explain processes of professional image construction occurring within given institutional contexts. We will specifically retrieve up to date literature on institutionalism that calls attention to institutional entrepreneurship, change, and power/knowledge dynamics. Institutional theory is one of the most prominent approaches for understanding organizational phenomena. What has been called neoinstitutionalism traces its origins in the seminal works of Meyer and Rowan [bib_ref] Institutionalized organizations: formal structure as myth and ceremony, Meyer [/bib_ref] , Meyer and Scott, Tolbert and Zucker [bib_ref] Institutional sources of change in the formal structure of organizations: the diffusion..., Tolbert [/bib_ref] , Zucker [bib_ref] Institutional theories of organizations, Zucker [/bib_ref] , and Powell and DiMaggio [bib_ref] The iron cage revisited: institutional isomorphism and collective rationality in organizational fields, Powell [/bib_ref] [bib_ref] Suddaby R: Introduction. In The SAGE Handbook of Organizational Institutionalism, Greenwood [/bib_ref]. The essential characteristics of institutional theory set up by these scholars can be summarized as follows [bib_ref] Suddaby R: Introduction. In The SAGE Handbook of Organizational Institutionalism, Greenwood [/bib_ref] [bib_ref] Institutional theory: contributing to a theoretical research program, Scott [/bib_ref] : all organizations undergo influences from their institutional (rationalized myths of appropriate conduct) and network/field contexts, although not all to the same extent. Isomorphism (coercive, normative or mimetic) would involve processes by which organizations try to conform to institutional pressures in order to gain legitimacy. In a particular context, a practice is institutionalized when it is widely accepted, followed, and enduring. Institutionalization is thus defined as the process by which something acquires the "rule-like status" [bib_ref] Institutionalized organizations: formal structure as myth and ceremony, Meyer [/bib_ref] , that is, it becomes taken-for-granted.
Until the 1990s, institutional scholars paid great attention to how institutional contexts influence organizations.
Following DiMaggio's remark [bib_ref] Interest and agency in institutional theory, Dimaggio [/bib_ref] that institutional theory should integrate 'agency', scholars have become more and more interested in understanding how organizations also shape their institutional contexts. This new trend in organizational institutionalism has been materialized in a number of new topics of interest, such as, among others, the examination of institutional change, institutional entrepreneurship and power. Institutional entrepreneurship has been defined as the set of activities displayed by "actors who have interest in particular institutional arrangements and who leverage resources to create new institutions or to transform existing ones" [bib_ref] Institutional entrepreneurship in emerging fields: HIV/AIDS treatment advocacy in Canada, Maguire [/bib_ref]. Institutional entrepreneurship therefore corresponds to the notion of institutional change. Hence, institutional entrepreneurs would therefore be those individual or collective actors to whom institutional change is attributed [bib_ref] Institutional Entrepreneurship. In The SAGE Handbook of Organizational Institutionalism, Hardy [/bib_ref]. Both exogenouslydriven institutional change [bib_ref] Theorizing change: the role of professional associations in the transformation of institutional..., Greenwood [/bib_ref] and endogenous source of change [bib_ref] Institutional change and the transformation of the U.S. radio broadcasting industry, Leblebici [/bib_ref] have been advocated. In this regard, Suddaby and Viale [bib_ref] Professionals and field-level change: institutional work and the professional project, Suddaby [/bib_ref] argue that "the dynamic of professionalization offers an endogenous explanation of institutional change" (p. 425). In other words, these authors emphasize the reciprocal dynamics between processes of institutionalization and processes of professionalization. Based on institutional and profession bodies of literature, these scholars sustain their thesis on two key assertions. First, due to their power attributes, i.e. expertise and legitimacy, professionals are key actors able to mobilize their social capital and skills in driving institutional change. Second, in their quest of securing/enhancing their privileged position, professional projects are closely related to other institutionalization projects, for instance, in universities [bib_ref] Exploring the limits of the new institutionalism: the causes and consequences of..., Kraatz [/bib_ref].
In sum, in the present study, institutional theory, as well as identity and impression management literature, will help us depict and explain: (1) why and how academic family physicians undertake processes of professional image reconstruction in their institutional contexts, i.e. motivation and construction of impression management strategies; and (2) how this professional project is recursively related to projects of academic institutional changesee also [fig_ref] Figure 1: Impression management in the construction of professional image [/fig_ref].
# Methods/design
## Research design
We have decided to adopt a case-based inquiry as a research strategy. Case study is a very appropriate research design when the researcher asks "why" or "how" research questions and is interested in examining in-depth contemporary phenomena in their naturally-occurring contexts. Our main unit of analysis, i.e. the "case", will be the medical school. Hence, this will be a multiple case study as 4 medical schools from 4 different countries will be involved in the study, namely Canada, France, Ireland, and Spain. It will also be an embedded case study as 4 levels of analysis will be considered: individual family physicians, interpersonal relationships, family physician professional group, and organizations (medical school). Furthermore, as longitudinal research appears crucial for attaining a rich understanding of organizational change [bib_ref] Organizational change: a review of theory and research in the 1990s, Armenakis [/bib_ref] [bib_ref] Improvising organizational transformation over time: a situated change perspective, Orlikowski [/bib_ref] , we will adopt a retrospective-prospective longitudinal design; hence our inquiry is better labelled as a longitudinal embedded multiple case study. Such a design appears fully consistent with the topic under investigation, as well as with the current phase of "the maturing of institutional theory" in organization studies, which asks for longitudinal designs and the consideration of multiple levels of analysis.
Approval
## Case selection
How were our cases selected? A qualitative researcher is not interested in statistical generalizations, but in studying in-depth relatively small samples (even single cases), which are selected purposively [bib_ref] Thousand Oaks: Sage, Patton [/bib_ref]. The logic and power of a purposive sampling lie in selecting information-rich cases; that is cases from which the researcher can intensively learn about the purpose of the investigation. Accordingly, the choice of our four cases responds first to theoretical concerns: cases included in the study offer a variety of situational, organizational and societal contexts regarding the topic under investigation. Consequently, to the extent that an international collective case study increases the amount of fieldwork, it will also enable us to elaborate deeper understandings of the complex process of identity construction, and thus, to maximize the possibility of elaborating richer theoretical explanations that may be useful in other contexts. Second, these cases have been selected because of practical concernsthat mostly relate to our prior research collaboration and the constitution of an international research team for the in-depth examination of processes of family physicians/general practitioners' identity construction at the international level.
## Data collection interviews
The main source of data in this study will be both individual and group interviewing techniques. Following a purposeful sampling rationale, participants in this investigation will always be academic family physicians. This being said, we will contemplate maximum variation sampling [bib_ref] Thousand Oaks: Sage, Patton [/bib_ref] with regard to variables such as age/sex, years of academic career and position occupied in the academic hierarchy. We plan to conduct a first round of individual interviews at the beginning of the fieldwork. Inspired by the conceptual framework adopted, the discussion with the participants will focus of the meaning of their professional identity, the awareness of image consistencies/discrepancies, their perceived need and motivations to adopt impression management strategies in order to reduce image discrepancies and enhance professional identity and reputation, and the description and rationale of impression management strategies effectively adopted. We plan to carry out about 60 face-to-face, one-to-one interviews, i.e. 15 interviews per medical school. Based on our prior research experience in these academic centers, we estimate that this volume of individual interviews will allow us to reach data saturation. Interviews will be mostly conducted by members of our research team. Participants will have to sign a written consent form for the interview to be carried out. With their permission, conversations will be tape-recorded, and immediately transcribed and analyzed with the support of the qualitative software package HyperRESEARCH 3.0. Further, we contemplate conducting a series of focus groups, also with academic family physicians, at least one year after having carried out individual interviews. Our aim with this technique will be to gather participants' convergent and divergent views with regard to the success or failure of impression management strategies adopted, as well as their intended and unintended consequences at the individual, interpersonal, group, and organizational levels. We plan to conduct a total of 8-12 focus groups; that is 2-3 focus groups per medical schools. Focus groups will also be taperecorded, and then transcribed and analyzed with the support of the HyperRESEARCH 3.0 package. They will be conducted by one of the three respective national researchers, with the support of the other two. In order to ensure validity of the data collected, debriefing meetings among the three members of the research team will take place immediately after each focus group.
## Documentary sources
Documents constitute another important method for collecting data in qualitative research. There are many different types of documents: minutes of meetings, books, manuals, administrative publications, newspapers and magazines, charts, tables, lists, and so on. Documents can also be photos, videotapes, films. And all these documents may already exist, or can be generated throughout the research period. The strengths of a documentary analysis would be the following: on the one hand, documents can help the researcher to elicit nuanced meanings that he or she is trying to understand and, on the other, documents help broaden the understanding of the context surrounding the phenomenon under examination [bib_ref] Thousand Oaks: Sage, Patton [/bib_ref] [bib_ref] The interpretation of documents and material culture, Hodder [/bib_ref]. In the proposed research, archival material will thus enrich our understanding of the academic context within which the family physician's professional identity is constantly constructed. The fact that all the applicants are professors and/or researchers in the faculties of medicine included in the study will facilitate access to pertinent documents and help identify those truly meaningful for the purpose of the study. Finally, diary techniques (i.e. description of any process and event related to the research question observed during fieldwork, summary of main points raised during interviews) complete the portrait of our methods for generating empirical material over the period of inquiry.
## Data analysis rhetorical analysis
For sake of coherence with the discursive nature of the phenomenon under examination here, we will first and foremost adopt discursive techniques for the analysis of the qualitative material generated. Discourse analysis is considered as the systematic study of texts [bib_ref] Discourse analysis in organizations: issues and concepts, Putnam [/bib_ref] ; in organizational studies, those texts compose and are composed by organizations. There is a great variety of discourse analysis traditions (e.g. discursive psychology, critical discourse analysis, conversation analysis, rhetoric, ethnography of communication, etc.) [bib_ref] Discourse analysis in organizations: issues and concepts, Putnam [/bib_ref] [bib_ref] The study of discourse, Van Dijk [/bib_ref]. In this study, as we are interested in examining how family physicians will try to discursively manage others' views about the family physician profession, that is how they will use persuasive speech in order to construct an enhance professional image, rhetoric is the discursive approach to adopt. Indeed, impression management will be nothing but rhetorical strategies adopted to promote institutional change [bib_ref] The contribution of a discursive view to understanding and managing organizational change, Heracleous [/bib_ref] [bib_ref] Organizational change as discourse: communicative actions and deep structures in the context..., Heracleous [/bib_ref] [bib_ref] Institutional field dynamics and the competition between institutional logics: the role of..., Green [/bib_ref] [bib_ref] Challenges for institutional theory, Suddaby [/bib_ref]. Furthermore, "language, particularly rhetoric, plays a central role in how professions reproduce social, cultural and symbolic capital within a field. Language is a crucial weapon in this process and professionals are skilled rhetoricians" [bib_ref] Professionals and field-level change: institutional work and the professional project, Suddaby [/bib_ref]. Rhetorical analysis, which implies the description and interpretation of how effectively a text persuades and convinces an intended audience [bib_ref] Corporate rhetoric as organizational discourse, Cheney [/bib_ref] [bib_ref] Rhetorical strategies of legitimacy, Suddaby [/bib_ref] will be applied to both texts from interviews and documents gathered. Our organizational rhetoric analysis, largely influenced by critical theory [bib_ref] Corporate rhetoric as organizational discourse, Cheney [/bib_ref] , will be performed by each national group, under the coordination of the principal investigator.
## Within-case and cross-case analysis
Due to the fact that our study involves more than one case, as well as different levels of analysis and different type of data, we will undertake two major analytical steps, i.e. within-and cross-case analysis stages. Withincase analysis will let us describe, understand and explain how family physicians operating in a particular institutional context construct their professional image, and with what immediate effects. Then, cross-case analysis will allow us recognize and explain regularities and variations among the four international medical schools involved in the investigation.
# Discussion
The results of our study will potentially have important implications for the practice of family medicine, medical education, and health and educational policies. The processes by which medical students' choose a residency program are complex, relying on a multiplicity of arguments that can be located at different levels, from personal to societal dimensions. Family medicine stakeholders (professionals, patients, policy decision-makers) of different health care systems have mostly focused on improving the working conditions of family physicians/general practitioners once they graduate. This being said, we argue that several interrelated initiatives could be undertaken in medical schools to help generate new institutional academic discourses that contribute to the formation of an enhanced family medicine professional identity. As our prior investigation has highlighted, unanimous agreement exists among medical students of several countries with regard to the lack of prestige of family medicine, reinforced through undergraduate training in academic contexts that clearly associate medical expertise and excellence with specialized medical knowledge. Such a low reputation of the profession is also reinforced by health care system and societal contexts where specialized medical knowledge is most valued, and may prevent even students who may initially be interested in this professional practice from finally choosing this path. To further reverse this trend and, therefore, support students' identification with this profession, more attention should be paid to family physicians' professional identity formation processes in medical schools; further research exploring such processes should thus be conducted. This is the commitment of our international research team, which in the present investigation aims to examine how academic family physicians reconstruct their professional image. As noted by Cooke and collaborators as closing remark of their 2010 book, "preparing physicians who have a firm professional identity, who continuously seek excellence through inquiry, and who are engaged as members of a moral community will ensure the highest-quality care for patients". Indeed, the results of our investigation will thus accomplish what Rist [bib_ref] Influencing the policy process with qualitative research, Rist [/bib_ref] qualifies as the "enlightenment function" of policy research: "Viewing policy research as serving an enlightenment function suggests that policy researchers work with policy makers and their staffs over time to create a contextual understanding about an issue, build linkages that will exist over time, and strive constantly to educate about new developments and research findings in the area" (p. 1003).
While we are deeply interested in supporting the practice of family medicine and education in the discipline, we also intend to further contribute to knowledge generation about professional identity and organizations. In this regard, the research proposed here is original and enhances the literature in organizational studies and health services and policy research in a number of ways. First, as pointed out in the literature review of this protocol, not only are theoretical contributions to professional image construct scant, but empirical research on family physicians professional image construct is inexistent. Second, the investigation is even more challenging and original as we propose to undertake an international research investigation that examines such processes in very different academic, institutional and national contexts. Third, the investigation is also innovative because it adopts a process approach and a longitudinal design. As noted by the organizers of the Symposium 'Doing Longitudinal Studies of Health Care Change: Studying Health Care Change' at the last Academy of Management Annual meeting: "In-depth longitudinal case studies of health care change are needed to better understand how these processes occur and how they might be more successfully managed" [bib_ref] Doing Longitudinal Studies of Health Care Change: Studying Health Care Change, Denis [/bib_ref]. In the end, the present investigation is also innovative in regard to the theoretical perspective adopted, i.e. institutional theory. More specifically, the originality of this study is related not only to the use of one of the most compelling theories in current organizational and management literature but also to its combination with identity and image corpus of knowledge, as well as our focus on the intertwinement between professions and organizations. As noted by Greenwood and collaborators [bib_ref] Suddaby R: Introduction. In The SAGE Handbook of Organizational Institutionalism, Greenwood [/bib_ref] : "In its own neglect of the more micro-dynamics of sensemaking, institutional theory has relinquished the opportunity to develop a richer theory of the intersubjective processes of perception, interpretation and interaction that establish the core of a micro-level understanding of institutionalization" (p.30).
[fig] Figure 1: Impression management in the construction of professional image. [/fig]
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A case report of renal calyceal diverticulum with hypertension in children and review of literature
Background: Renal calyx diverticulum refers to a cystic lesion covered with the transitional epithelium in the renal parenchyma. Although there is no clear evidence that calyx diverticulum can cause hypertension, there exists a close association between the two, and there are few related reports. Herein, we reported the case of a child with renal calyx diverticulum complicated with hypertension and summarized the diagnosis and treatment.Case presentation: Physical examination of the patient, an 11-year-old child, revealed a left renal cyst with hypertension (155/116 mmHg). There were no related symptoms. Routine urine and blood biochemical examinations showed no abnormalities. Imaging revealed left renal cyst compression causing the hypertension. She underwent renal cyst fluid aspiration and injection of a sclerosing agent into the capsule, but her blood pressure increased again 3 days postoperatively. Color Doppler ultrasonography showed that the size of the left renal cyst was the same as that preoperatively. To further confirm the diagnosis, cystoscopic retrograde ureteropyelography was performed to confirm the diagnosis of renal calyx diverticulum. Subsequently, renal calyceal diverticulum resection and calyx neck enlargement were performed. The operation went smoothly and the blood pressure returned to normal postoperatively. No abnormalities were noted at the 7-month postoperative follow-up.Conclusion:There exists an association between renal calyx diverticulum and hypertension. Therefore, hypertension can be considered a surgical indication for renal calyx diverticulum. Moreover, renal calyceal diverticulum in children can be easily misdiagnosed as a renal cyst. Therefore, it is important to be vigilant to prevent a series of complications, such as postoperative urine leakage, in such cases.BackgroundRenal calyceal diverticulum refers to a cystic lesion covered with the transitional epithelium in the renal parenchyma [1], which is connected to the calyceal or renal pelvis through a narrow passage. It is difficult to differentiate between this lesion, the renal pelvis, and paracal cystic diseases [2], which could easily lead to misdiagnosis. Presently, reports on hypertension caused by renal calyceal diverticulum in children are rare. Here we report a case of renal calyceal diverticulum with hypertension in The Fourth Hospital of Baotou.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
## Case presentation
This study was approved by the ethics committee of The Fourth Hospital of Baotou (ethics approval number is not applicable for our ethics committee). Written informed consent was obtained from the patient's parents.
The patient was an 11-year-old girl whose physical examination revealed left renal cyst with hypertension (blood pressure, 155/116 mmHg). She had no back pain and no other symptoms such as frequent, acute, or painful urination. Her blood pressure upon admission was 153/113 mmHg. Oral amlodipine besylate (benzenesulfonic acid amlodipine) was used to normalize the blood pressure, following which routine urine and blood biochemical tests were performed. Urological CT revealed normal size, shape, and location of both kidneys. The left kidney was round, had a clear outline, had low density, and measured about 47.2 × 46.1 × 59.3 mm in size, with clear boundaries on CT at approximately 6 HU. No significant enhancement was noted in enhanced CT for any of the phases. There was also no abnormal density in the fatty capsule surrounding both kidneys, no separation of the bilateral assembly system, and no thickening sign in the perirenal fascia [fig_ref] Figure 1: Preoperative urological CT [/fig_ref]. Upon CTA of both renal arteries, both arteries were found to be of normal shape at their origins. There was no obvious stenosis and expansion. The left renal vein was normal and there was no obvious compression stenosis. Serum renin, serum angiotensin, serum aldosterone, thyroid function, plasma cortisol, and blood catecholamine levels were normal.
Hypertension due to compression of the renal cyst was considered on the basis of imaging. She was admitted to the hospital and was monitored using color ultrasonography. Renal cyst fluid aspiration was performed, followed by intracapsular injection of a sclerosing agent. Her blood pressure returned to normal, but increased again on the third day. Repeat ultrasonography showed that the left renal cyst had returned to its pre-aspiration size. Hence, we noted the following: 1. The injection of a sclerosing agent in the renal cyst is ineffective in children; soon after aspiration, the cyst fills again; hence, it is important to be vigilant for renal calyx diverticulum. 2. Hypertension in children is associated with cysts. Cystoscopic retrograde ureteropyelography was performed to further clarify the diagnosis. After anesthesia, cystoscopic retrograde intubation of the left ureter showed normal left upper and lower renal calyces and a spherically dilated middle calyceal contrast entry with a cyst attached to the renal pelvis [fig_ref] Figure 2: Intraoperative C-arm view [/fig_ref]. The diagnosis of diverticulum of the renal calyces was confirmed. Open resection of the calyx diverticulum and enlargement of the calyx neck were performed. Upon intraoperative incision of the thin diverticular wall of the renal calyces, the diverticulum was observed to originate from the middle calyces of the kidney and the neck of the calyces was apparently narrowed. The narrowed calyces were enlarged to a diameter of approximately 1 cm by probing, and the incision was sutured continuously with locked edges to prevent restenosis. Subsequently, most of the dilated diverticular wall was excised and the thicker residual part of the calyx wall was closed. The 8-Fr catheter was left in the calyx neck as a stent tube, and the operation went smoothly. The patient's blood pressure decreased to normal on the postoperative day without the use of oral antihypertensive The left superior and inferior calyces were normal, the middle calyces were spherically dilated, and the diverticulum was attached to the renal pelvis drugs, and the stent tube was removed 1 week postoperatively. At the 7-month postoperative follow-up and urological CT [fig_ref] Figure 3: Seven-month postoperative urological CT [/fig_ref] , the patient showed good recovery, with no blood pressure abnormalities since discharge.
## Literature review
The PubMed database was searched using the search term "calyceal diverticulum" for articles published between January 2010 and December 2020. A total of 36 patients with calyceal diverticulum were reported in the literature, which, combined with this report, makes 37 cases. The average age of the patients was 21.2 years (1-82 years), and the average age of the children was 8.5 years (1-17 years) in 21 cases. There were four cases with no symptoms. No cases of hypertension in combination with renal diverticulum were reported. Fifteen cases were treated surgically, as described in [fig_ref] Table 1: General information of patients with renal calyx diverticulum [/fig_ref] [bib_ref] Challenges in the diagnosis of calyceal diverticulum: a report of two cases..., Zhang [/bib_ref] [bib_ref] Treatment options for Calyceal diverticula, Smyth [/bib_ref] [bib_ref] Squamous cell carcinoma in a Calyceal diverticulum detected by percutaneous Nephroscopic biopsy, Mitome [/bib_ref] [bib_ref] Rupture of a Calyceal diverticulum secondary to Ureteroscopy: a rare complication, Yamasaki [/bib_ref] [bib_ref] A Petal-like Calyceal Diverticulum, Zhang [/bib_ref] [bib_ref] Calyceal diverticulum mimicking simple parapelvic cyst: a case report, Peng [/bib_ref] [bib_ref] Laparoscopic calyceal diverticulectomy, Bonastre [/bib_ref] [bib_ref] Robotic treatment of a type 2 calyceal diverticulum in a child: is..., Sripathi [/bib_ref] [bib_ref] The left ureterocele and stone of calyceal diverticulum in the patient with..., Pan [/bib_ref] [bib_ref] Calyceal diverticulum: a benign imitator of serious pathology, Pareek [/bib_ref] [bib_ref] Retrograde intrarenal surgery in atretic calyceal diverticular stone, a case report, Ng [/bib_ref] [bib_ref] Percutaneous holmium laser fulguration of calyceal diverticula, Alwaal [/bib_ref] [bib_ref] Bacterial sepsis after extracorporeal shock-wave lithotripsy (ESWL) of calyceal diverticular stone, Oh [/bib_ref] [bib_ref] Invasive urothelial carcinoma within a calyceal diverticulum associated with renal stones: a..., Nakano [/bib_ref] [bib_ref] Calyceal diverticulum with a large extra-renal extension in a 5-year-old child, Ferroni [/bib_ref] [bib_ref] Calyceal diverticulum of the kidney -diagnostic imaging dilemma in pediatric patients -case..., Bombiński [/bib_ref] [bib_ref] Potential role of Tc-99m DTPA diuretic renal scan in the diagnosis of..., Lin [/bib_ref] [bib_ref] Calyceal diverticulum of the kidney in pediatric patients -is it as rare..., Alaygut [/bib_ref]. Two of these cases could not be treated owing to the severity of the disease.
# Discussion and conclusion
The prevalence of hypertension in children in China is 14.5% and is higher in males (16.1%) than in females (12.9%) [bib_ref] The association of overweight and obesity with blood pressure among Chinese children..., Dong [/bib_ref]. Hypertension in early childhood often has no obvious symptoms. Common causes of this condition include congenital aortic stenosis, congenital renal hypoplasia, congenital urinary tract malformations, renal artery stenosis, latent glomerulonephritis, and adrenal disease. There are very few reports stating that hypertension in childhood is caused by renal calyceal diverticulum. Diverticula of the renal calyces in children is clinically rare, with a documented incidence of 0.6% [bib_ref] Management of stones in patients with anomalously sited kidneys, Gross [/bib_ref]. A renal calyceal diverticulum is a sac-like structure that is located in the renal parenchyma and connected to the renal calyx. According to the different connection positions of the passage, it can be divided into type I and type II diverticulum. The former is connected to the minor renal calyx, mostly at one pole of the kidney, and the latter is connected to the major renal calyx, mostly at the central part of the kidney [bib_ref] Incision and internal drainage treatment of renal cyst and calyceal diverticulum by..., Yu [/bib_ref].
Secondary stones are more common in cases of calvarial diverticula, with a reported clinical incidence of 9.5-50.0% among cases of calvarial diverticula caused by outflow tract obstruction and urinary reflux [bib_ref] Outcomes of septoplasty in young adults: the nasal obstruction Septoplasty effectiveness study, Gandomi [/bib_ref]. In the last decade, 10 of 36 cases of renal calyx diverticulum were complicated by stones. Further, among other clinical manifestations, pain was the most common symptom (17/36), and concomitant symptoms, such as fever or bladder irritation, were often present when secondary urinary tract infection was present. In children, abdominal pain was the most common symptom (6/21). Four children had a combination of stones, and the diverticulum was rarely diagnosed in children. Considering that it resembles other cystic lesions of the kidney, further evaluation of children with renal cysts should be done in the presence of back pain, recurrent urinary tract infections, hematuria, and stones [bib_ref] Calyceal diverticulum of the kidney in pediatric patients -is it as rare..., Alaygut [/bib_ref]. Per our experience, this disease may be misdiagnosed as renal cysts using ultrasonography and plain or even enhanced CT. Therefore, delayed contrast or delayed enhanced CT should be performed when the diagnosis is not confirmed or when renal calyx diverticulum is suspected. In addition, retrograde urography may also be performed to clarify the diagnosis. This CT + pyelography approach was used to confirm the diagnosis in 16 of 36 cases of renal calyx diverticulum. In cases for which imaging is not possible, cyst fluid aspiration can be performed under ultrasoundguided localization to assist in the diagnosis based on the cyst fluid composition [bib_ref] Challenges in the diagnosis of calyceal diverticulum: a report of two cases..., Zhang [/bib_ref] , which has not been reported in the literature in the last decade. Renal calyceal diverticulum is not common among children, and only 20% of the cases eventually present symptoms [bib_ref] Robotic treatment of a type 2 calyceal diverticulum in a child: is..., Sripathi [/bib_ref]. In the last decade, 36 cases of renal calyx diverticulum were investigated, among which 21 were of children. Further, 15 patients were treated surgically for more obvious complications and 2 were treated symptomatically because they could not tolerate surgery. The treatment of renal calyx diverticulum needs to be determined by clinical symptoms. Symptomatic treatment can be given to children with no symptoms or mild clinical symptoms and for small-sized diverticula. Regular follow-up and surgery are recommended for children with large diverticula (> 4 cm) or complications. Kavukcu et al. [bib_ref] Diagnosis of caliceal diverticulum in two pediatric patients: a comparison of sonography,..., Kavukcu [/bib_ref] proposed that the treatment of the diverticulum depends on the complications, including repeated kidney infections, hematuria, and symptomatic kidney stones. Considering our experience, we believe that for children with hypertension and renal calyceal diverticulum, if the cause of hypertension cannot be determined, surgery should be performed in time. In our case, retrograde ureterography was performed preoperatively and a ureteral stent tube was left in place as a marker. The wall of the diverticulum was incised, the stenotic opening was found, the opening of the diverticulum was enlarged, the ureteral stent tube was visible and was determined to be connected to the renal pelvis, and the enlarged diverticulum opening was sutured with locked edges, with the stent tube left in place for support. The diverticulum wall was excised and the portion with the remaining renal tissue was sutured closed. The stent tube was removed 1 week postoperatively, and the patient's blood pressure returned to normal. The diverticulum did not recur on repeat ultrasonography and enhanced CT performed 7 months postoperatively, and the patient's blood pressure continued to remain normal.
Long et al. [bib_ref] Pediatric calyceal diverticulum treatment: an experience with endoscopic and laparoscopic approaches, Long [/bib_ref] suggested that ureteroscopy should be selected for middle and upper renal calyceal diverticula, whereas laparoscopic treatment should be considered first for larger exogenous lesions and lower pole diverticula. In well-equipped hospitals, robot-assisted technology can be used to help accurately identify and ligate the opening of the renal calyceal diverticulum to achieve better surgical outcomes [bib_ref] Robotic treatment of a type 2 calyceal diverticulum in a child: is..., Sripathi [/bib_ref] [bib_ref] 84 robot assisted laparoscopic calyceal diverticulum repair, Naeyer [/bib_ref]. If diverticulum-induced stones are found in the preoperative examination, endoscopic percutaneous nephrolithotomy or laparoscopic treatment can be performed according to the location. However, ESWL is not ideal in the treatment of such stones [bib_ref] Percutaneous Management of Stone Containing Calyceal Diverticula: associated factors and outcomes, Parkhomenko [/bib_ref] [bib_ref] Management of stones in calyceal diverticulum, Gross [/bib_ref].
The diagnosis in this case was renal calyceal diverticulum with hypertension. Although there is no clear evidence stating that renal calyceal diverticulum can cause hypertension, there exists a close association between the two. During kidney transplantation, high blood pressure can also be "transplanted" along with the kidney, which sufficiently proves the important role of the kidney in blood pressure regulation. Any kidney disease could cause abnormal blood pressure [bib_ref] Hypertension after kidney transplant, Mangray [/bib_ref] [bib_ref] The kidney and hypertension: over 70 years of research, Campese [/bib_ref] [bib_ref] Hypertension and kidney disease: is renalase a new player or an innocent..., Malyszko [/bib_ref]. Presently, the mechanisms by which kidney diseases can increase blood pressure mainly include sodium ion retention, renin-angiotensin system (RAS) dysregulation, sympathetic nervous system dysfunction, and endothelial cell-mediated vasodilation impairment [bib_ref] The kidney and hypertension: over 70 years of research, Campese [/bib_ref]. In recent years, some scholars have discovered that the renal enzyme system is closely related to blood pressure [bib_ref] Renalase in hypertension and kidney disease, Desir [/bib_ref] , and its way of regulating blood pressure might be related to the metabolism of NADH [bib_ref] Renalase deficiency aggravates ischemic myocardial damage, Wu [/bib_ref] and catecholamines and the transport of sodium in the proximal renal tubules [bib_ref] Role of renalase in the regulation of blood pressure and the renal..., Desir [/bib_ref]. Malyszko et al. [bib_ref] Renalase, hypertension, and kidney -the discussion continues, Malyszko [/bib_ref] pointed out that the mechanism underlying blood pressure regulation by renal enzymes and the relationship with dopamine receptors and endothelial function need further confirmatory evidence, and the specific mode of action is still unclear. Our patient had type II renal calyceal diverticulum with a large cyst. The cause of hypertension in this case could not be determined preoperatively, and the blood pressure immediately returned to normal postoperatively. Therefore, we considered the increase in blood pressure to be caused by cyst compression. The major reasons for this could be as follows: (1) Expansion of the cyst connected with the renal calyx compressed the sympathetic nerve branch, thereby causing constriction of the afferent arteriole of the corresponding nephron. (2) Compression caused renal ischemia and RAS activation. The formation of bladder cysts and long-term compression of the kidney might result in the loss of some nephrons and affect blood pressure regulation. (3) Abnormalities in the RAS might lead to abnormal cholesterol function, thereby affecting blood pressure regulation.
There exists an association between renal calyceal diverticulum and hypertension; hypertension can thus be a surgical indication in cases of renal calyceal diverticulum. In addition, renal calyceal diverticulum can be easily misdiagnosed as a renal cyst in children. Hence, it is important to be vigilant to prevent a series of complications, such as postoperative urine leakage, in such cases.
Abbreviations CT: Computed tomography; CTA : Computed tomography angiography; RAS: Renin-angiotensin system; NADH: Nicotinamide adenine dinucleotide.
[fig] Figure 1: Preoperative urological CT. A rounded hypointense shadow (approximately 47.2 × 46.1 × 59.3 mm) with clear borders observed in the left kidney [/fig]
[fig] Figure 2: Intraoperative C-arm view. [/fig]
[fig] Figure 3: Seven-month postoperative urological CT. The left kidney was slightly larger, with irregular morphology of the superior parenchyma, slightly enlarged local renal pelvis, and normal renal structure, considering postoperative changes [/fig]
[table] Table 1: General information of patients with renal calyx diverticulum [/table]
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Wool Keratin-Associated Protein Genes in Sheep—A Review
The importance of sheep's wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses.
# Introduction
Wool is a natural fibre with unique attributes. It is widely used in the apparel, insulation and carpet industries, and humans have long recognised the benefits of wool, including its thermal attributes, breathability and fire resistance.
Wool fibre is primarily composed of proteins called hard α-keratins [bib_ref] Hair-The most sophisticated biological composite material, Popescu [/bib_ref]. These have a high sulphur content, consistent with having a high relative abundance of the amino acid cysteine. In wool, the α-keratins are assembled into keratin intermediate filaments (KIFs), before being embedded in an inter-filamentous matrix containing keratin-associated proteins (KAPs). There are also other protein components in wool, such as trichohyalin, which is located in the inner root sheath and the medulla of the fibre, but these proteins are not regarded as essential to the fibre structure [bib_ref] Hair-The most sophisticated biological composite material, Popescu [/bib_ref].
The earliest attempt to identify and classify wool proteins was made in 1935 [bib_ref] Derivatives of keratin, Goddard [/bib_ref] , and it divided the major wool components into two classes of extractable protein: S-carboxy methyl kerateine A (SCMK-A) and S-carboxy methyl kerateine B (SCMK-B). These were of lower and higher sulphur
## The complexity of wool kaps
Electrophoretic analyses of the SCMK-Bs revealed that a large number of KAPs existed [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. Seven KAPs with a high sulphur content (B2A or KAP1-1, B2B or KAP1-2, B2C or KAP1-3, BIIIA3 or KAP2-n, BIIIB2 or KAP3-1, BIIIB3 or KAP3-3 and BIIIB4 or KAP3-3) were initially isolated from wool and their amino acid sequences were determined [bib_ref] Studies on the high-sulfur proteins of reduced Merino wool. Part III: The..., Haylett [/bib_ref] [bib_ref] Amino-acid sequence of a high-sulphur protein from wool, Elleman [/bib_ref] [bib_ref] Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence..., Haylett [/bib_ref] [bib_ref] Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence..., Swart [/bib_ref] [bib_ref] The amino acid sequence of protein SCMK-B2A from the high-sulphur fraction of..., Elleman [/bib_ref] [bib_ref] The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of..., Elleman [/bib_ref] [bib_ref] The sequence of SCMK-B2B, a high-sulfur protein from wool keratin, Elleman [/bib_ref] [bib_ref] Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence..., Swart [/bib_ref] and two KAPs with a high glycine-tyrosine content were also isolated and characterised [bib_ref] The primary structure of a protein, component 0.62, rich in glycine and..., Dopheide [/bib_ref].
With the advent of DNA cloning techniques, 16 gene sequences encoding KAPs of a high sulphur content [bib_ref] Mammalian keratin gene families: Organisation of genes coding for the B2 high-sulphur..., Powell [/bib_ref] [bib_ref] The keratin BIIIB gene family: Isolation of cDNA clones and structure of..., Frenkel [/bib_ref] [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] Dietary cysteine regulates the levels of messenger-rnas encoding a family of cysteine-rich..., Fratini [/bib_ref] [bib_ref] Differential expression of genes encoding a cysteine-rich keratin family in the hair..., Jenkins [/bib_ref] and a high glycine-tyrosine content [bib_ref] Sheep keratins: Characterization of cDNA clones for the glycine + tyrosine-rich wool..., Kuczek [/bib_ref] [bib_ref] Sheep wool (glycine + tyrosine)-rich keratin genes, Kuczek [/bib_ref] [bib_ref] Sequence, expression, and evolutionary conservation of a gene encoding a glycine tyrosine-rich..., Fratini [/bib_ref] were discovered through the isolation of their cDNA or genomic sequences. The subsequent development of further gene technologies, together with the sequence information derived from other species, has led to the identification of another five high sulphur KAP genes [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref] [bib_ref] Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep, Gong [/bib_ref] [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref] and four high glycine-tyrosine [bib_ref] Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (KAP6) family in sheep, Gong [/bib_ref] [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref] KAP genes in sheep.
Sequence analyses suggest that the KAP sequences identified to date probably represent 27 different KAP family members [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref]. However, the possibility cannot be ruled out that some of these 27 may represent different variants of the same gene, rather than being different family members. This was shown for the human KAP1 [bib_ref] Polymorphisms in the human high sulfur hair keratin-associated protein 1, KAP1, gene..., Shimomura [/bib_ref] and KAP4 [bib_ref] Size polymorphisms in the human ultrahigh sulfur hair keratin-associated protein 4, KAP4,..., Kariya [/bib_ref] genes. This suggests that further investigation is required to confirm both the number and constituency of the families, especially as our current understanding of the wool KAPs is still limited. For example, there may be ovine KAP genes that have not been identified yet, especially given that 88 functional and 17 pseudo-KAP genes have now been described in the human genome [bib_ref] Human KAP genes, only the half of it? Extensive size polymorphisms in..., Rogers [/bib_ref] [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref] [bib_ref] Characterization and expression analysis of the hair keratin associated protein KAP26.1, Rogers [/bib_ref]. This likely represents the complete number of KAPs in humans, although sequencing of more human genetic material from different races and genetic backgrounds may result in minor changes to this thinking.
## The categorisation and classification of wool kaps
Following the original classification into SCMK-A and SCMK-B, the KAPs were subsequently categorised into three groups according to their amino acid composition: the high sulphur (HS; ď30 mol% cysteine), the ultra-high sulphur (UHS; >30 mol% cysteine) and the high glycine/tyrosine (HGT; 35-60 mol% glycine and tyrosine) groups [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. They were then assigned into families based on amino acid sequence homology.
In general terms, the individual KAP families are distinguished by the cysteine, or glycine and tyrosine content, the type and number of amino acid repeat structures, and the occurrence of unique sequence motifs [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref]. In humans, the 88 functional KAP genes identified have been assigned to 25 families: KAP1 to KAP27, but with the absence of KAP14 and KAP18, which have only been found in mice [bib_ref] Human KAP genes, only the half of it? Extensive size polymorphisms in..., Rogers [/bib_ref] [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref] [bib_ref] Characterization and expression analysis of the hair keratin associated protein KAP26.1, Rogers [/bib_ref].
In sheep, 27 putative wool KAPs having been identified to date and these have been assigned into the following 11 families: KAP1: KAP1 is a HS-KAP family that contains four members (KAP1-1, KAP1-2, KAP1-3 and KAP1-4) [bib_ref] The amino acid sequence of protein SCMK-B2A from the high-sulphur fraction of..., Elleman [/bib_ref] [bib_ref] The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of..., Elleman [/bib_ref] [bib_ref] Mammalian keratin gene families: Organisation of genes coding for the B2 high-sulphur..., Powell [/bib_ref] [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref]. The number of members equals that described in humans [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref]. The KAP1 family members are similar to each other and differ mainly in the number of conserved tandem decapeptide "QTSCCQPXXX" repeats in the N-terminal half of the proteins [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. There are between three to five decapeptide repeats in KAP1-1 [bib_ref] Polymorphism in two genes for B2 high sulfur proteins of wool, Rogers [/bib_ref] , whereas KAP1-2, KAP1-3 and KAP1-4 have three, two and five repeats, respectively [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Polymorphism of the KAP1.1, KAP1.3 and K33 genes in Merino sheep, Itenge-Mweza [/bib_ref] [bib_ref] Polymorphism of the ovine keratin-associated protein 1-4 gene (KRTAP1-4), Gong [/bib_ref]. There is an apparent loss of a five residue C-terminus tail in KAP1-2, compared to the other KAP1 proteins [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] and KAP1-1 and KAP1-4 are acidic KAPs, while KAP1-2 and KAP1-3 are neutral [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP2: KAP2 is a HS-KAP family. Five family members have been described in humans [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref] , but only two protein sequences (BIIIA3A and BIIIA3) have been reported in sheep [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. These ovine sequences share 95% homology.
BIIIA3A is referred to as the orthologue of human KAP2-1 [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] , although the same human sequence was previously called KAP2.12 [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. BIIIA3 is identical to the predicated amino acid sequence of a partial DNA sequence referred to as KAP2-3 (GenBank U60024). To date no ovine KAP2-2 gene has not been reported. Given that the KAP2 family is conserved in humans, with over 97% sequence identity between the family members [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref] , it is likely that these two wool KAP2 sequences represent two members of the same family. The KAP2 family members possess several cysteine-rich pentameric repeat structures (CCXPX) and length differences are also observed among family members in both sheep and humans. The KAP2 proteins are weakly basic [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP3: There are three major (originally designated as BIIIB2, BIIIB3 and BIIIB4) and one minor (originally called BIIIB1) proteins described for ovine KAP3 [bib_ref] Studies on the high-sulfur proteins of reduced Merino wool. Part II1: The..., Swart [/bib_ref]. The three major proteins have been isolated and sequenced [bib_ref] Studies on the high-sulfur proteins of reduced Merino wool. Part III: The..., Haylett [/bib_ref] [bib_ref] Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence..., Haylett [/bib_ref] [bib_ref] Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence..., Swart [/bib_ref] and the gene sequences thought to encode the major proteins have also been identified [bib_ref] The keratin BIIIB gene family: Isolation of cDNA clones and structure of..., Frenkel [/bib_ref]. These have been named KAP3-1, KAP3-2 and KAP3-3 representing BIIIB2, BIIIB3 and BIIIB4, respectively. Minor differences between the protein sequences and the gene sequences have been reported for each family member, which may reflect nucleotide variation in the genes, or alternatively sequencing errors. The BIIIB2 protein sequence (labelled as KAP3-1A) [bib_ref] Studies on the high-sulfur proteins of reduced Merino wool. Part III: The..., Haylett [/bib_ref] appears to be different to the predicted amino acid sequence derived from translation of the BIIIB2 gene (labelled as KAP3-1B) [bib_ref] The keratin BIIIB gene family: Isolation of cDNA clones and structure of..., Frenkel [/bib_ref] , and shares only 94% homology at the amino acid sequence level. Interestingly, these two proteins would appear to have different pI values [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref] , so whether these two BIIIB2 sequences represent different family members, or are variant forms of the same family member, requires further investigation. The human KAP3 family contains three functional genes and one pseudogene [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref]. KAP3-2 and KAP3-3 are very similar to each other, but different to KAP3-1 [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. Little repetitiveness of structure is seen in this family and the proteins are generally neutral. The dashes represent amino acids identical to the top sequence and the dots act as spacers to improve alignments. The QTSCCQPXXX decapeptide repeat sequence found in KAP1-n and the CCVPX pentameric repeat sequence found in KAP2-n are shown in boxes. The central region consisting of contcatenates of nonocysteine-and dicysteine-containing pentameric repeats found in KAP4-n is shaded. In KAP5-n, the glycine-rich decapeptide repeat sequence is boxed, whereas the cycteine/serine-rich decapeptide repeat sequence is both boxed and shaded. All the amino acid sequences are predicated from DNA sequences, except for those indicated by * which were derived directly from protein sequencing. # indicates a partial sequence. The sheep, goat, cattle and human sequences are indicated with a prefix of "s", "g", "c" and "h", respectively. Accession numbers or references for the ovine sequences are shown in [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref] , and those from other species are: AB096962 (hKAP7-1); AY510121 (gKAP7-1); AP001709 (hKAP8-1); AY510123 (gKAP8-2); NM_001080740 (cKAP11-1); NW_001838706 (hKAP11-1); ENSBTAG00000040032 (cKAP13-3); NM_181622.1 (hKAP13-3); XM_002684598.1 (cKAP24-1) and NM_001085455 (hKAP24-1). The dashes represent amino acids identical to the top sequence and the dots act as spacers to improve alignments. The QTSCCQPXXX decapeptide repeat sequence found in KAP1-n and the CCVPX pentameric repeat sequence found in KAP2-n are shown in boxes. The central region consisting of contcatenates of nonocysteine-and dicysteine-containing pentameric repeats found in KAP4-n is shaded. In KAP5-n, the glycine-rich decapeptide repeat sequence is boxed, whereas the cycteine/serine-rich decapeptide repeat sequence is both boxed and shaded. All the amino acid sequences are predicated from DNA sequences, except for those indicated by * which were derived directly from protein sequencing. # indicates a partial sequence. The sheep, goat, cattle and human sequences are indicated with a prefix of "s", "g", "c" and "h", respectively. Accession numbers or references for the ovine sequences are shown in [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref] , and those from other species are: AB096962 (hKAP7-1); AY510121 (gKAP7-1); AP001709 (hKAP8-1); AY510123 (gKAP8-2); NM_001080740 (cKAP11-1); NW_001838706 (hKAP11-1); ENSBTAG00000040032 (cKAP13-3); NM_181622.1 (hKAP13-3); XM_002684598.1 (cKAP24-1) and NM_001085455 (hKAP24-1).
KAP4: KAP4 is a UHS-KAP family. This family is reported to contain 11 members in humans [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref] , but only three members have been identified in sheep [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref] [bib_ref] Dietary cysteine regulates the levels of messenger-rnas encoding a family of cysteine-rich..., Fratini [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref]. The ovine KAP4 proteins possess a repeat structure that covers a large portion of the middle section of the protein and consists of concatenates of a monocysteine-and dicysteine-containing pentameric repeat structure [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. The proteins are weakly basic [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP5: KAP5 belongs to the UHS group and is one of the largest KAP families, with 12 family members described in humans [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Human KAP genes, only the half of it? Extensive size polymorphisms in..., Rogers [/bib_ref]. In sheep, three complete and one partial DNA sequences have been identified [bib_ref] Hair keratin: Composition, structure and biogenesis, Powell [/bib_ref] [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] Differential expression of genes encoding a cysteine-rich keratin family in the hair..., Jenkins [/bib_ref] and these have been designated KAP5-1, KAP5-2, KAP5-4 and KAP5-5. Sheep KAP5 proteins possess repeat structures that are either cysteine/serine-rich, or glycine-rich, and the repeat number varies between family members [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. The proteins are weakly basic [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP6: KAP6 is a HGT-KAP family. Three family members have been identified in the human genome [bib_ref] Characterization of a first domain of human high glycine-tyrosine and high sulfur..., Rogers [/bib_ref]. Southern-hybridisation analysis revealed that there were potentially several family members in sheep and as many as twenty in mice [bib_ref] Sequence, expression, and evolutionary conservation of a gene encoding a glycine tyrosine-rich..., Fratini [/bib_ref]. Early research identified two ovine members designated KAP6-1 and KAP6-2 [bib_ref] Sequence, expression, and evolutionary conservation of a gene encoding a glycine tyrosine-rich..., Fratini [/bib_ref] , but three additional members (designated KAP6-3, KAP6-4 and KAP6-5) have recently been identified in the sheep genome and submitted to GenBank [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. The KAP6 proteins consist of repetitive units of glycine-tyrosine and glycine-tyrosine-glycine [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref] , and they are weakly basic [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP7: KAP7 belongs to a HGT-KAP family. This KAP family contains only one family member designated KAP7-1 [bib_ref] Sheep wool (glycine + tyrosine)-rich keratin genes, Kuczek [/bib_ref]. There is high sequence similarity between the orthologs from sheep and humans, and the number of glycine-tyrosine repeat structures is small compared to the KAP6 family [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. Ovine KAP7-1 is basic, with a pI value of 8.7 [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP8: KAP8 is a HGT-KAP family. In sheep, it was thought to contain only one member [bib_ref] Sheep wool (glycine + tyrosine)-rich keratin genes, Kuczek [/bib_ref] , but another potential member called KAP8-2has recently been identified. In humans only one KAP8 gene has been identified [bib_ref] Characterization of a first domain of human high glycine-tyrosine and high sulfur..., Rogers [/bib_ref]. KAP8 proteins exhibit high sequence similarity across species, but have fewer glycine-tyrosine repeat structures when compared to the KAP6 family [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. KAP8-2 contains a high content (4.8 mol%) of aspartic acid and glutamic acid, which is not common in other HGT-KAPs, and hence it has a low pI value of 6.3. In sheep, KAP8-1 is weakly basic, while KAP8-2 is weakly acidic [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP11: KAP11 is a HS-KAP family consisting of a single member categorised as KAP11-1 [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref]. While KAP11-1 belongs to the HS group, the protein contains a low frequency of the "CCXP" motif that is common in the other cysteine-rich KAPs, and that is often found as part of longer repeat elements [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. This motif is present in a single copy in KAP11-1 from sheep, cattle and humans, but it is absent in mouse KAP11-1. The KAP11-1 protein does not have a high content of glycine, but instead has proportionally more serine and threonine residues [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. These two amino acids make up over 30 mol% of the residues in ovine KAP11-1 [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref] , but the biological function of having higher concentrations of serine and threonine is not known. As serine and threonine residues can be post-translationally phosphorylated, it may be associated with the phosphorylation of this protein. KAP11-1 is a weakly basic protein [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP13: Belonging to the HS group, the KAP13 family has four members in humans [bib_ref] The human type I keratin gene family: Characterization of new hair follicle..., Rogers [/bib_ref] , but only one member (KAP13-3) has been described in sheep [bib_ref] Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep, Gong [/bib_ref]. Ovine KAP13-3 has a low sequence similarity to the human orthologues, and only a poorly conserved repeat structure is found [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref]. Like KAP11-1, the ovine KAP13-3 protein contains a high content of serine and threonine, and many of these residues are potentially post-translationally modified. The ovine KAP13-3 protein contains more positively charged amino acids (arginine, lysine and histidine) and fewer negatively charged ones (aspartic acid and glutamic acid), and hence it has a pI value of 9.4 [bib_ref] Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep, Gong [/bib_ref]. It is the most basic KAP protein identified to date in sheep [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP24: There is only one member (KAP24-1) reported in sheep [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] and in humans [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref]. While probably belonging to the HS group, the KAP24-1 protein contains a moderate level of cysteine, but a high content of serine and tyrosine. High tyrosine contents are typically not observed in other HS or UHS KAPs and additionally, the tyrosine in KAP24-1 is not part of the dimeric glycine-tyrosine repeats that are found in other high glycine-tyrosine KAPs [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref]. Wool KAP24-1 is a basic protein [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref] and it has a low (~64%) sequence similarity to the human orthologue. The human KAP24-1 protein possesses a series of tandem decameric repeat structures at the C-terminal end [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref] , but similar structures are not obvious in the ovine KAP24-1 sequence [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref].
## The chromosomal location of wool kap genes
The detection of multiple KAP genes in single genomic clones from a variety of mammalian species, immediately suggested the grouping of KAP genes in the genome [bib_ref] Mammalian keratin gene families: Organisation of genes coding for the B2 high-sulphur..., Powell [/bib_ref] [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] A cluster of keratin-associated proteins on mouse chromosome 10 in the region..., Cole [/bib_ref] [bib_ref] Pmg-1 and pmg-2 constitute a novel family of KAP genes differentially expressed..., Kuhn [/bib_ref]. More recent bioinformatic analyses of genome sequences have confirmed this clustering in humans [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref] [bib_ref] Characterization of a first domain of human high glycine-tyrosine and high sulfur..., Rogers [/bib_ref] [bib_ref] Hair keratin associated proteins: Characterization of a second high sulfur KAP gene..., Rogers [/bib_ref] [bib_ref] A cluster of 21 keratin-associated protein genes within introns of another gene..., Shibuya [/bib_ref] [bib_ref] Identification of two novel clusters of ultrahigh-sulfur keratin-associated protein genes on human..., Yahagi [/bib_ref] and in a variety of others species [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] [bib_ref] Molecular evolution of the keratin associated protein gene family in mammals, role..., Wu [/bib_ref].
The ovine KAP genes identified to date have been mapped to, or located on, three chromosomes. The KAP1-n, KAP3-n and KAP4-n genes are located on OAR11 [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] [bib_ref] An Updated Nomenclature for Keratin-Associated Proteins (KAPs), Gong [/bib_ref] ; the genes for the KAP6-KAP8, KAP11, KAP13 and KAP24 families are located on OAR1 [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref] [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref] [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] ; and the KAP5-n genes are located on OAR21 [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] [bib_ref] An Updated Nomenclature for Keratin-Associated Proteins (KAPs), Gong [/bib_ref] [fig_ref] Figure 2: Clustering of the ovine KAP genes in three chromosome regions [/fig_ref]. The location of the KAP2-3 and KAP5-5 genes has not been determined, as only partial DNA sequences are available. The clustering and relative position of the various ovine KAP genes identified appears to correspond closely with that reported in humans [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref]. charged ones (aspartic acid and glutamic acid), and hence it has a pI value of 9.4 [bib_ref] Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep, Gong [/bib_ref]. It is the most basic KAP protein identified to date in sheep [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref].
KAP24: There is only one member (KAP24-1) reported in sheep [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] and in humans [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref]. While probably belonging to the HS group, the KAP24-1 protein contains a moderate level of cysteine, but a high content of serine and tyrosine. High tyrosine contents are typically not observed in other HS or UHS KAPs and additionally, the tyrosine in KAP24-1 is not part of the dimeric glycine-tyrosine repeats that are found in other high glycine-tyrosine KAPs [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref]. Wool KAP24-1 is a basic protein [fig_ref] Table 1: Ovine keratin-associated proteins [/fig_ref] and it has a low (~64%) sequence similarity to the human orthologue. The human KAP24-1 protein possesses a series of tandem decameric repeat structures at the C-terminal end [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref] , but similar structures are not obvious in the ovine KAP24-1 sequence [fig_ref] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families... [/fig_ref].
## The chromosomal location of wool kap genes
The detection of multiple KAP genes in single genomic clones from a variety of mammalian species, immediately suggested the grouping of KAP genes in the genome [bib_ref] Mammalian keratin gene families: Organisation of genes coding for the B2 high-sulphur..., Powell [/bib_ref] [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] A cluster of keratin-associated proteins on mouse chromosome 10 in the region..., Cole [/bib_ref] [bib_ref] Pmg-1 and pmg-2 constitute a novel family of KAP genes differentially expressed..., Kuhn [/bib_ref]. More recent bioinformatic analyses of genome sequences have confirmed this clustering in humans [bib_ref] Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded..., Rogers [/bib_ref] [bib_ref] Characterization of a first domain of human high glycine-tyrosine and high sulfur..., Rogers [/bib_ref] [bib_ref] Hair keratin associated proteins: Characterization of a second high sulfur KAP gene..., Rogers [/bib_ref] [bib_ref] A cluster of 21 keratin-associated protein genes within introns of another gene..., Shibuya [/bib_ref] [bib_ref] Identification of two novel clusters of ultrahigh-sulfur keratin-associated protein genes on human..., Yahagi [/bib_ref] and in a variety of others species [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] [bib_ref] Molecular evolution of the keratin associated protein gene family in mammals, role..., Wu [/bib_ref].
The ovine KAP genes identified to date have been mapped to, or located on, three chromosomes. The KAP1-n, KAP3-n and KAP4-n genes are located on OAR11 [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] [bib_ref] An Updated Nomenclature for Keratin-Associated Proteins (KAPs), Gong [/bib_ref] ; the genes for the KAP6-KAP8, KAP11, KAP13 and KAP24 families are located on OAR1 [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref] [bib_ref] Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue..., Zhou [/bib_ref] [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref] [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] ; and the KAP5-n genes are located on OAR21 [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] [bib_ref] An Updated Nomenclature for Keratin-Associated Proteins (KAPs), Gong [/bib_ref] [fig_ref] Figure 2: Clustering of the ovine KAP genes in three chromosome regions [/fig_ref]. The location of the KAP2-3 and KAP5-5 genes has not been determined, as only partial DNA sequences are available. The clustering and relative position of the various ovine KAP genes identified appears to correspond closely with that reported in humans [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Characterization of human KAP24. 1, a cuticular hair keratin-associated protein with unusual..., Rogers [/bib_ref].
## Nucleotide polymorphism within the kap genes
While the KAP genes (denoted as KRTAPs) have been identified in humans, only limited efforts have been made to fully understand the presence of nucleotide variation within those genes. To date, it has only been described for the KAP1 and KAP4 families and studies only undertaken in Caucasian and Japanese populations [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] [bib_ref] Polymorphisms in the human high sulfur hair keratin-associated protein 1, KAP1, gene..., Shimomura [/bib_ref] [bib_ref] Size polymorphisms in the human ultrahigh sulfur hair keratin-associated protein 4, KAP4,..., Kariya [/bib_ref]. In contrast, variation has been extensively looked for in ovine KAP genes, including KAP1, KAP3, KAP5, KAP6-KAP8, KAP11, KAP13 and KAP24 genes. This likely reflects the importance of wool in the production of apparel and interior textile products.
In the KAP1 family, three variant sequences containing 13 SNPs and length variation (having between three to five repeats of a 30 bp sequence) have been described for KRTAP1-1 [bib_ref] Polymorphism in two genes for B2 high sulfur proteins of wool, Rogers [/bib_ref] [bib_ref] Polymorphism of the KAP1.1, KAP1.3 and K33 genes in Merino sheep, Itenge-Mweza [/bib_ref] , 11 variant sequences containing 10 SNPs have been described for KRTAP1-2 [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref] [bib_ref] Association of wool traits with variation in the ovine KAP1-2 gene in..., Gong [/bib_ref] , nine variant sequences containing 20 SNPs have been described for KRTAP1-3 [bib_ref] Polymorphism in two genes for B2 high sulfur proteins of wool, Rogers [/bib_ref] [bib_ref] Polymorphism of the KAP1.1, KAP1.3 and K33 genes in Merino sheep, Itenge-Mweza [/bib_ref] and nine variant sequences containing 14 SNPs have been reported for KRTAP1-4 [bib_ref] Polymorphism of the ovine keratin-associated protein 1-4 gene (KRTAP1-4), Gong [/bib_ref].
The SNPs described in KRTAP1-1 and KRTAP1-4 are predominantly non-synomynous, whereas those decribed in KRTAP1-2 and KRTAP1-3 are predominantly synomynous. This appears to reflect the chromosomal locations of these genes, where KRTAP1-1 and KRTAP1-4 are close to each other on the chromosome and further removed from KRTAP1-2 and KRTAP1-3 [bib_ref] Identification of the ovine keratin-associated protein KAP1-2 gene (KRTAP1-2), Gong [/bib_ref]. It should however be noted that the sequence variants described for KRTAP1-1 have been detected by PCR and agarose gel electrophoresis. This approach is inadequate for detecting SNPs and thus the varation described to date for KRTAP1-1 may be an under-estimation of what actually exists in sheep. Further investigation using other nucleotide variation screening techniques is required to reveal the full extent of variation in this gene, if it exists.
In the KAP3 family, variation has only been revealed in KRTAP3-2 [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref]. Two variants of ovine KRTAP3-2 detected by PCR-SSCP have been reported, but as no sequence information describing the nucleotide varation has been reported, the nature of the variation is currently unknown.
In the KAP5 family, five sequnec variants have been decribed for ovine KRTAP5-4 [bib_ref] Analysis of variation in the ovine ultra-high sulphur keratin-associated protein KAP5-4 gene..., Gong [/bib_ref]. There are six SNPs and one length polymorphism in this gene. Non-synonymous SNPs predominate and there is variation in copy number of a 30-bp repeat sequence encoding a cysteine-rich decapeptide "RPCCSQSSCC" in the C-terminal region. Either one, or two copies of the repeat sequence are present in each varaint.
In the KAP6 family, variaton has been investigated in all of the family members [bib_ref] Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (KAP6) family in sheep, Gong [/bib_ref] [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref] [bib_ref] A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter, Zhou [/bib_ref]. KRTAP6-1 has been described to have three sequence variants that are comprised of three SNPs and a 57-bp insertion/deletion [bib_ref] Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (KAP6) family in sheep, Gong [/bib_ref] [bib_ref] A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter, Zhou [/bib_ref]. KRTAP6-2 has six sequence variants containing five SNPs [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. Five sequence variants are detected for KRTAP6-3. There are three SNPs and a 45-bp insertion/deletion observed for KRTAP6-3 [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. KRTAP6-4 has been reported to have three SNPs resulting in three sequence variants [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. There are six sequence variants described for KRTAP6-3 that contain five SNPs and an 18-bp insertion/delection [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. The SNPs found in KRTAP6-3 and KRTAP6-5 are predominantly non-synonymous, whereas synonymous SNPs predominate in KRTAP6-1, KRTAP6-2 and KRTAP6-4. The nature of the polymorphism does not appear to coincide with the physical locations of the genes on the chromosome, but it is in some respects consistent with KRTAP6-1, KRTAP6-2 and KRTAP6-4 being located in the middle of a region that is flanked by KRTAP6-5 and KRTAP6-3.
Variation in KRTAP7-1 has been investigated by two groups [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] [bib_ref] Search for variation in the ovine KAP7-1 and KAP8-1 genes using polymerase..., Gong [/bib_ref]. McLaren et al. [bib_ref] Linkage mapping of wool keratin and keratin-associated protein genes in sheep, Mclaren [/bib_ref] reported two sequence variants using the restriction endonuclease BglII and four sequence variants using the restriction endonuclease MspI in Southern hydridisation-Restriction Fragment Length Polymorphism (RFLP) analysis. No specific information on nucleotide sequence variation was given. Gong et al. [bib_ref] Search for variation in the ovine KAP7-1 and KAP8-1 genes using polymerase..., Gong [/bib_ref] used PCR-SSCP to screen the entire coding region of KRTAP7-1 and reported two sequence variants resulting from one non-synonymous SNP. This SNP is not in either a nominal BglII or MspI restriction endonuclease recognition site. What is more, there are no BglII recognition sequences present, or that can be created by a single nucleotide substitution in the coding region of ovine KRTAP7-1. This suggests that these restriction endonuclease recognition sites were located outside of the coding region of the KRTAP7-1 gene.
In the KAP8 family, varation has been investigated in both KRTAP8-1 and KRTAP8-2 [bib_ref] Search for variation in the ovine KAP7-1 and KAP8-1 genes using polymerase..., Gong [/bib_ref]. KRTAP8-1 has five sequnce variants containing four SNPs, whereas KRTAP8-2 has only two sequnce variants containing one SNP. The majority of SNPs found in KRTAP8-1 are synonymous [bib_ref] Search for variation in the ovine KAP7-1 and KAP8-1 genes using polymerase..., Gong [/bib_ref] , while the sole SNP found to date in KRTAP8-2 is located 21 bp upstream of the nominal TATA box sequence.
Six and five sequnce variants are identified in ovine KRTAP11-1 and KRTAP13-3, respectively [bib_ref] Identification of the ovine KAP11-1 gene (KRTAP11-1) and genetic variation in its..., Gong [/bib_ref] [bib_ref] Identification of the keratin-associated protein 13-3 (KAP13-3) gene in sheep, Gong [/bib_ref]. There are five and four SNPs in these genes respectively, and four of the five SNPs in KRTAP11-1 are synonymous, while three of the four SNPs in KRTAP13-3 are non-synonymous.
Using PCR-stem loop conformational polymorphism (PCR-SLCP), Zhou et al. [bib_ref] Detection of sequence variation and genotyping of polymorphic genes using polymerase chain..., Zhou [/bib_ref] reported four sequnce variants of ovine KRTAP24-1. Seven SNPs were described in ovine KRTAP24-1 and four of them are non-synonymous.
In summary, variation has been found in all of the ovine KAP genes investigated to date. The apparent higher degree of variation found in sheep when compared to humans, is possibly due to a greater number of individuals having been screened. Genetic variation in human KAP genes could therefore potentially be significantly higher than previously thought based on comparison with ovine KAP genes, although this remains to be revealed.
## The expression of kaps
In humans, all the KAP genes that have been identified to date are expressed in the hair follicle, with the exception of families . These KAP genes exhibit a uniform hair follicle expression pattern that is characteristic of the chromosome domain in which these gene are located. The exception is for the KAP genes on chromosome domain 21q22.1 and a single KAP gene (KRTAP17-1) on chromosome 17q21.2 [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref]. The detailed expression of human KAP genes has been reviewed [8] and will not be described here.
The expression of ovine KAPs is not well understood. Of the ovine KAP families investigated, the HGT-KAPs (KAP6, KAP7 and KAP8) are the first expressed in growing wool follicles and appear in the orthocortical cells [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref]. The HS-KAPs (KAP1, KAP2 and KAP3) are initially expressed in the orthocortical cells, but soon after this are expressed in all cortical cells [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. The UHS-KAP4 family is expressed later in follicle growth and only in paracortical cells [bib_ref] Dietary cysteine regulates the levels of messenger-rnas encoding a family of cysteine-rich..., Fratini [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref]. The UHS-KAP5 family is expressed during cuticle differentiation [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] Differential expression of genes encoding a cysteine-rich keratin family in the hair..., Jenkins [/bib_ref]. A schematic summary of wool KAP expression is shown in [fig_ref] Figure 3: Schematic representation of the sites of KAP gene expression in the wool... [/fig_ref].
them are non-synonymous.
In summary, variation has been found in all of the ovine KAP genes investigated to date. The apparent higher degree of variation found in sheep when compared to humans, is possibly due to a greater number of individuals having been screened. Genetic variation in human KAP genes could therefore potentially be significantly higher than previously thought based on comparison with ovine KAP genes, although this remains to be revealed.
## The expression of kaps
In humans, all the KAP genes that have been identified to date are expressed in the hair follicle, with the exception of families . These KAP genes exhibit a uniform hair follicle expression pattern that is characteristic of the chromosome domain in which these gene are located. The exception is for the KAP genes on chromosome domain 21q22.1 and a single KAP gene (KRTAP17-1) on chromosome 17q21.2 [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref]. The detailed expression of human KAP genes has been reviewed [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] and will not be described here.
The expression of ovine KAPs is not well understood. Of the ovine KAP families investigated, the HGT-KAPs (KAP6, KAP7 and KAP8) are the first expressed in growing wool follicles and appear in the orthocortical cells [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref]. The HS-KAPs (KAP1, KAP2 and KAP3) are initially expressed in the orthocortical cells, but soon after this are expressed in all cortical cells [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. The UHS-KAP4 family is expressed later in follicle growth and only in paracortical cells [bib_ref] Dietary cysteine regulates the levels of messenger-rnas encoding a family of cysteine-rich..., Fratini [/bib_ref] [bib_ref] Expression patterns of keratin intermediate filament and keratin associated protein genes in..., Yu [/bib_ref]. The UHS-KAP5 family is expressed during cuticle differentiation [bib_ref] Structure and expression of genes for a class of cysteine-rich proteins of..., Mackinnon [/bib_ref] [bib_ref] Differential expression of genes encoding a cysteine-rich keratin family in the hair..., Jenkins [/bib_ref]. A schematic summary of wool KAP expression is shown in [fig_ref] Figure 3: Schematic representation of the sites of KAP gene expression in the wool... [/fig_ref]. The expression of the KAP genes in sheep is consistent with that reported in humans, but some differences are observed. The most notable is the refined localisation of expression zones observed in wool follicles. For example, human KAP1 to KAP4, KAP6 and KAP7 are all expressed in the hair cortex [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] , but in the wool follicle, KAP6 and KAP7 are only expressed in the orthocortex, and the expression of KAP4 is restricted to the paracortex [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref] [bib_ref] Dietary cysteine regulates the levels of messenger-rnas encoding a family of cysteine-rich..., Fratini [/bib_ref] [bib_ref] Identification of four new gene members of the KAP6 gene family in..., Zhou [/bib_ref]. While KAP1 and KAP2 are expressed across the entire cortex of wool follicles, their expression starts in the orthocortical half [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref]. Such a well-defined bilateral expression pattern is not found in human hair follicles. In humans, KAP8 is expressed earlier than KAP6 and KAP7 [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] , but they appear to be co-expressed in the wool follicle [bib_ref] The role of keratin proteins and their genes in the growth, structure..., Powell [/bib_ref].
The weak expression of KAP6 in human hair [bib_ref] Human hair keratin-associated proteins (KAPs), Rogers [/bib_ref] is of interest as it contrasts the high level of expression of KAP6 in the wool follicle. It should however be noted that the level of expression reported for the human KAP genes was based on the quantitation of mRNA level and not on the amount of protein observed in fibre [bib_ref] Characterization of a first domain of human high glycine-tyrosine and high sulfur..., Rogers [/bib_ref]. It has been observed that there is only a moderate correlation between mRNA transcript levels and protein translation levels [bib_ref] Protein functions and biological contexts, Miklos [/bib_ref] , and that the amount of mRNA present in a cell or tissue can be a poor predictor of protein levels, especially for genes with low mRNA expression. Equally, it has been reported that for some genes with similar levels of mRNA expression, the protein levels can vary 20-to 30-fold in difference [bib_ref] Correlation between protein and mRNA abundance in yeast, Gygi [/bib_ref].
## Kap genes and wool traits
There have been a number of studies describing associations between wool traits and variation in KAP genes.
Wool crimp is thought to be affected by the composition of KAPs in the wool fibre. Felting lustre (FL) mutant wool has a very low relative abundance of HGT-KAP proteins in the fibre itself [bib_ref] Relation between the tyrosine content of various wools and their content of..., Gillespie [/bib_ref] , but the genes encoding the HGT-KAPs are present [bib_ref] Special biochemical features of the hair follicle, Rogers [/bib_ref]. This suggests a reduction or failure of transcription or translation of the HGT-KAPs genes in FL mutant wool. Research suggested originally that the FL mutation was inherited in a single autosomal dominant fashion [bib_ref] A dominant felting lustre mutant fleece-type in the Australian Merino sheep, Short [/bib_ref] , which was later confirmed by Blair [bib_ref] Inheritance of a major gene for excessively lustrous wool in sheep, Blair [/bib_ref]. However, examination of transcript prevalence, suggests that the KAP6-1, KAP7-1 and KAP8-1 genes are down-regulated, while the KAP2-12 and KAP4-2 genes are up-regulated in mutant follicles [bib_ref] Characterization of the structural and molecular defects in fibres and follicles of..., Li [/bib_ref]. In the FL mutant follicles, there is only one type of cortical cell present (paracortical) and the orthocortical cell type where the HGT-KAPs are usually expressed appear to be absent [bib_ref] Characterization of the structural and molecular defects in fibres and follicles of..., Li [/bib_ref]. A recent report by Wang et al. [bib_ref] Genome-wide association study for wool production traits in a Chinese Merino sheep..., Wang [/bib_ref] describes an OAR11 QTL for crimp that is approximately 30 MB from the known KAP genes on that chromosome.
Parson et al. [bib_ref] Evidence of linkage between high-glycine-tyrosine keratin gene loci and wool fiber diameter..., Parsons [/bib_ref] reported that variation in a KAP6 gene was associated with Mean Fibre Diameter (MFD) in medium wool Peppin Merinos. The KAP6 gene maps to ovine chromosome 1 [bib_ref] Linkage relationships between keratin-associated protein (KRTAP) genes and growth hormone in sheep, Parsons [/bib_ref] in a chromosome region where Beh et al. [bib_ref] A genome scan for QTL affecting fleece and wool traits in Merino..., Beh [/bib_ref] detected a QTL affecting MFD in medium wool Merinos. On this chromosome, Roldan et al. [bib_ref] Merino sheep: A further look at quantitative trait loci for wool production, Roldan [/bib_ref] did not find any QTL for MFD, but instead they found a QTL for other wool traits including curvature and wool yield. Allain et al. [bib_ref] QTL detection with DNA markers for wool traits in a sheep backcross..., Allain [/bib_ref] also detected a QTL on chromosome 1 for the "objectionable fibre content" (being defined as a large medulated fibres with a latticed medulla deficient in sulphur) in backcross SardaˆLacaune sheep. Wang et al. [bib_ref] Genome-wide association study for wool production traits in a Chinese Merino sheep..., Wang [/bib_ref] describe two chromosome 1 QTLs for fibre diameter, but they are at least 100 MB from the known OAR1 KAP genes.
Recently, variation in KRTAP6-1 was found to be associated with variation in wool fibre diameter associated traits, and a 57-bp deletion in the gene was associated with the occurrence of coarser wool with a greater Fibre Diameter Standard Deviation (FDSD), a greater Coefficient of Variation of Fibre Diameter (CVFD) and increased Prickle Factor (percentage of fibres over 30 microns; PF) [bib_ref] A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter, Zhou [/bib_ref].
On ovine chromosome 11, Rogers et al. [bib_ref] A Potential QTL for Wool Strength Located on Ovine Chromosome 11, Rogers [/bib_ref] reported a putative QTL for wool staple strength in the region spanning KRTAP1-1, KRTAP1-3 and KRT33A (KRT1-2) in Romney sheep. On this chromosome, Roldan et al. [bib_ref] Merino sheep: A further look at quantitative trait loci for wool production, Roldan [/bib_ref] detected a QTL for wool weight, staple strength and CVFD. Itenge-Mwezareported an association between variation in KRTAP1-1 and wool yield in one half-sib family and with Mean Staple Length (MSL) and wool brightness in another half-sib family. An association between variation in KRTAP1-2 and Greasy Fleece Weight (GFW) and Clean Fleece Weight (CFW) was revealed in a recent study, suggesting KRTAP1-2 mainly affects wool weights [bib_ref] Association of wool traits with variation in the ovine KAP1-2 gene in..., Gong [/bib_ref]. This is in agreement with a study in goats reporting that KRTAP1-1 which is near KRTAP1-2, affects cashmere yield [bib_ref] Sequence variant in the KAP1.1 gene associate with cashmere trait in two..., Zhang [/bib_ref].
Finally, Raadsma et al. [bib_ref] QTL and association analysis for skin and fibre pigmentation in sheep provides..., Raadsma [/bib_ref] report that OAR1 contains a QTL for wool pigmentation. The QTL (LOD score 2.5) is located near markers MAF64 and CSSM4 (192-256 cM) on chromosome 1 and while these are proximal to the KAP genes on that chromosome, it would be difficult to mount an argument of how the KRTAPs affect wool pigmentation specifically. This is not to say they do not affect pigmentation.
These findings suggest that KAP genes have a significant effect on wool traits, with further investigation required to fully explore this potential. While Wang et al. [bib_ref] Genome-wide association study for wool production traits in a Chinese Merino sheep..., Wang [/bib_ref] found SNPs that might be associated with specific wool traits as described above, another more recent study using a SNP chip across 84 predicted KAP and keratin genes (59 SNPs were tested), did not reveal any association with fleece weight, wool fibre diameter and fibre curvature [bib_ref] Preliminary linkage studies in sheep of keratin and keratin-associated protein genes with..., Phuaa [/bib_ref]. A SNP chip of these size may not have sufficient resolving power to precisely identify variation in ovine KAP genes, as many of the genes are highly polymorphic, having multiple variant forms as a consequence of containing many SNPs and insertions/deletions.
Given that KAP genes are clustered and that all the ovine KAP genes described to date are polymorphic and potentially expressed in the wool fibre, it will undoubtedly be difficult to unravel and determine the effects of individual genes on wool traits. However, the discovery of associations between variation in KAP genes and fibre traits, may ultimately allow for the future development of gene-markers for improving wool quality and, thus, enable the production of wool with specifications and performance more closely matched to targeted end-uses.
[fig] Figure 1: Amino acid sequence alignments of representative sequences from the ovine KAP families and orthologous sequences from other species. (a) KAP1-n; (b) KAP2-n; (c) KAP3-n; (d) KAP4-n; (e) KAP5-n; (f) KAP6-n; (g) KAP7-1; (h) KAP8-n; (i) KAP11-1; (j) KAP13-3; and (k) KAP24-1. [/fig]
[fig] Figure 2: Clustering of the ovine KAP genes in three chromosome regions. The bolded vertical bars represent known KAP genes and the arrows indicate the direction of transcription. The numbers below these bars are the KAP gene names (i.e., 11.1 refers to KRTAP11-1). The spacing of the genes is only approximate and is based on the Ovine Genome Sequence Assembly v4.0 sequence, where coordinates are given for the boundaries of the clusters, based on the first and last gene identified in the group. [/fig]
[fig] Figure 3: Schematic representation of the sites of KAP gene expression in the wool follicle. Modified from Powell and Rogers[5]. [/fig]
[table] Table 1: Ovine keratin-associated proteins (KAP) protein and gene sequences identified. [/table]
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Mediastinal undifferentiated pleomorphic sarcoma with pleural effusion cytopathologically misdiagnosed as epithelial malignant pleural mesothelioma: An autopsy case report
Undifferentiated pleomorphic sarcoma (UPS) is a new disease in the World Health Organization's classification of tumors of soft tissue and bone published in 2013. Primary mediastinal UPS is rare, especially with pleural effusion. Herein, we describe the pathological findings of pleural effusion followed by mediastinal UPS, which was initially misdiagnosed as epithelial malignant pleural mesothelioma (MPM). The cytopathological findings of the pleural effusion cell block often contribute to the diagnosis of various malignant tumors. However, these findings may lead to misdiagnosis of highly invasive mediastinal tumors such as UPS. A biopsy for primary mediastinal lesions should be performed because MPM rarely mimics mediastinal tumors with pleural effusion.K E Y W O R D S cell block, malignant pleural mesothelioma, mediastinum, pleural effusion, undifferentiated pleomorphic sarcoma
# Introduction
Undifferentiated pleomorphic sarcoma (UPS), previously known as malignant fibrous histiocytoma (MFH), was reclassified into an unclassifiable/undifferentiated sarcoma (US) in the fourth edition of the World Health Organization's classifications published in 2013.UPS is common in malignant soft tissue tumors and occurs in the extremities and retroperitoneum.Primary mediastinal UPS is rare, especially with pleural effusion. Therefore, the characteristics of pleural effusion with UPS remain unclear.We report a case of mediastinal UPS with pleural effusion cytopathologically misdiagnosed as epithelial malignant pleural mesothelioma (MPM).
## Case report
A 50-year-old man with acute onset of dyspnea visited another hospital. He had an unremarkable medical history. However, he had smoked two packets of cigarettes per day for 25 years and had been in the construction industry with significant asbestos exposure since he was 20 years old. Chest radiography showed extensive abnormal shadows in the right lower lung field and cardiac expansion . Chest computed tomography (CT) revealed moderate right pleural effusion, two pleural nodules, and a large mediastinal tumor invading the pericardium . Aside from the mediastinal tumor and pleural nodules, no further abnormal fluorodeoxyglucose uptakes were detected by positron emission tomography CT . The pleural effusion was drained. The cell block sample showed some multinucleated and atypical mesothelial cells with hump-like cytoplasmic processes among lymphocyte inflammation upon hematoxylin-eosin (HE) staining . On immunohistochemistry (IHC) staining, calretinin, D2-40, WT-1, and HEG1 were positive while TTF-1, CEA, claudin-4, and desmin were negative. Based on these findings, MPM was suspected. The patient was then referred to our hospital.
The patient was urgently hospitalized because of severe tachycardia and hypoxia at his initial visit. Laboratory findings, including tumor markers, such as CEA, CYFRA, and ProGRP, were almost within normal limits, except for high levels of D-dimer (1.2 × 10 4 ng/ml) and CRP (0.15 g/l). Transthoracic echocardiography showed that both ventricles were pushed directly by the tumor and had almost collapsed.
A diagnostic percutaneous needle biopsy was performed. Polymorphic variant cells with nonspecific differentiation were observed and almost all markers, including calretinin, D2-40, and WT-1, were negative. The histological findings of the mediastinal tumor were completely different from those of pleural effusion. This did not lead to a definitive diagnosis of MPM. After a few days, the patient died of untreated cardiac tamponade. An autopsy revealed that atypical fibroblast-like cells were arranged in a storiform pattern. This was associated with a pleomorphic feature . Immunohistochemically, tumor cells were negative for almost all markers such as CK-AE1/AE3, CK-CAM5.2, EMA, calretinin, desmin, α-SMA, D2-40, CD34, S-100, and neurofilament. This finding was similar to the needle biopsy sample. Thus, we diagnosed the patient with UPS.
# Discussion
MFH was first described in 1963 as a malignant soft tissue tumor arising from histiocytes and characterized by a storiform pattern with less differentiated and pleomorphic formation.MFH was regarded as the most common soft tissue sarcoma in adults for many years. However, Fletcher and Daugaard reported that most tumors diagnosed as MFH turned out to be other types of sarcomas upon using techniques such as immunochemistry. 5-7 Therefore, the term MFH was completely removed from the 2013 WHO Classification and replaced with a new category, US, which pertained to tumors that did not show a specific differentiation pattern despite various examinations. Moreover, US was classified into five subtypes. The UPS subtype displays polymorphism.
Since the revision of the WHO classification in 2013, only four cases of primary mediastinal UPS have been reported. All cases had undifferentiated and pleomorphic patterns on HE and IHC staining.Generally, UPS is an exclusion diagnosis. Our case was consistent with the F I G U R E 3 (a) Autopsy revealed that the specimen resected from the mediastinum was 160 × 150 × 140 mm in size and invaded the pericardium. aforementioned findings. Although one of the four cases had pleural effusion, pathological evaluation was not performed. Mehta and Guardiola reported a case of pleural MFH with pleural effusion, and the CT findings resembled MPM closely. However, the cell block of pleural effusion was not evaluated.Therefore, we discussed the histological findings of pleural effusion associated with mediastinal UPS. In this case, although MPM was not generally suspected from the radiological findings, both MTAP and BAP1 on IHC were positive and homozygous deletion of 9p21 on fluorescence in situ hybridization was difficult to determine in the cell block sample; despite being rare, mediastinal MPM was also identified.The autopsy revealed few asbestos bodies, and the tumor directly invaded the mesothelium of the pericardium and pleura, producing reactive mesothelial cells . Therefore, the reason for differences between the findings of the cell block sample and the mediastinal tumor was revealed, and MPM was almost ruled out.
The general prognosis for mediastinal UPS is poor because of its rapid progression and the difficulty of complete resection of the primary lesion in the mediastinum.Therefore, early diagnosis and treatment are required. Thoracentesis is easier to perform than biopsy of the primary mediastinal lesion, especially for a patient with severe cardiopulmonary status such as the current case. The pathological findings of the pleural effusion cell block often contribute to the diagnosis of various malignant tumors. However, these findings may lead to misdiagnosis of highly invasive mediastinal tumors such as UPS. Moreover, MPM rarely mimics mediastinal tumors with pleural effusion and a biopsy of the primary mediastinal lesion should be performed.
## Conflict of interest
Dr Tamiya reported grants and personal fees from AstraZeneca, Ono Pharmaceutical, and Bristol-Myers Squibb; personal fees from Eli Lilly Japan, Chugai Pharmaceutical, Boehringer Ingelheim, MSD, Pfizer, Taiho Pharmaceutical, and Kissei Pharmaceutical. Dr Atagi reported grants and personal fees from AstraZeneca, Ono Pharmaceutical, Bristol-Myers Squibb, Eli Lilly Japan, Boehringer Ingelheim, MSD, Pfizer, Taiho Pharmaceutical, Chugai Pharmaceutical, and Merck Pharmaceutical; grants from F. Hoffmann-La Roche; personal fees from Kyowa Hakko Kirin, and Hisamitsu Pharmaceutical. The remaining authors state that they have no conflict of interest.
ORCID Kinnosuke Matsumoto https://orcid.org/0000-0002-4930-6997 |
Reduced circulating levels of chemokine CXCL14 in adolescent girls with polycystic ovary syndrome: normalization after insulin sensitization
## Significance of this study
What is already known about this subject? ► Treatment with a low-dose combination of one mixed antiandrogen and antimineralocorticoid (spironolactone), and two insulin sensitizers (pioglitazone/metformin) (SPIOMET) is particularly effective in improving the endocrine-metabolic derangements in adolescent girls with polycystic ovary syndrome (PCOS). ► CXCL14 (C-X-C motif chemokine ligand-14) is a chemokine released by active brown fat and protective against insulin resistance in experimental models.
What are the new findings?
► Serum CXCL14 levels are abnormally reduced in patients with PCOS. ► SPIOMET treatment for 1 year normalized CXCL14 concentrations and improved the endocrine-metabolic status of patients with PCOS. ► Pioglitazone and spironolactone, drug components of SPIOMET, induce CXCL14 expression and release in human adipocytes, in parallel with the induction of marker genes of brown adipogenesis.
How might these results change the focus of research or clinical practice? ► CXCL14 may become a novel biomarker for PCOS. In addition, CXCL14 appears as a potential mediator of the beneficial effects of the SPIOMET combination and may hold the capacity of serving as therapeutic modulator of the disorder.
# Abstract
Objective CXCL14 (C-X-C motif chemokine ligand-14) is a chemokine released by active brown fat, showing protective effects against insulin resistance in experimental models. Polycystic ovary syndrome (PCOS) in adolescent girls is usually related to hepato-visceral fat excess and insulin resistance, and associates with comorbidities such as type 2 diabetes. Treatment with a low-dose combination of one antiandrogen and antimineralocorticoid drug (spironolactone) and two insulin sensitizers (pioglitazone/ metformin) (SPIOMET) is particularly effective in improving these metabolic derangements. Adipose tissue may be involved in the metabolic alterations of PCOS, and it is a likely target of therapeutic action. We investigated the alterations in CXCL14 levels and the effects of drugs composing SPIOMET treatment on CXCL14 in human adipocytes.
Research design and methods We studied 51 adolescent patients with PCOS and 21 age-matched healthy controls. Thirty-one adolescent patients with PCOS under SPIOMET or oral contraception-based treatment were also studied. For studies in vitro, Simpson Golabi Behmel Syndrome (SGBS) adipose cells were used. Gene expression for CXCL14 and other genes was quantified using quantitative real-time PCR. The levels of CXCL14 and adipokines in serum and cell culture media were determined by ELISA.
Results Serum CXCL14 levels are reduced in patients with PCOS. One-year SPIOMET treatment normalized CXCL14 concentrations and improved the metabolic status of patients with PCOS. Pioglitazone induced CXCL14 expression in differentiating human SGBS adipocytes, in parallel with the induction of marker genes of brown adipogenesis. Spironolactone induced CXCL14 expression and release in differentiated human adipocytes. Conclusion Insulin sensitization with SPIOMET normalizes the abnormally low levels of CXCL14 in girls with PCOS. This is consistent with the effects of pioglitazone and spironolactone inducing CXCL14 expression and promoting a brown-like phenotype in adipocytes. CXCL14 may be a novel biomarker for PCOS as well as a potential mediator of the beneficial effects of the SPIOMET combination and may hold promise as a therapeutic modulator of the disorder. Trial registration numbers ISRCTN29234515 and ISCRCTN11062950.
# Introduction
Adipose tissue plasticity is growingly recognized as a relevant factor for the development of metabolic syndrome, independently of the presence or absence of obesity. The acquisition of a 'brown' or 'beige' phenotype by adipose tissue is considered protective against hyperglycemia and hyperlipidemia and, indeed, the relative protection against these alterations Metabolism in young versus elder individuals is associated with the known prevalence of the brown/beige phenotype in early human development. 1 CXCL14 (C-X-C motif chemokine ligand-14) is a chemokine produced by active brown/beige adipose tissue, capable of improving glucose metabolism in insulin-resistant rodent models. [bib_ref] CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation, Cereijo [/bib_ref] Central (hepato-visceral) fat excess and insulin resistance are common metabolic comorbidities in girls and young women with polycystic ovary syndrome (PCOS). Treatment with a low-dose combination of one mixed antiandrogen and antimineralocorticoid (spironolactone) and two insulin sensitizers (pioglitazone plus metformin) (SPIOMET) has been shown to improve the metabolic condition of these patients to a better extent than oral contraception (OC). [bib_ref] Normalizing ovulation rate by preferential reduction of hepato-visceral fat in adolescent girls..., Ibáñez [/bib_ref] However, the specific cellular and tissue targeting, and the relative role of each of the SPIOMET components in the context of PCOS is poorly understood. It has been recently reported that women with PCOS have lower brown adipose tissue (BAT) activity as compared with healthy controls, and experimental data suggest that BAT activation might be a promising therapeutic option for PCOS. [bib_ref] Brown adipose tissue transplantation ameliorates polycystic ovary syndrome, Yuan [/bib_ref] Here, we report abnormally low levels of CXCL14 in girls with PCOS, the differential effects of SPIOMET and OC on circulating CXCL14 in relation to metabolic improvement, and the effects of SPIOMET components on the expression and release of CXCL14 in a cellular model of human adipocytes.
## Research design and methods study population and design
The study population consisted of 52 adolescent girls with PCOS (age, 15.6 years; body mass index (BMI), 24.3 kg/m 2 ) who were enrolled into two randomized, open-label, controlled trials (with identical design) exploring the effects of OC versus SPIOMET treatment for 1 year, with post-treatment ovulation rate as primary outcome. The results of the first trial (ISRCTN29234515) have been already published, 5 and the second trial (ISCRCTN11062950) will be completed in 2019. Twenty-one out of the 52 girls belonged to the first study and 31 to the second trial; 31 of the 52 had available samples for CXCL14 assessment at 0 and 12 months (online supplementary [fig_ref] Figure 1: A [/fig_ref]. Both trials were performed at Sant Joan de Déu University Hospital, Barcelona, Spain. Inclusion and exclusion criteria have been described previously. [bib_ref] Normalizing ovulation rate by preferential reduction of hepato-visceral fat in adolescent girls..., Ibáñez [/bib_ref] OC treatment consisted of 20 µg of ethinylestradiol plus 100 mg of levonorgestrel for 21/28 days and placebo for 7/28 days; SPIOMET is a low-dose combination of spironolactone 50 mg/day, pioglitazone 7.5 mg/day, and metformin 850 mg/day. Twenty-one agematched healthy girls recruited in nearby schools served as controls. All had regular menstrual cycles and none was hirsute or taking medications.
Clinical, endocrine-metabolic, and imaging assessments Birth weight and BMI (and their Z-scores) were retrieved, and endocrine-metabolic variables were assessed in the early morning, in the follicular phase (days 3-7) of the cycle, or after 2 months of amenorrhea. Serum glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), lipids, ultrasensitive C reactive protein (usCRP), sex hormone-binding globulin, androgens, and high-molecular-weight (HMW) adiponectin were assessed as reported. 5 CXCL14 levels in serum and cell culture medium were determined using a specific ELISA kit (RayBiotech), 2 whose sensitivity was 0.7 ng/mL, and interassay and intra-assay coefficients of variation less than 12%.
Body composition was assessed by dual X-ray absorptiometry with a Lunar Prodigy and Lunar software (version 3.4/3.5; Lunar, Madison, Wisconsin, USA); abdominal (subcutaneous and visceral) and hepatic fat were assessed by MRI using a multiple-slice MRI 1.5 Tesla scan (Signa LX Echo Speed Plus Excite; General Electric, Milwaukee, Wisconsin, USA). [bib_ref] Normalizing ovulation rate by preferential reduction of hepato-visceral fat in adolescent girls..., Ibáñez [/bib_ref] Central fat was arbitrarily defined as the sum of visceral fat (in squared centimeter) and hepatic fat (in per cent).
## Studies in human adipocytes in culture
The effects of pioglitazone, spironolactone, and metformin on adipocytes were studied in human Simpson Golabi Behmel Syndrome (SGBS) cells, a cell model of human beige (brown-like) adipogenesis. SGBS preadipocytes were maintained in Dulbecco's modified Eagle's (DMEM)/F12 medium, 10% FBS. Adipogenic differentiation was initiated by incubating confluent cell cultures for 4 days in serum-free medium plus 20 nM insulin, 0.2 nM triiodothyronine, 100 nM cortisol, 25 nM dexamethasone, 500 µM 3-isobutyl-1-methyl-xanthine, and 2 µM rosiglitazone. Subsequently, the cells were switched to DMEM/F12, 20 nM insulin, 0.2 nM triiodothyronine, and 100 nM cortisol and maintained for up to 10 days, when more than 90% cells have acquired differentiated adipocyte morphology. Two experimental designs were followed: (1) cells were treated with the medications across their differentiation process, in the absence of rosiglitazone and maintaining the drugs throughout the 10 days of differentiation and (2) adipocytes were treated with the drugs acutely (24 hours), once the cells have been differentiated. Controls included a 1:1 methanol/ dimethyl sulfoxide mixture (drug solvents) at <1/1000 concentration. Cell culture reagents and drugs were from Sigma. The cell culture medium was collected and concentrated 1:5 prior to measurement of CXCL14 levels. RNA was extracted from cells using an affinity columnbased method (Machery-Nagel). Real-time quantitative reverse transcription PCRs were performed using 0.5 µg RNA and employing TaqMan reagents and probes (Life Technologies), according to supplier indications. PCR was conducted in an ABI/Prism-7700 Sequence Detector System. The following TaqMan probes were used: CXCL14, Hs01557413; uncoupling protein-1 (UCP1), Hs00222453; fatty acid-binding protein-4 (FABP4), Hs00609791, and ribosomal protein lateral stalk subunit 0 (RPLP0) mRNA, Hs99999902. Each sample was run in duplicate and the mean value was used to calculate the relative amount of individual mRNAs. Each mean value was normalized to that of the RPLP0 mRNA using the comparative (2-ΔCT) method.
## Statistical analysis and ethics
Statistical analyses were performed with SPSS V.23.0 (SPSS, Chicago, Illinois, USA). Baseline differences in CXCL14 concentrations between patients and controls were tested with unpaired t-test; covariance analysis was used to adjust for age and BMI. Longitudinal changes between groups were compared by repeated-measures general linear model. Differences in longitudinal changes between groups were tested by the interaction term among between-and within-subject effects. Associations were sought by Pearson correlation analysis. P value <0.05 was considered statistically significant. Results are expressed as mean±SD.
The study was conducted after approval by the Institutional Review Board of Sant Joan de Déu University Hospital, after written consent by the parents and assent by each of the participants.
## Results serum cxcl14 levels are reduced in adolescent girls with pcos
At baseline, PCOS girls showed lower sex hormone-binding protein (SHGB) levels, higher total testosterone concentrations and free androgen index, increased central and hepatic fat, and a trend toward higher insulin and lower HMW adiponectin levels versus controls, as expected (online supplementary table 1). None of the girls were obese (BMI ≥30 kg/m 2 ); however, mean BMI was higher in girls with PCOS as compared with control girls (p=0.01). Fasting glucose levels were within the normal range in both subgroups and were marginally higher in control girls (online supplementary table 1). Serum CXCL14 concentrations were reduced in girls with PCOS close to twothirds of the normal levels (figure 1A), did not correlate with markers of adiposity or glucose homeostasis, and were positively associated with HMW adiponectin levels .
Serum CXCL14 concentrations were reduced in girls with PCOS close to two-thirds of the normal levels (figure 1A) and were positively associated with HMW adiponectin levels (online supplementary table 2). CXCL14 did not correlate with other markers of adiposity or glucose homeostasis; however, there was a trend toward a positive correlation with circulating glucose and a negative correlation with hepatic fat .
sPIOmeT, but not OC, normalizes serum CXCl14 levels in adolescent girls with PCOs SPIOMET treatment was associated with more benefits than OC on endocrine-metabolic and imaging markers, including on fasting insulin, HOMA-IR, HMW adiponectin, usCRP, and hepato-visceral (central) fat, consistent with previous observations [bib_ref] Normalizing ovulation rate by preferential reduction of hepato-visceral fat in adolescent girls..., Ibáñez [/bib_ref]. Serum CXCL14 levels increased significantly after SPIOMET, reaching levels similar to those in controls and remained significantly decreased after OC, as compared with controls and with SPIOMET-treated girls [fig_ref] Figure 1: A [/fig_ref]. The increase in CXCL14 levels in the SPIOMET-treated patients correlated significantly with the extent of reduction in fasting insulin and HOMA-IR (online supplementary table 4).
## Effects of the spiomet components on cxcl14 expression and release by human adipocytes
The effects of the three components of SPIOMET on human adipocyte differentiation were assessed individually and in combination (figure 2). Pioglitazone had a doseresponse dramatic effect inducing CXCL14 mRNA expression, whereas neither spironolactone nor metformin did. No concentration higher than 10 µM could be tested for spironolactone, because of cell toxicity on preadipocytes. Indeed, pioglitazone at the lowest concentration tested (0.1 µM) already caused a close to 10-fold induction of CXCL14 mRNA expression [fig_ref] Figure 2: Effects of pioglitazone [/fig_ref]. This effect paralleled a strong positive effect of pioglitazone on adipogenic differentiation, as evidenced by lipid droplet accumulation (figure 2C) and expression of gene markers of general adipogenic (FABP4) and beige adipogenic (UCP1) differentiation [fig_ref] Figure 2: Effects of pioglitazone [/fig_ref].
Addition of metformin to pioglitazone did not modify the extent of pioglitazone induction of CXCL14 mRNA expression [fig_ref] Figure 2: Effects of pioglitazone [/fig_ref]. However, the addition of spironolactone reduced significantly the effects of pioglitazone on CXCL14 mRNA expression. The induction of CXCL14 mRNA expression caused by the three drugs in combination was similar to that elicited by pioglitazone plus spironolactone. These data also paralleled the observations on adipogenic differentiation after combined treatments, according to cell morphology and expression of FABP4 Neither pioglitazone nor metformin alone had any effect on CXCL14 mRNA expression on already differentiated human adipocytes [fig_ref] Figure 3: Effects of pioglitazone [/fig_ref]. However, 10 µM of spironolactone caused a significant induction of CXCL14 mRNA expression (figure 3A). The combination of three components (with spironolactone dosed at 10 µM) induced CXCL14 mRNA to the same extent as treatment with spironolactone alone. Only spironolactone, either alone or in combination with pioglitazone plus metformin, elicited a significant increase (close to eightfold) of the CXCL14 protein levels released by adipocytes to the cell culture medium [fig_ref] Figure 3: Effects of pioglitazone [/fig_ref].
# Discussion
Our study shows that PCOS associates with reduced levels of CXCL14, a chemokine recently proposed to be secreted preferentially by brown/beige adipose tissue. [bib_ref] CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation, Cereijo [/bib_ref] The restoration of normal levels of CXCL14 with SPIOMET, the capacity of pioglitazone to increase CXCL14 expression in preadipocytes, and the capacity of spironolactone to increase the release of CXCL14 in adipocytes, respectively, suggest that SPIOMET treatment could target adipose tissue to improve the metabolic profile of patients with PCOS. In addition, the normalization of central fat after SPIOMET treatment may indicate that circulating CXCL14 relates to ectopic fat rather than to BMI per se.
High CXCL14 levels have been shown to improve insulin sensitivity in adipocytes in vitro 12 and in rodent models 2 ; however, not all reports agree in the antidiabetic actions of CXCL14, and even deleterious prodiabetogenic effects have been proposed in vitro [bib_ref] CXCL14 inhibits insulin secretion independently of CXCR4 or CXCR7 receptor activation or..., Atanes [/bib_ref] and in vivo. [bib_ref] Disruption of CXC motif chemokine ligand-14 in mice ameliorates obesity-induced insulin resistance, Nara [/bib_ref] Our study, which is, to our knowledge, the first exploring CXCL14 in human metabolic pathology, indicates that normalization of CXCL14 after SPIOMET associates with decreased insulin resistance. Pioglitazone increases insulin sensitivity as well as adipogenic differentiation and also promotes the Metabolism acquisition of a brown/beige phenotype in adipose cells. [bib_ref] Induction of uncoupling protein in brown adipose tissue. synergy between norepinephrine and..., Foellmi-Adams [/bib_ref] The induction of CXCL14 expression by pioglitazone is consistent with data in experimental models highlighting increased expression of CXCL14 with brown/beige adipogenic differentiation. [bib_ref] CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation, Cereijo [/bib_ref] The positive effects of spironolactone-inducing CXCL14 in differentiated adipocytes are also consistent with previous reports indicating that spironolactone induces adipose tissue browning in rodents [bib_ref] Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of..., Armani [/bib_ref] and activates brown fat in humans. [bib_ref] Mineralocorticoid antagonism enhances brown adipose tissue function in humans: a randomized placebo-controlled..., Thuzar [/bib_ref] Recent reports disclosed impaired BAT activity in PCOS subjects and the capacity of brown fat activation to ameliorate PCOS in experimental models. [bib_ref] Brown adipose tissue transplantation ameliorates polycystic ovary syndrome, Yuan [/bib_ref] Considering that CXCL14 is preferentially released by brown adipocytes, 2 it may be speculated that SPIOMET treatment drives a shift in adipose tissue plasticity to a more brown/beige phenotype resulting in enhanced CXCL14 release, potentially improving glucose homeostasis and possibly reducing diabetes risk. This sequence may be especially relevant in young girls, where the brown/beige adipose tissue amount is particularly significant. [bib_ref] Relevance of brown adipose tissue in infancy and adolescence, Gilsanz [/bib_ref] Our study has obvious limitations. First, the effect of SPIOMET on CXCL14 levels in tissues other than adipose tissue cannot be ruled out. Second, the physiological significance of the drug concentrations used in the in vitro studies is unclear. The concentrations of pioglitazone increasing CXCL14 levels in differentiated adipocytes (from at least 0.1 µM) were in the range of those in plasma from SPIOMET-treated healthy women. [bib_ref] Optimizing the formulation an the study design to assess the comparative bioavailability..., Ballester [/bib_ref] However, although spironolactone concentrations used here were the same as those employed in previous studies in adipose cells, plasma spironolactone levels in SPIOMET-treated controls are lower. [bib_ref] Optimizing the formulation an the study design to assess the comparative bioavailability..., Ballester [/bib_ref] Finally, evidence for a direct role of CXCL14 downregulation and reinduction on the metabolic status of patients with PCOS after SPIOMET is obviously lacking, as it would imply intervention experiments beyond the ethical standards of human studies. Nevertheless, our data support a role for CXCL14 as a novel potential biomarker and molecular mediator in the improvement of PCOS-associated metabolic alterations following SPIOMET intervention. Contributors CG-B, RC, and TQ-L researched data, contributed to data interpretation, and reviewed/edited manuscript. RM and AL-B contributed to data interpretation and reviewed/edited manuscript. FdZ contributed to study design and data interpretation, and reviewed/edited the manuscript. LI contributed to study design and data interpretation, wrote the manuscript, and reviewed/edited manuscript. FV contributed to study design and data interpretation, wrote the manuscript, and reviewed/edited manuscript.
[fig] Figure 1: A) Baseline circulating CXCL14 (C-X-C motif chemokine ligand-14) concentrations in girls with polycystic ovary syndrome (PCOS, n=52) and in healthy age-matched controls (n=21). Data are mean±SD. * P=0.007, for patients versus controls at baseline. (B) Longitudinal results of CXCL14 serum concentrations in girls with PCOS who received an oral contraceptive (OC, white circles, n=16) or low-dose spironolactone, pioglitazone, and metformin (SPIOMET, black circles, n= 15). The dotted line is the mean in healthy controls (n=21); the shaded area represents the mean±SD in those healthy controls. *P=0.004, for patients versus controls at baseline; §p=0.02, on treatment differences between controls and the OC subgroup; #p=0.01, for changes 0-1 year in the SPIOMET subgroup; &p=0.04 for changes 0-1 year between the OC subgroup and the SPIOMET subgroup. [/fig]
[fig] Figure 2: Effects of pioglitazone (Pio), spironolactone (Spi), and metformin (Met) on adipogenic differentiation of SGBS human preadipocytes in culture. SGBS human preadipocytes were treated chronically (10 days) with pioglitazone, spironolactone, or metformin alone (A) or in combination (B, C, D) at the indicated doses across the adipogenic differentiation process. (A, B) CXCL14 (C-X-C motif chemokine ligand-14) transcript levels are presented as means±SD from four to five independent experiments and are expressed relative to values from untreated control cells. (C) Representative photomicrographs of adipocyte cell cultures differentiating in the presence of the indicated components: top: control; next to top: pioglitazone (10 µM); middle: pioglitazone (10 µM) and metformin (10 µM); next to bottom: spironolactone (10 µM); bottom: pioglitazone (10 µM), spironolactone (10 µM), and metformin (10 µM). Each column represents duplicate representative pictures per treatment condition. (D) Fatty acid-binding protein-4 (FABP4) and uncoupling protein-1 (UCP1) transcript levels are presented as means±SD from three to four independent experiments and are expressed relative to values from untreated control cells. *P<0.05, **p<0.01, and ***p<0.001 for each component versus control; # p<0.05 relative to pioglitazone alone. ND, not detected; SGBS, Simpson Golabi Behmel Syndrome. [/fig]
[fig] Figure 3: Effects of pioglitazone (Pio), spironolactone (Spi), and metformin (Met) on CXCL14 (C-X-C motif chemokine ligand-14) gene expression and CXCL14 protein release in human adipocytes. SGBS cells were treated acutely (24 hours, at the indicated doses) when already differentiated adipocytes. CXCL14 transcript level (A) and CXCL14 protein levels in cell culture medium (B) are presented as means±SD from four to five independent experiments and are expressed relative to values from untreated control cells. *P<0.05, **p<0.01, and ***p<0.001 for each component versus control. SGBS, Simpson Golabi Behmel Syndrome.and UCP1 (figure 2C,D). CXCL14 protein was only detectable in the cell culture medium of differentiating cells under pioglitazone treatment, either alone (0.185 ng/mL) or in combination with spironolactone and metformin (0.179 ng/mL). [/fig]
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Tau Pathology in Chronic Traumatic Encephalopathy is Primarily Neuronal
Millions of individuals are exposed to repetitive head impacts (RHI) each year through contact sports, military blast, and interpersonal violence. RHI is the major risk factor for developing chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy. Recent consensus criteria defined the pathognomonic lesion in CTE as perivascular, hyperphosphorylated tau (p-tau) in neuronal aggregates. Astroglial p-tau is an inconsistent supporting feature and not in itself diagnostic of CTE. This study quantitated the spatial and cellular distribution of p-tau pathology in postmortem dorsolateral frontal cortex of 150 individuals with CTE, from ages 21 to 80 years old, without comorbid pathology. p-Tau-immunoreactive cells were quantitated in the gray matter sulcus, crest, subpial region, and within pathognomonic CTE lesions. Significantly more neuronal p-tau than astrocytic p-tau was found across all cortical regions (p < 0.0001). Sulcal astrocytic ptau was primarily (75%, p < 0.0001) localized to subpial regions as thorn-shaped astrocytes, a form of age-related tau astrogliopathy. Neuronal p-tau was significantly associated with age, years of RHI exposure, and CTE severity; astrocytic p-tau pathology was only significantly associated with age. These findings strongly support neuronal degeneration as a driving feature of CTE and will help inform future research and the development of fluid biomarkers for the detection of neuronal degeneration in CTE.
# Introduction
Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease associated with a history of exposure to repetitive head impacts (RHI) sustained through contact sports, such as American football, hockey, soccer, and boxing, military service, or interpersonal violence [bib_ref] Duration of American football play and chronic traumatic encephalopathy, Mez [/bib_ref]. Currently, CTE can only be diagnosed after death, and there are no therapeutic treatments or interventions available. CTE is neuropathologically defined by the accumulation of hyperphosphorylated tau (p-tau) around small blood vessels and concentrated at the depths of the cortical sulcus. In early-stage CTE, tau pathology is confined to the cortical gray matter, but as the disease progresses, p-tau pathology is also observed in medial temporal lobe structures, such as the hippocampus, entorhinal cortex, amygdala, the diencephalon, and brainstem [bib_ref] The second NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis..., Bieniek [/bib_ref]. The 2015 CTE consensus conference defined the pathognomonic lesion as "an abnormal accumulation of p-tau in neurons and astrocytes distributed around small blood vessels at the depth of the cortical sulcus in an irregular pattern" [bib_ref] TBI/CTE group. The first NINDS/NIBIB consensus meeting to define neuropathological criteria for..., Mckee [/bib_ref]. The revised 2021 CTE consensus criteria state that "a pathognomonic lesion consists of ptau aggregates in neurons with or without glial tau at the depth of a cortical sulcus around a small blood vessel" [bib_ref] Validity of the 2014 traumatic encephalopathy syndrome criteria for CTE pathology, Mez [/bib_ref] , highlighting that p-tau containing neurons (i.e. neurofibrillary tangles , pretangles, and neurites) are the primary p-tau containing cell type observed in CTE. Furthermore, our previous analysis of 99 contact sport athletes diagnosed with CTE with no comorbidities, ranging in age at death from ages 20 to 89, found that the presence of p-tau containing astrocytes within the pathognomonic CTE lesion seemed to be associated with age at death and that most p-tau containing cells in the CTE pathognomonic lesions were neurons [bib_ref] Evolution of neuronal and glial tau isoforms in chronic traumatic encephalopathy, Cherry [/bib_ref]. However, this previous analysis was restricted to morphologic assessment. New techniques and methods that utilize machine learning and artificial intelligence platforms to count cells are superior. Additionally, to truly identify cell population that contains p-tau, multiplex immunofluorescence labeling (mIF) is required.
Subtypes of astrocytic p-tau have been described principally using morphologic assessment of p-tau-immunolabeled histological sections [bib_ref] Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Kovacs [/bib_ref]. Tufted astrocytes are hallmarks of progressive supranuclear palsy [bib_ref] Astroglia and tau: New perspectives, Kovacs [/bib_ref] ; astrocytic plaques are found in corticobasal degeneration [bib_ref] A new type of neuritic plaque without amyloid in corticonigral degeneration without..., Mattiace [/bib_ref] ; ramified astrocytes are present in Pick disease [bib_ref] Astroglia and tau: New perspectives, Kovacs [/bib_ref] ; and globular astroglial inclusions are seen in globular glial tauopathy [bib_ref] Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Kovacs [/bib_ref]. Astrocytic pathology that is related to brain aging is broadly termed age-related tau astrogliopathy (ARTAG) and includes the thorn-shaped astrocytes and granular-fuzzy astrocytes that can be found in the white and gray matter, and in subpial, subependymal, and perivascular locations [bib_ref] Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Kovacs [/bib_ref]. ARTAG pathology is typically observed in individuals over the age of 60 years old and due to its subpial and perivascular localization can sometimes be confused with CTE [bib_ref] Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Kovacs [/bib_ref]. The 2021 consensus criteria for CTE clarified that subpial TSA pathology, or ARTAG, is not a diagnostic feature of the CTE pathognomonic lesion. Recently, however, a study of 22 aged individuals variably diagnosed with CTE, whose age range at death was narrow (most individuals over the age of 70 [16 of whom were diagnosed with a comorbid neurodegenerative disease]), suggested that astrocytes, not neurons, better define CTE pathology [bib_ref] Astroglial tau pathology alone preferentially concentrates at sulcal depths in chronic traumatic..., Arena [/bib_ref]. Further, another study of 12 aged individuals (most individuals >70 years old) diagnosed with CTE and other comorbid neurodegenerative diseases suggested that labeling of astrocytes facilitates the diagnosis of CTE [bib_ref] CONNECT-TBI Investigators. Detection of astrocytic tau pathology facilitates recognition of chronic traumatic..., Ameen-Ali [/bib_ref]. Both of these studies called for a revision of the NINDS CTE criteria to include astrocytic p-tau pathology in the pathognomonic lesion of CTE. To further inform this debate, we undertook a comprehensive, quantitative analysis of p-tau-positive cells in CTE in a large cohort of brain donors, representing a wide age range at death and with no comorbid neurodegenerative conditions, using mIF and cell-type specific markers. The goal was to determine the role of astrocytic p-tau pathology in the progression of CTE.
Overall, determining the spatial, temporal, and cellular distribution of p-tau deposition in CTE over the lifespan is critical to understanding the pathogenesis of CTE, and identifying novel biomarkers and therapeutic intervention strategies.
# Materials and methods
## Participant selection and neuropathologic assessment
Cases were selected from the BU CTE Brain Bank. At the time of the study, there were approximately 1200 cases that were donated to the CTE brain bank. Each donated case is assessed by a board-certified neuropathologist for a neuropathologic diagnosis of CTE, Alzheimer's disease, neocortical Lewy bodies, frontotemporal lobar degeneration, or motor neuron disease, based on established neuropathologic criteria for each disease [bib_ref] TBI/CTE group. The first NINDS/NIBIB consensus meeting to define neuropathological criteria for..., Mckee [/bib_ref] [bib_ref] Alzheimer's Association. National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment..., Montine [/bib_ref] [bib_ref] Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy..., Mckeith [/bib_ref] [bib_ref] Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: An update, Mackenzie [/bib_ref]. Cases were excluded if they carried a neuropathologic diagnosis of any neuropathologic disease, except CTE. Additionally, to minimize confounding effects of neuritic plaque pathology, cases were excluded if they received an amyloid beta CERAD score (Consortium to Establish a Registry for Alzheimer's Disease, semiquantitative neuritic plaque score) >1. Using these exclusion criteria, 235 cases were neuropathologically diagnosed with CTE without the presence of any other comorbid neurodegenerative disease diagnosis. Of the 235, 81 had missing tissue regions or were not immediately available for use, and 4 did not have sufficient clinical exposure records, leaving 150 available for analysis as represented in [fig_ref] TABLE 1: Group Demographics [/fig_ref]. A randomized representative subset of 51 individuals from the initial groups was used for mIF analysis. Neuropathologic diagnosis was obtained from formalin-fixed paraffin-embedded tissue as previously described [bib_ref] TBI/CTE group. The first NINDS/NIBIB consensus meeting to define neuropathological criteria for..., Mckee [/bib_ref]. Briefly, sections of paraffin-embedded tissue were stained for p-tau, alpha-synuclein, amyloid beta, TAR DNA-Binding Protein-43 (TDP-43), Luxol fast blue, and hematoxylin and eosin using previously described methods [bib_ref] Chronic traumatic encephalopathy in athletes: Progressive tauopathy after repetitive head injury, Mckee [/bib_ref]. A neuropathologic diagnosis of CTE was made using National Institute of Neurological Disorders and Stroke (NINDS) / National Institute of Biomedical Imaging and Bioengineering (NIBIB) consensus criteria [bib_ref] TBI/CTE group. The first NINDS/NIBIB consensus meeting to define neuropathological criteria for..., Mckee [/bib_ref]. Individuals with CTE were diagnosed based on the presence, extent, and severity of p-tau deposition through the brain. All evaluations were reviewed by 4 neuropathologists (VEA, BH, TDS, and ACM); discrepancies in the diagnosis were resolved by a consensus conference. Demographics, athletic history (type of sports played, level, position, age of first exposure to sports, and years playing contact sports), military history (branch, location of service, and duration of combat exposure), and traumatic brain injury history (including the number of concussions) were queried during a telephone interview, as detailed previously [bib_ref] Clinical presentation of chronic traumatic encephalopathy, Stern [/bib_ref]. Institutional review board approval for brain donation was obtained through the Boston University Alzheimer's Disease and CTE Center, Human Subjects Institutional Review Board of the Boston University School of Medicine, and VA Bedford Healthcare System (Bedford, MA). One individual from the Australian Sports Brain Bank was used for a representative image in [fig_ref] FIGURE 3: Neuronal tau defines the CTE perivascular lesion [/fig_ref] but not included in the data analysis. The Australian Sports Brain Bank operates under RPA Human Research Ethics Committee approval X19-0010.
## Immunohistochemistry and immunofluorescence staining
Tissue was obtained from the dorsolateral frontal cortex (DLFC) and processed as previously described [bib_ref] Microglial neuroinflammation contributes to tau accumulation in chronic traumatic encephalopathy, Cherry [/bib_ref]. The DLFC was chosen because it is one of the first regions affected in CTE. Since pathologic alterations are found in the DLFC early, it is easier to identify more subtle changes and observe a wider range of CTE-related effects. All cases used in this study had p-tau pathology in the DLFC. Both brightfield immunohistochemistry (IHC) staining and mIF were performed. Ten-micrometer thick sections were stained with anti-Phospho-Tau Ser202, Thr205 (AT8) (Invitrogen, Waltham, MA, 1:500) and visualized for IHC with 3,3'-diaminobenzidine (DAB), as previously described [bib_ref] Microglial neuroinflammation contributes to tau accumulation in chronic traumatic encephalopathy, Cherry [/bib_ref]. mIF staining was performed using the Akoya Bioscience Opal Polaris 7 color manual IHC detection kit per the manufacturer's protocol and as previously described [bib_ref] Tau isoforms are differentially expressed across the hippocampus in chronic traumatic encephalopathy..., Cherry [/bib_ref]. Sections were incubated with antibodies to AT8 (Invitrogen, 1:500, Mouse, cat # MN1020), microtubule assocaited protein 2 (MAP2) (BioLegend, San Diego, CA, 1:500, Mouse, cat # 801801), and glial fibrillary acidic protein (GFAP) (BioLegend, 1:750, mouse, cat # 837604) and stained with DAPI to label cell nuclei. Sections were imaged using an Akoya Bioscience Vectra Polaris Digital Slide Scanner. Automated spectral unmixing algorithms were created using inForm software (Akoya Biosciences, Marlborough, MA) and applied to whole-slide scans in order to remove tissue autofluorescence and spectral overlap. Images were analyzed with HALO image processing software (Indica Laboratory, Albuquerque, NM).
# Image analysis
The gray matter of the depths of the cortical sulci (bottom third of two connecting gyri) and crests of the gyri (top third of the gyrus adjacent to the selected sulcus) were defined and annotated in HALO (Indica Laboratory). The gray/ white matter boundary was used as the outer edge of the annotation region. Initial counting of NFTs and p-tau-positive astrocytes was carried out by morphologic assessment using IHC-stained AT8-labeled sections. All counting was performed in HALO. The sulcus and crest were annotated and analyzed separately as previously described [bib_ref] Microglial neuroinflammation contributes to tau accumulation in chronic traumatic encephalopathy, Cherry [/bib_ref]. First, using HALO AI (Indica Laboratory), an image recognition algorithm was trained to identify and count NFTs. The algorithm was trained using 4236 individual neurons and allowed to iterate over 75, 000 times. As astrocytes have a diverse morphologic phenotype, automated AI-based counting techniques were not able to reliably and consistently identify p-tau-positive astrocytes. Therefore, they were manually counted by an unbiased, blinded observer as previously described [bib_ref] Evolution of neuronal and glial tau isoforms in chronic traumatic encephalopathy, Cherry [/bib_ref]. Briefly, these cells were defined as granular, less-defined cytoplasmic p-tau staining accompanied by delicate radially oriented processes surrounding a small nucleus.
Cells that were morphologically ambiguous were not counted. Cell counts were standardized to area measured and were presented as p-tau-positive cells/mm 2 , log of cell density, or percent of total cells. For mIF section analysis, the sulcus and crest were annotated using the same methods as the IHC analysis. Additionally, subpial regions were annotated using the HALO marginal partitioning tool and defined as the 200-mm strip of tissue below the pial surface. Lesions were identified with the help of a neuropathologist and annotated with the ellipse tool and defined as 0.4 mm 2 of pathognomonic CTE lesions surrounding small cortical blood vessels. This size captured lesion-associated cells, while avoiding neighboring AT8-positive cells. All lesions observed were included in the analysis. Of the 51 total mIF cases, CTE lesions were identified in 17 cases and 38 total lesions were sampled for analysis. If multiple lesions were identified within an individual, the number of cells was averaged across lesions for that one case. The total number of cells double positive for MAP2/AT8 (neurons) and GFAP/ AT8 (astrocytes) were manually counted by a blinded observer and expressed either as the number of double positive cells or standardized to the total annotation area and expressed as cells/mm 2 .
## Statistics
Statistical analysis was performed using Prism (v9, GraphPad Software, San Diego, CA) or SPSS (v27). Individuals were either partitioned into groups based on CTE stage (CTE I/II/III/IV) or CTE severity [low CTE (CTE I/II) or high CTE (CTE III/IV)]. One-way ANOVA used to determine group differences in age at death and years of RHI exposure. Paired t-test was used to determine the difference between NFT-and p-tau-positive astrocyte densities. Linear regressions were used on log-transformed data to determine the relationship between p-tau containing cell densities and RHI exposure. Analyses examining associations with RHI exposure included only individuals with football as their primary method of exposure to control for differences in exposure type. Linear regression was also used to determine the relationship between percentages of p-tau containing cells in CTE lesions and age. Analysis of covariance (ANCOVA) tests were used to determine the relationship between astrocytic p-tau, NFT densities, CTE stage, and years of RHI exposure with age as a covariate. Data expressed as mean 6 standard deviation. Age at death and years of exposure analyzed with one-way ANOVA.
# Results
## Neuronal p-tau is predominant in cte and both neuronal and astrocytic p-tau are elevated in the sulcus
To examine the spatial and cellular distribution of p-taupositive cells in CTE, IHC AT8 immunostaining was performed in the DLFC of 150 individuals pathologically diagnosed with CTE. NFTs and astrocytes were counted in the sulcus and crest from each individual. p-Tau-positive cells were found to be predominantly neuronal [fig_ref] FIGURE 1: Neuronal p-tau is predominant in CTE and both neuronal and astrocytic p-tau... [/fig_ref]. NFT density was significantly elevated in the cortical sulcus compared to the gyral crest in low-stage CTE (p < 0.0001) and highstage CTE [fig_ref] FIGURE 1: Neuronal p-tau is predominant in CTE and both neuronal and astrocytic p-tau... [/fig_ref]. Additionally, there was significantly elevated sulcal astrocytic p-tau density in both low (p < 0.001) and high CTE (p < 0.0001, [fig_ref] FIGURE 1: Neuronal p-tau is predominant in CTE and both neuronal and astrocytic p-tau... [/fig_ref]. Density heatmap plots of the total p-tau containing neurons and astrocytes demonstrated that both cell types were found predominantly in the sulcus compared to the crest, however, the distribution within the gray matter and prevalence differed between cell types [fig_ref] FIGURE 1: Neuronal p-tau is predominant in CTE and both neuronal and astrocytic p-tau... [/fig_ref].
## Astrocytic p-tau in cte is primarily age-related and subpial, while neuronal p-tau is associated with rhi exposure
Next, the relationship between p-tau containing cell types and age and years of RHI exposure was determined. Linear regression demonstrated that neuronal p-tau and astrocytic p-tau were both associated with age (p < 0.0001, b ¼ 0.343, . However, only neuronal ptau densities were associated with years of exposure to American football play (p ¼ 0.0005, b ¼ 0.199); astrocytic densities were not (p ¼ 0.329, b ¼ 0.094, .
[formula] p ¼ 0.0014, b ¼ 0.324, [/formula]
In order to provide a robust measure of p-tau containing cell types in CTE, mIF analysis was performed in a subset of the larger groups used for IHC analysis. Neurons were labeled with MAP2, astrocytes with GFAP, and p-tau with AT8 . To investigate the localization of p-tau containing cells within the sulcus, the sulcus was separated into parenchymal and subpial regions. Paired t-test demonstrated that a significant proportion (75.25%, p < 0.0001) of p-tau-positive astrocytes was localized to the subpial region .
ANCOVA analyses comparing astrocytic p-tau densities to CTE stage with age as a covariate showed that astrocytic p-tau was significantly associated with age at death (p ¼ 0.007) but not CTE stage or severity (p ¼ 0.44, p ¼ 0.105). Subpial TSAs were observed in individuals with CTE stage II and higher, with no p-tau-positive astrocytes found in CTE stage I individuals . There was no significant association between FIGURE 2. Astrocytic p-tau in CTE is primarily age-related and subpial, while neuronal p-tau is associated with RHI exposure. (A) Scatter plot of the log-transformed neuronal and astrocytic p-tau densities compared to age demonstrate a linear relationship between age and neuronal p-tau density (p < 0.0001, b ¼ 0.343) and astrocytic p-tau density (p ¼ 0.0014, b ¼ 0.324), n ¼ 149. subpial and parenchymal astrocytic p-tau densities and CTE stage . In contrast, both subpial and parenchymal astrocytic p-tau consistently increased and were significantly associated with age (p < 0.001, p ¼ 0.048). Sparse subpial TSAs were found in one individual as young as 36 years old and were found in all cases over the age of 50. Their prevalence remained a consistent proportion of the total p-tau pathology in individuals above the age of 50. Parenchymal p-tau-positive astrocytes were present in individuals as young as 30 (one ptau-positive astrocyte), in all cases over the age of 60, and their prevalence increased with each decade of age .
## Neuronal tau defines the cte perivascular lesion
Next, analysis was focused on the CTE lesion to further characterize the primary p-tau containing cell found within the perivascular lesion. Qualitative morphological assessment demonstrated that the cell type primarily observed around the CTE lesion was neuronal [fig_ref] FIGURE 3: Neuronal tau defines the CTE perivascular lesion [/fig_ref]. Multiplex immunofluorescence was used to quantitate the number of neurons (MAP2-positive) and astrocytes (GFAP-positive) within the CTE lesion [fig_ref] FIGURE 3: Neuronal tau defines the CTE perivascular lesion [/fig_ref]. Significantly more neuronal p-tau was observed in pathognomonic lesions compared to astrocytic ptau in all the individuals studied, with 95% of p-tau containing cells being neurons [fig_ref] FIGURE 3: Neuronal tau defines the CTE perivascular lesion [/fig_ref]. Only 29% of pathognomonic lesions contained astrocytic p-tau. Finally, the proportion of ptau containing cells found within CTE lesions was not found to have a significantly increased proportion of astrocytes with increasing age [fig_ref] FIGURE 3: Neuronal tau defines the CTE perivascular lesion [/fig_ref]. These findings demonstrate that there is a distinct neuronal predominance of p-tau within CTE lesions in individuals across a range of ages and CTE stages.
# Discussion
This study demonstrated that the neuron is the primary cell type with p-tau pathology within the CTE lesion and at the depth of the cortical sulcus. Although p-tau containing astrocytes were observed in individuals with CTE, they were predominantly found as subpial TSA and not primary cell components of the deeper gray matter or perivascular CTE lesions. Furthermore, astrocytic p-tau was associated with age and not years of exposure to RHI. Overall, this work supports the 2021 consensus requirement for neuronal p-tau in the pathognomonic lesion of CTE.
Past studies have attempted to determine the cellular distribution of p-tau across neurons and astrocytes in CTE neuropathology. However, these studies solely relied on morphologic assessment of p-tau staining or assumed that 4R p-tau immunostaining was astrocyte-specific [bib_ref] Astroglial tau pathology alone preferentially concentrates at sulcal depths in chronic traumatic..., Arena [/bib_ref] [bib_ref] A quantitative study of tau pathology in eleven cases of chronic traumatic..., Armstrong [/bib_ref]. Although morphologic assessment of p-tau in the shape of neurons or astrocytes is useful, neurons and astrocytes do not always conform to traditional morphology and can be misidentified. Recent approaches utilizing 3R and 4R p-tau isoforms to distinguish neurons and astrocytes also failed to consider the evolution of neuronal p-tau from 4R to 3R isoforms throughout the progression of CTE [bib_ref] CONNECT-TBI Investigators. Detection of astrocytic tau pathology facilitates recognition of chronic traumatic..., Ameen-Ali [/bib_ref] [bib_ref] Tau isoforms are differentially expressed across the hippocampus in chronic traumatic encephalopathy..., Cherry [/bib_ref]. This study is the first to utilize quantitative mIF to directly coloc-alize cell-type-specific markers in a large group of individuals with CTE, and no comorbidities over a wide age range.
Age-related diseases such as ARTAG and primary agerelated tauopathy (PART) share some pathologic features with CTE. Subpial TSA pathology or ARTAG is common in CTE and bears similarities to the depth of sulcus perivascular lesion in CTE but is not a diagnostic feature [bib_ref] Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Kovacs [/bib_ref]. While perivascular CTE lesions may include p-tau-positive astrocytes, overlapping ARTAG pathology observed in this study was predominantly confined to the subpial surface at the depth of the cortical sulcus and was visually distinct from the CTE neuropathologic lesion. Importantly, neuronal pathology in CTE is directly related to years of exposure to RHI, whereas ARTAG pathology is not related to RHI exposure and is agerelated [bib_ref] Chronic traumatic encephalopathy (CTE) is absent from a european community-based aging cohort..., Forrest [/bib_ref]. In a community-based aging cohort, Forrest et al [bib_ref] Chronic traumatic encephalopathy (CTE) is absent from a european community-based aging cohort..., Forrest [/bib_ref] found ARTAG on neuropathological examination of 117 out of 310 individuals (38%) ranging in age from 76 to 91 (mean ¼ 83 6 3 years) and no instances of CTE, further supporting ARTAG as an age-related, non-RHI-related pathologic feature.
RHI exposure has been associated with several neurodegenerative pathologies, including b-amyloid deposition and Lewy body disease [bib_ref] Beta-amyloid deposition in chronic traumatic encephalopathy, Stein [/bib_ref] [bib_ref] Association of probable REM sleep behavior disorder with pathology and years of..., Adams [/bib_ref] [bib_ref] Lewy body pathology and chronic traumatic encephalopathy associated with contact sports, Adams [/bib_ref]. Additionally, subpial TSA and perivascular astrocytic tau pathology have been reported after head trauma in several case reports [bib_ref] The aftermath of boxing revisited: Identifying chronic traumatic encephalopathy pathology in the..., Goldfinger [/bib_ref] [bib_ref] Characterisation of interface astroglial scarring in the human brain after blast exposure:..., Shively [/bib_ref] [bib_ref] Space-occupying brain lesions, trauma-related tau astrogliopathy, and ARTAG: A report of two..., Bachstetter [/bib_ref]. It has been proposed that ARTAG, specifically subpial TSA, is associated with exposure to serum proteins and other peripherally circulating factors as a result of blood-brain barrier (BBB) dysfunction with age. BBB disruption is altered after RHI and might be exacerbated by CTE pathology [bib_ref] Blood-brain barrier dysfunction as a hallmark pathology in chronic traumatic encephalopathy, Doherty [/bib_ref]. In this study, 69% of the RHI-exposed brain donors displayed subpial TSA, however, no association was found between subpial TSA, CTE severity, or RHI exposure. Comparison of an RHIexposed group to an age-matched non-RHI control group is necessary to directly determine the effect of RHI exposure and CTE on the presence of ARTAG. The data presented here do not support ARTAG as a diagnostic feature of RHI exposure or CTE, consistent with the existing NINDS/NIBIB consensus criteria [bib_ref] The second NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis..., Bieniek [/bib_ref]. Overall, the current study demonstrates that p-tau containing astrocytes are observed in CTE, yet are not numerous, and are associated with age not CTE severity. While the driving factor for astrocytic p-tau in CTE is age, astrocytic degeneration likely plays a role in the pathobiology and progression of CTE pathology along with other factors such as cellular senescence, BBB breakdown, and increases in baseline inflammation as features of normal aging [bib_ref] Evolution of neuronal and glial tau isoforms in chronic traumatic encephalopathy, Cherry [/bib_ref].
Much of the discrepancy in previous studies regarding the role of astrocytic p-tau in CTE pathology can be attributed to examination of a narrow or limited older age range of individuals with extensive comorbid neurogenerative diseases and reliance on cellular morphology to discriminate p-tau in astrocytes from p-tau in neurons [bib_ref] CONNECT-TBI Investigators. Detection of astrocytic tau pathology facilitates recognition of chronic traumatic..., Ameen-Ali [/bib_ref]. The inclusion of cases with extensive overlapping disease makes identification of a CTEspecific features challenging. The strengths of the current study are the inclusion of a large number of individuals without neuropathological evidence of comorbid disease from a wide range of ages (21-80 years old), and the use of quantitative mIF to determine cell type. In both low and high CTE, neurons represented significantly more (p < 0.0001) of the total p-tau containing cells in a lesion compared to astrocytes. Statistics generated using paired t-tests. (F) Neither neuronal or astrocytic p-tau was significantly associated with age within the pathognomonic lesion (p ¼ 0.34).
# Limitations
This was a convenience sample limited to brain donors neuropathologically diagnosed with CTE that is not representative of the general population. Additionally, only 2 cell-type markers were used, MAP2 and GFAP, which might not have captured all neurons or astrocytes in the tissue samples, as, for example, astrocytes are known to change their expression of GFAP in disease [bib_ref] Reactive astrocyte nomenclature, definitions, and future directions, Escartin [/bib_ref]. For the CTE lesion analysis, as p-tau pathology becomes more severe, the confluent nature of p-tau distribution at end-stage disease makes it difficult to identify clearly defined lesion borders. It is possible that some lesions were missed in very severe cases of CTE in older individuals. However, this would represent only a small fraction of the cases used in the current study and is unlikely to affect the results. A larger sample of CTE lesions may reveal an association of increased perivascular astrocytes with age. Future work with additional neuronal or astrocytic markers would be useful to identify possible additional p-tau containing cellular subtypes. Furthermore, the goal of this study was to characterize the progression of p-tau pathology in CTE; therefore, healthy aged controls and RHI with no disease controls were not included. Future studies are needed to determine how the cell type profile of neuronal and astrocytic p-tau might change across all control and non-CTE, comorbid neurodegenerative disease conditions.
# Conclusion
This study demonstrates, through the use of a large group of brain donors neuropathologically diagnosed with CTE, across a wide range of age at death and CTE stage, without comorbid neuropathology, that the primary p-tau containing cell type in CTE is neuronal, and that the presence of astrocytic p-tau is a factor of age, not CTE severity or years of exposure to RHI. This work provides additional evidence that CTE p-tau pathology evolves over the lifespan. Overall, this work validates the recent observations and consensus statements made by the NINDS/NIBIB consensus panels on CTE and provides a better understanding of the pathobiology of CTE as a basis for future diagnostic and therapeutic strategies.
[fig] FIGURE 1: Neuronal p-tau is predominant in CTE and both neuronal and astrocytic p-tau are elevated in the sulcus. (A) Quantitation of all morphologically identified neurofibrillary tangles (NFTs) and p-tau containing astrocytes. Paired t-test (****p < 0.0001). (B) Neuronal p-tau densities are elevated in the sulcus in low and high CTE. Paired t-tests (***p < 0.001, ****p<0.0001). (C) Astrocytic p-tau densities are predominant in sulci in low and high CTE. Paired t-tests (***p < 0.001, ****p < 0.0001). (D, E) Representative brightfield images of AT8-immunolabeled sections in low and high CTE and corresponding density heatmap demonstrating sulcal predominance of neuronal and astrocytic p-tau. All graphs represent mean 6 standard error of the mean. [/fig]
[fig] FIGURE 3: Neuronal tau defines the CTE perivascular lesion. (A, B) Representative brightfield images from young donors demonstrating consistent identification of neuronal p-tau at the CTE pathognomonic lesion. The CTE Brain Bank (A) and Australian Sports Brain Bank (B) both demonstrate the appearance of p-tau found around the CTE neuropathologic lesion in young individuals is primarily neuronal. (B) Panel is included as a representative image only and not included in the quantitative analysis with the goal of demonstrating the CTE lesion appears consistent across separate brain banks. Inserts shows a magnified view on individual neurofibrillary tangles from numbered inset boxes. Scale bar ¼ 200 mm. (C) Representative image of an immunofluorescence labeled section with CTE pathognomonic lesion at the depth of the cortical sulcus. White box indicates right panel inset zoom of the lesion. White arrows indicate neuronal p-tau co-labeled with MAP2 and AT8, asterisks indicate a blood vessel at the center of the lesion, and scale bar ¼ 1 mm, 250 mm, n ¼ 17 individuals. (D) Quantification of total cells per CTE lesion demonstrating neuronal predominance (p < 0.0001). (E) [/fig]
[table] TABLE 1: Group Demographics [/table]
|
Recommendations for COVID‐19 vaccination in people with rheumatic disease: Developed by the Singapore Chapter of Rheumatologists
Aim: People with rheumatic diseases (PRD) remain vulnerable in the era of the COVID-19 pandemic. We formulated recommendations to meet the urgent need for a consensus for vaccination against SARS-CoV-2 in PRD.Methods: Systematic literature reviews were performed to evaluate: (a) outcomes in PRD with COVID-19; (b) efficacy, immunogenicity and safety of COVID-19 vaccination; and (c) published guidelines/recommendations for non-live, non-COVID-19 vaccinations in PRD. Recommendations were formulated based on the evidence and expert opinion according to the Grading of Recommendations Assessment, Development and Evaluation methodology. Results: The consensus comprises 2 overarching principles and 7 recommendations.Vaccination against SARS-CoV-2 in PRD should be aligned with prevailing national policy and should be individualized through shared decision between the healthcare provider and patient. We strongly recommend that eligible PRD and household contacts be vaccinated against SARS-CoV-2. We conditionally recommended that the COVID-19 vaccine be administered during quiescent disease if possible.Immunomodulatory drugs, other than rituximab, can be continued alongside vaccination. We conditionally recommend that the COVID-19 vaccine be administered prior to commencing rituximab if possible. For patients on rituximab, the vaccine should be administered a minimum of 6 months after the last dose and/or 4 weeks prior to the next dose of rituximab. Post-vaccination antibody titers against SARS-CoV-2 need not be measured. Any of the approved COVID-19 vaccines may be used, with no particular preference. Conclusion: These recommendations provide guidance for COVID-19 vaccination in PRD. Most recommendations in this consensus are conditional, reflecting a lack of evidence or low-level evidence. | 747 SANTOSA eT Al.
## | introduc ti on
Novartis (Novavax® or NVX-CoV2373)and Bharat Biotech (BB-152 or Covaxin®),
## | me thods
## | review of the literature
The CWG sent out preselected topics to the TFP and sought their input on additional clinically important topics. Considering the TFP's input, the CWG selected the following core topics relevant to clinical decision-making for COVID-19 vaccination:
1. Are PRD at increased risk of adverse outcomes from COVID-19?
A recent systematic review and meta-analysis of global data showed that PRD remain vulnerable, with substantial rates of severe outcomes. 3 The overall rates of hospitalization, oxygen support, ICU admission and fatality among COVID-19 infected patients with rheumatic diseases were 58% (95% CI 48%-67%), 33% (95% CI 21%-47%), 9% (95% CI 5%-15%) and 7% (95% CI 3%-11%), respectively, which are comparable with data from the COVID-19 Global Rheumatology Alliance (GRA) physician registry. The fatality rate was higher both in this meta-analysis and the COVID-19 GRA (7.0% and 6.7%, respectively) than that (3.4%) of general population infected with COVID-19 in the WHO database, although age, gender and comorbidities were not matched. 3 D'Silva et al reported a higher risk of hospitalization, ICU admission, mechanical ventilation, acute kidney injury, renal replacement therapy and death based on TriNetX, a multi-center research network with real-time electronic health record data across 35 healthcare organizations in the US.The authors concluded that these outcomes were likely mediated by a higher comorbidities burden in PRD, such as hypertension, diabetes mellitus, chronic kidney disease and asthma.
2. Are existing approved vaccines against SARS CoV2 safe, immunogenic and efficacious in PRD?
## K e y w o r d s
COVID-19, immunosuppression, people with rheumatic diseases, SARS-CoV-2, vaccination Two mRNA vaccines are currently approved by the US FDA and Singapore HSA. It is known that selected DNA and RNA molecules have the unique property to activate the immune system, through activation of Toll-like receptors.It has been shown that the innate immune response would be suppressed by nucleoside modification of RNA, as the innate immune system detects RNA lacking nucleoside modification as a means of selectively responding to bacteria or viruses.vaccine trials to date are summarized in .There are currently no available data on the immunogenicity and efficacy of COVID-19 vaccination in PRD. .The definitions of PRD and immunomodulatory drugs considered in this recommendation are summarized in .
## | creation of preliminary statements and rating
The CWG met to formulate and finalize preliminary statements
## | finalizing consensus statements
The final consensus statement was circulated to the TFP after the consensus meeting and was approved by all members.
## | re sults
The final consensus statements consist of 2 overarching principles and 7 recommendations. They are summarized in .
## | overarching principles
1. Vaccination in PRD should be aligned with prevailing national policy.
The knowledge on COVID-19 vaccination is rapidly evolving protection is not well established at present, we conditionally recommend that titers not be measured.. We strongly recommend that household contacts be vaccinated against SARS-CoV-2. Level of evidence as defined: High -very unlikely to change confidence in the estimate of effect by an additional study; Moderate -likely to change confidence in the estimate of effect by an additional study; Low -highly likely to change confidence in the estimate of effect by an additional study; Very low -not sure about confidence in the estimate of effect c Level of evidence as defined: 1a -meta-analysis of randomized controlled trials (RCT); 1b -RCT; 2 -prospective controlled intervention study without randomization; 3 -descriptive/analytic study (including case-control, cross-sectional, case series); 4 -expert committee reports or opinion or clinical experience of respected authorities or both d GRADE level of evidence. 32 e Level of evidence as defined: High -consistent evidence from well performed RCTs or exceptionally strong evidence from unbiased observational studies; Moderate -evidence from RCTs with important limitations (inconsistent results, methodological flaws, indirect, or imprecise) or exceptionally strong evidence from unbiased observational studies; Low -evidence for at least 1 critical outcome from observational studies, RCTs with serious flaws or indirect evidence; Very low -evidence for at least 1 critical outcome from unsystematic clinical observations or very indirect evidence.
## Efficacy
Timing /DMARD cessation
## 7.
We conditionally recommend that any of the approved COVID-19 vaccines may be used, with no particular preference.
The various SARS-CoV-2 vaccines in development are non-live vaccines. The anticipated risk-benefit ratio should therefore be similar for vaccinations to be recommended without preference for any particular vaccine. However, long-term follow-up in PRD will be needed to ascertain longer term efficacy and safety of the various vaccines.
## | d iscuss i on
# Strength of recommendation
Overarching principles Vaccination in people with rheumatic diseases should be aligned with prevailing national policy.
## 100 -
The decision for vaccination should be individualized, and should be explained to the patient, to provide a basis for shared decision-making between the healthcare provider and the patient. |
Increased generosity under COVID-19 threat
In the face of crises-wars, pandemics, and natural disasters-both increased selfishness and increased generosity may emerge. In this paper, we study the relationship between the presence of COVID-19 threat and generosity using a four-year longitudinal dataset (N = 696,942) capturing real donations made before and during the pandemic, as well as allocations from a 6-month dictator game study (N = 1003 participants) during the early months of the pandemic. Consistent with the notion of "catastrophe compassion" and contrary to some prior research showing a tendency toward self-interested behavior under threat, individuals across both datasets exhibited greater financial generosity when their county experienced COVID-19 threat. While we find that the presence of threat impacted individual giving, behavior was not sensitive to threat level. Our findings have significant societal implications and advance our understanding of economic and psychological theories of social preferences under threat.During major crises, such as natural disasters, wars, and now the COVID-19 pandemic, two conflicting behaviors may emerge: increased selfishness or increased generosity. Selfishness is innate to our survival instincts 1,2 . Evidence suggests that when facing adverse circumstances, individuals' may shift away from other-regarding practices 3 , arguably because fear and uncertainty resulting from increased risk perceptions 4 drive self-preservation 5 . Along these lines, research finds that the presence of threat can decrease individuals' willingness to engage in charitable activities and civic duties (e.g., paying taxes and reporting a crime 6 ) and that generosity toward in-group and out-group members may decrease following a natural disaster 7 . Considering these findings, one might expect that individuals experiencing COVID-19 threat would, on average, behave more selfishly than those not experiencing threat. Indeed, at an early stage of the COVID-19 pandemic, survey data representing 35 countries showed that, despite government appeals, those who felt more threatened were more likely to engage in selfish stockpiling 8 , putting the health and well-being of others at risk 9 . Across the world, many of the widespread product shortages during the early months of the COVID-19 pandemic were triggered by consumers purchasing resources in excess of their actual need (e.g., hoarding toilet paper 10 and masks 11 ).On the other hand, there is evidence suggesting that groups facing a common threat often demonstrate stronger social cohesion 12,13 and more cooperative, communal behaviors 14-17 . As proposed by Jamil Zaki's "catastrophe compassion" theory 18 , disasters may promote an increased sense of community and altruism 19 . Indeed, experiencing high (vs. low) stress can increase trust and sharing behaviors 20,21 . Perceived threat may also promote the expansion of social connections, as observed in monkeys in response to the environmental instability caused by Hurricane Maria 22 . Considering these findings, one might predict that experiencing and bearing witness to the devastating effects of the COVID-19 pandemic would promote generosity. Consistent with this proposition, a survey conducted by the Lilly Family School of Philanthropy 23 during the COVID-19 pandemic found that nearly half of respondents supported their communities in a variety of ways, for example by continuing to pay individuals and businesses for services that could not be rendered 24 .Interestingly, research suggests that the impact of threat could go either way-increasing or decreasing generosity. For example, when primed with resource scarcity, individuals became more competitive, causing some to become more selfish while others to exhibit greater generosity, depending on the context (e.g., behavior observability) 25 . Additional research finds evidence that communities experiencing disasters could simultaneously undergo positive and negative behavioral change 26 .We examine the relationship between COVID-19 threat and generosity using two independent longitudinal datasets. The first dataset, provided by Charity Navigator (CN), the world's largest independent charity evaluator, consisted of actual charitable-giving data spanning July 2016 through December 2020 (N = 696,942 donations). For each donation, the data included the donation amount, the charities benefited, each charity's assigned category (e.g., environment and human services), and the donor's location. In addition, CN assigned each donor OPEN
## Increased generosity under covid-19 threat ariel fridman * , rachel gershon & ayelet gneezy
In the face of crises-wars, pandemics, and natural disasters-both increased selfishness and increased generosity may emerge. In this paper, we study the relationship between the presence of COVID-19 threat and generosity using a four-year longitudinal dataset (N = 696,942) capturing real donations made before and during the pandemic, as well as allocations from a 6-month dictator game study (N = 1003 participants) during the early months of the pandemic. Consistent with the notion of "catastrophe compassion" and contrary to some prior research showing a tendency toward self-interested behavior under threat, individuals across both datasets exhibited greater financial generosity when their county experienced COVID-19 threat. While we find that the presence of threat impacted individual giving, behavior was not sensitive to threat level. Our findings have significant societal implications and advance our understanding of economic and psychological theories of social preferences under threat.
During major crises, such as natural disasters, wars, and now the COVID-19 pandemic, two conflicting behaviors may emerge: increased selfishness or increased generosity. Selfishness is innate to our survival instincts [bib_ref] Altruism and selfishness, Rachlin [/bib_ref]. Evidence suggests that when facing adverse circumstances, individuals' may shift away from other-regarding practices [bib_ref] Loss of communality at Buffalo Creek, Erikson [/bib_ref] , arguably because fear and uncertainty resulting from increased risk perceptions 4 drive self-preservation [bib_ref] The influence of stress hormones on fear circuitry, Rodrigues [/bib_ref]. Along these lines, research finds that the presence of threat can decrease individuals' willingness to engage in charitable activities and civic duties (e.g., paying taxes and reporting a crime [bib_ref] The neurogenetics of nice: Receptor genes for oxytocin and vasopressin interact with..., Poulin [/bib_ref] and that generosity toward in-group and out-group members may decrease following a natural disaster [bib_ref] Property damage and exposure to other people in distress differentially predict prosocial..., Vardy [/bib_ref]. Considering these findings, one might expect that individuals experiencing COVID-19 threat would, on average, behave more selfishly than those not experiencing threat. Indeed, at an early stage of the COVID-19 pandemic, survey data representing 35 countries showed that, despite government appeals, those who felt more threatened were more likely to engage in selfish stockpiling [bib_ref] Influence of perceived threat of Covid-19 and HEXACO personality traits on toilet..., Garbe [/bib_ref] , putting the health and well-being of others at risk. Across the world, many of the widespread product shortages during the early months of the COVID-19 pandemic were triggered by consumers purchasing resources in excess of their actual need (e.g., hoarding toilet paper 10 and masks 11 ).
On the other hand, there is evidence suggesting that groups facing a common threat often demonstrate stronger social cohesion [bib_ref] Civil war and social cohesion: Lab-in-the-field evidence from Nepal, Gilligan [/bib_ref] and more cooperative, communal behaviors [bib_ref] Conflict, sticks and carrots: War increases prosocial punishments and rewards, Gneezy [/bib_ref] [bib_ref] Can war foster cooperation, Bauer [/bib_ref] [bib_ref] Sudden solidarity and the rush to normalization: Toward an alternative approach, Turkel [/bib_ref]. As proposed by Jamil Zaki's "catastrophe compassion" theory 18 , disasters may promote an increased sense of community and altruism [bib_ref] Effect of a national disaster on blood supply and safety: The September..., Glynn [/bib_ref]. Indeed, experiencing high (vs. low) stress can increase trust and sharing behaviors [bib_ref] The social dimension of stress reactivity: Acute stress increases prosocial behavior in..., Von Dawans [/bib_ref] [bib_ref] Effects of stress and commonality of fate on helping behavior, Dovidio [/bib_ref]. Perceived threat may also promote the expansion of social connections, as observed in monkeys in response to the environmental instability caused by Hurricane Maria [bib_ref] Rhesus macaques build new social connections after a natural disaster, Testard [/bib_ref]. Considering these findings, one might predict that experiencing and bearing witness to the devastating effects of the COVID-19 pandemic would promote generosity. Consistent with this proposition, a survey conducted by the Lilly Family School of Philanthropy 23 during the COVID-19 pandemic found that nearly half of respondents supported their communities in a variety of ways, for example by continuing to pay individuals and businesses for services that could not be rendered.
Interestingly, research suggests that the impact of threat could go either way-increasing or decreasing generosity. For example, when primed with resource scarcity, individuals became more competitive, causing some to become more selfish while others to exhibit greater generosity, depending on the context (e.g., behavior observability) [bib_ref] On the psychology of scarcity: When reminders of resource scarcity promote selfish..., Roux [/bib_ref]. Additional research finds evidence that communities experiencing disasters could simultaneously undergo positive and negative behavioral change [bib_ref] The norm of solidarity: Experiencing negative aspects of community life after a..., Nurmi [/bib_ref].
We examine the relationship between COVID-19 threat and generosity using two independent longitudinal datasets. The first dataset, provided by Charity Navigator (CN), the world's largest independent charity evaluator, consisted of actual charitable-giving data spanning July 2016 through December 2020 (N = 696,942 donations). For each donation, the data included the donation amount, the charities benefited, each charity's assigned category (e.g., environment and human services), and the donor's location. In addition, CN assigned each donor www.nature.com/scientificreports/ a unique identifier, which allowed us to observe within-person differences in donation behavior in both the presence and absence of COVID-19 threat. The second dataset, which sheds light on the relationship between COVID-19 threat and generosity in a more controlled setting, consisted of individuals' (N = 1003 U.S. participants) allocations from an incentivized dictator game. In the dictator game, one player (the dictator) receives $10 and makes a unilateral decision on how to divide it between themselves and another, typically unknown, individual [bib_ref] What's in a name? Anonymity and social distance in dictator and ultimatum..., Charness [/bib_ref]. Rather than maximizing their own financial payoff (i.e., allocating $0), experimental evidence shows that "dictators" often choose to give some of their money to recipients [bib_ref] On the interpretation of giving in dictator games, List [/bib_ref] [bib_ref] Dictator games: A meta study, Engel [/bib_ref]. In our study, participants played an incentive-compatible dictator game monthly, from March to August 2020. Importantly, at the start of this period, COVID-19 threat was only present in 10% of participants' counties (see SI Appendix, , allowing us to observe their behavior when threat was first introduced in most counties. Notably, while the dictator game has previously been used to capture generosity at a single point in time, there are few cases of its use in a longitudinal setting [bib_ref] Property damage and exposure to other people in distress differentially predict prosocial..., Vardy [/bib_ref] [bib_ref] Learning to be selfish? A large-scale longitudinal analysis of Dictator games played..., Arechar [/bib_ref]. Finally, our research is unique in the use of longitudinal dictator game data jointly with longitudinal archival data to show convergent evidence of changes in giving behavior.
While our observational datasets do not lend themselves to causal claims, it is reasonable to infer that the presence of threat would increase generosity [bib_ref] Civil war and social cohesion: Lab-in-the-field evidence from Nepal, Gilligan [/bib_ref] [bib_ref] Conflict, sticks and carrots: War increases prosocial punishments and rewards, Gneezy [/bib_ref] [bib_ref] Can war foster cooperation, Bauer [/bib_ref] [bib_ref] Catastrophe compassion: Understanding and extending prosociality under crisis, Zaki [/bib_ref] [bib_ref] Effect of a national disaster on blood supply and safety: The September..., Glynn [/bib_ref] [bib_ref] The social dimension of stress reactivity: Acute stress increases prosocial behavior in..., Von Dawans [/bib_ref] [bib_ref] Effects of stress and commonality of fate on helping behavior, Dovidio [/bib_ref] [bib_ref] Rhesus macaques build new social connections after a natural disaster, Testard [/bib_ref] , while reverse causality is highly unlikely. See "Discussion" section for a more detailed explanation.
Our large-scale longitudinal datasets provide real-world evidence that people exhibited greater generosity during a time where some theories and experts predicted the opposite due to the economic downturn associated with the pandemic. While our analyses consider various levels of threat, we found that only the presence (vs. absence) of threat was associated with greater generosity. Our findings contribute to economic and psychological theories of social preferences, suggesting that people come together in the presence of a shared threat and demonstrate a willingness to support others, despite the uncertainty surrounding their own health and financial well-being.
# Results
Across both datasets, we observe increased generosity in the presence of COVID-19 threat in participants' geographic location.
Our analyses controlled for potential confounds at the national level (e.g., stimulus payouts and the Black Lives Matter movement) by including date fixed effects in all regressions. See "Materials and methods" section for details. Charity Navigator. We first analyzed the data with a relatively simplified approach by treating COVID-19 threat as a binary variable-whether any COVID-19-related deaths (vs. no COVID-19 deaths) occurred in each calendar month. In this analysis, we compared the proportion of counties that increased their overall donation amount as a function of whether the county had experienced COVID-19 threat. Compared with March 2019, 78% of counties that experienced threat increased the total amount donated in March 2020. Of the counties that did not face threat, 55% increased giving (χ 2 (1, N = 440) = 24.75, P < .001; see [fig_ref] Figure 1: Counties by COVID-19 threat and donation changes-March 2019 versus March 2020 [/fig_ref]. We found similar results for the comparison between April 2019 and April 2020 (see SI Appendix, [fig_ref] Figure 1: Counties by COVID-19 threat and donation changes-March 2019 versus March 2020 [/fig_ref]. After April 2020, only 33 counties or fewer did not face threat, though results were directionally the same for all months except August and October. While we treat counties with no COVID-19 deaths as "no threat", it is possible that some people in these counties still experienced COVID-19 threat, suggesting that our analysis is conservative. These results present initial model-free evidence for our main finding-that a greater proportion of counties experiencing threat increased giving, compared with those facing no threat.
To examine the relationship between COVID-19 threat level and county-level donation amount, we ran a regression analysis using county-level data aggregated by month-year. We captured the COVID-19 threat level using a 7-day average of daily new deaths per million in each county, which we averaged over all days in each month and binned into four categories by quantiles (no, low, medium, or high threat; log transformation generated similar results, see SI Appendix, Tables S2 and S3). We binned the threat variable to allow for non-linear relationships between threat and charitable giving. We regressed log-transformed aggregated giving amounts on threat level and included county and month-year fixed effects. This analysis showed that, overall, giving through CN's platform increased across all threat levels compared with no threat. On average, county-level giving increased 31.6% under low threat (SE = 0.06, t = 4.94, P < .001), 28.5% under medium threat (SE = 0.07, t = 3.86, P < .001), and 32.9% under high threat (SE = 0.05, t = 6.10, P < .001), relative to periods of no threat in the county. All pairwise comparisons across low, medium, and high threat levels were non-significant (P > .32), suggesting insensitivity to threat magnitude.
To examine whether our findings held within individuals (i.e., among repeat donors), we analyzed individuallevel data, which allows us to rule out potential selection bias (i.e., changes in donor characteristics before and during the COVID-19 pandemic). Of those who donated in 2020, 32% were repeat donors, meaning they made donations through the platform more than once. We captured the level of COVID-19 threat using the same seven-day lagged moving average of daily new deaths per million (without month-level averaging), considering "no threat" as our baseline. A regression of log-transformed giving amounts on threat level, including individual and date fixed effects, revealed that repeat donors' giving increased significantly across all charity categories by 3.4% under high threat (SE = 0.02, t = 2.11, P = .040), and non-significantly by 1.3% under medium threat (P = .348), and 2.8% under low threat (P = .063). All pairwise comparisons across low, medium, or high threat levels were non-significant (P > .07).
Although this effect is smaller than the effect we observe with the county-level model, including interaction terms with the charity category revealed a highly significant increase in donations to human services charitiesorganizations that provide direct services to those in need (see SI Appendix, [fig_ref] Figure 2: Charity Navigator donations [/fig_ref]. All pairwise comparisons across low, medium, or high threat levels were non-significant (P > .29). Collapsing all charity categories except human services revealed no significant difference in donations in response to the presence of COVID-19 threat (Ps > .55 for low, medium, and high threat). Notably, our analysis revealed a significant interaction of COVID-19 threat with human services charities (Ps < .001), indicating that under COVID-19 threat in one's county, repeat donors were significantly more likely to donate to human services charities than to other types of charities (see SI Appendix,. Together, these findings suggest giving to human services charities increased under threat but not at the expense of donations to other charity categories.
In an additional analysis, we found that the observed county-level increases in giving did not vary by countylevel median household income (see SI Appendix, for results).
Dictator game. An analysis of our six-wave longitudinal dictator-game data also showed that participants gave significantly more under the presence of COVID-19 threat. We used the same threat measure and individual-level regression model as the CN analysis. Our outcome measure was participants' allocation decisions, and we used wave fixed effects instead of date fixed effects. We found that within-person giving increased by approximately $0.25 (8.6%) under low threat (SE = 0.10, t = 2.58, P = .013), $0.38 (13.1%) under medium threat (SE = 0.11, t = 3.45, P = .001), and $0.24 (8.3%) under high threat (SE = 0.09, t = 2.56, P = .014), relative to periods of no threat in the participant's county [fig_ref] Figure 3: Dictator game allocations [/fig_ref] and SI Appendix, . Percentages were calculated relative to a mean allocation of $2.92. All pairwise comparisons across low, medium, and high threat levels were nonsignificant (P > .10). Our analysis further revealed a significant interaction between allocation amount and gender, indicating women gave more than men under threat (collapsed across low, medium, and high threat levels, P = .033). We found no interaction between allocation amount and age. Unlike some other COVID-19-related behaviors [bib_ref] Political ideology predicts perceptions of the threat of covid-19 (and susceptibility to..., Calvillo [/bib_ref] [bib_ref] COVID-19 and vaccine hesitancy: A longitudinal study, Fridman [/bib_ref] , we found no difference in giving patterns based on political affiliation.
# Discussion
Researchers have long argued that experiencing threat influences social preferences [bib_ref] Catastrophe compassion: Understanding and extending prosociality under crisis, Zaki [/bib_ref] , but offer different predictions for how. While some propose that those facing threat will become more generous, others predict increased selfishness. Leveraging data from a naturally-occurring state of emergency-the COVID-19 pandemic-we investigated the relationship between local COVID-19 threat and generosity. The present work offers several important findings. First, analyses of both datasets show that individuals exhibited greater financial generosity under COVID-19 threat. The CN data indicates contributions were directed primarily toward charities in the human services category-organizations that help mitigate the effects of COVID-19. Second, although we examined local COVID-19 threat, increased generosity often emerged in support of non-local organizations (CN data) or unidentified individuals (dictator game data). CN's data also show that the increase in donations was exhibited by both repeat and new donors, suggesting an overall increase www.nature.com/scientificreports/ in giving, as opposed to a mere allocation shift. We note that although both datasets demonstrate an association between the presence of threat and generosity, we found insensitivity to the level of threat. Although merely speculative, this pattern is consistent with research demonstrating scope insensitivity in emotionally charged settings [bib_ref] Music, pandas, and muggers: On the affective psychology of value, Hsee [/bib_ref] [bib_ref] Scope insensitivity and the "mere token" effect, Urminsky [/bib_ref] [bib_ref] Mindful judgment and decision making, Weber [/bib_ref]. Finally, the increased generosity observed across both datasets is particularly intriguing in light of expert predictions, based on historical data, that the economic downturn caused by the pandemic would lead to reduced giving, and the fact that a record-high majority of Americans reported a worsening financial situation during the same period. Prior work suggests that when people experience such financial scarcity, they may engage in extreme, even immoral, behaviors to acquire financial wealth [bib_ref] Financial deprivation selectively shifts moral standards and compromises moral decisions, Sharma [/bib_ref] [bib_ref] Resource scarcity and antisocial behavior, Prediger [/bib_ref]. Yet analyses of both our datasets clearly shows that in this particular circumstance, individuals were, on average, more willing to part with their financial resources. From a methodological perspective, our results lend credibility to the dictator game as a reliable measure of real-world generosity [bib_ref] What do laboratory experiments measuring social preferences reveal about the real world, Levitt [/bib_ref] , because our dictator-game findings are consistent with CN's field data. Thus, this work adds to a growing discussion in the literature regarding the validity of lab findings [bib_ref] Experimental methods: Extra-laboratory experiments-extending the reach of experimental economics, Charness [/bib_ref] [bib_ref] The External Validity of Laboratory Experiments: The Misleading Emphasis on Quantitative Effects, Kessler [/bib_ref]. The present research is also unique with respect to the longitudinal nature of our data, which, as noted in a recent call for the integration of such data 47 , is largely absent from behavioral research, and particularly rare in the context of major crises [bib_ref] Rhesus macaques build new social connections after a natural disaster, Testard [/bib_ref].
Although both datasets show that the presence of local COVID-19 threat is associated with increased generosity, our observational data does not necessarily lend itself to causal claims. With that in mind, we believe it is reasonable to infer that the presence of threat would influence generosity [bib_ref] Civil war and social cohesion: Lab-in-the-field evidence from Nepal, Gilligan [/bib_ref] [bib_ref] Conflict, sticks and carrots: War increases prosocial punishments and rewards, Gneezy [/bib_ref] [bib_ref] Can war foster cooperation, Bauer [/bib_ref] [bib_ref] Catastrophe compassion: Understanding and extending prosociality under crisis, Zaki [/bib_ref] [bib_ref] Effect of a national disaster on blood supply and safety: The September..., Glynn [/bib_ref] [bib_ref] The social dimension of stress reactivity: Acute stress increases prosocial behavior in..., Von Dawans [/bib_ref] [bib_ref] Effects of stress and commonality of fate on helping behavior, Dovidio [/bib_ref] [bib_ref] Rhesus macaques build new social connections after a natural disaster, Testard [/bib_ref] ; it is improbable, however, that the increase in generosity would trigger an increase in local COVID-19 threat. While we cannot rule out the possibility that there could be another variable that influences both factors to produce the observed correlation, our analyses and contemplation do not point to any likely candidates. However, it is possible that the presence of COVID-19 threat influences other variables (e.g., local lockdowns, media attention, etc.), which in turn lead to an increase in generosity. Nonetheless, we cautiously conclude that the presence of local COVID-19 threat led, either directly or indirectly, to an increase in generosity.
We are unable to discern what mechanism underlies the observed increase in generosity. Individuals may have been motivated to give more when experiencing threat as a result of increased feelings of sympathy 48 , a desire to regain a sense of agency and self-efficacy [bib_ref] Avoiding overhead aversion in charity, Gneezy [/bib_ref] [bib_ref] Goal gradient in helping behavior, Cryder [/bib_ref] , mortality salience 51,52 , or simply to experience positive emotions (e.g., warm glow 53,54 ) during a stressful period. It is also possible our findings reflect self-interested behavior if people increased their donations to benefit themselves, thinking that doing so would help to mitigate the negative effects of COVID-19 in general. However, it is unlikely that this motive explains the dictator game findings, where giving benefits a randomly chosen anonymous individual. Based on our CN data, it also seems likely individuals donated to honor those who passed away during the pandemic or were otherwise affected (e.g., sick, lost a loved one, etc.). Indeed, a closer look at the CN data shows that the proportion of donations made "in memory of " someone was significantly greater in 2020 than every prior year, although this motivation is likely less salient in our longitudinal dictator-game findings.
Since our CN data represents individuals with financial means to give, we cannot rule out the possibility that our findings apply to a subset of the population. However, we found similar results using the dictator-game data, which likely represents a less affluent demographic [bib_ref] Data collection in a flat world: The strengths and weaknesses of Mechanical..., Goodman [/bib_ref] , and an analysis comparing changes in giving on CN by county-level median household income also found no significant differences.
While our work focuses on the early period of the COVID-19 pandemic, additional research is needed to understand the dynamics of the relationship between threat and generosity in the longer term, as well as once the crisis has ended (e.g., see 7 for a dictator-game study before and after Cyclone Pam in 2015).
Finally, while our results show an increase in financial generosity under COVID-19 threat, it is possible that the pandemic also resulted in selfish behavior unobserved in our data (e.g., hoarding resources). Future research can investigate when, why, and for whom these divergent reactions may occur.
This work adds to our understanding of human behavior during times of crisis. Amidst the uncertainty, fear, and tragedy of the pandemic, we find a silver lining: people became more financially generous toward others in the presence of COVID-19 threat.
# Materials and methods
Human subject protections. The longitudinal dictator game received ethical approval from the UC San Diego Institutional Review Board (Project #191273XX) and was carried out in accordance with the guidelines and regulations for a human research study. Informed consent was obtained for all participants. No personal identifying information was collected to ensure participants' privacy.
## Covid-19 threat.
To capture COVID-19 threat, we used the Center for Systems Science and Engineering at Johns Hopkins University (https:// github. com/ CSSEG ISand Data) [bib_ref] An interactive web-based dashboard to track COVID-19 in real time, Dong [/bib_ref] time-series data of daily new deaths at the county level, which also includes U.S. Census Bureau population data by county. Our measure represented a seven-day lagged moving average of daily new deaths per million, which we binned into four categories based on population-weighted quantiles, taken over the entire timespan of the COVID-19 data in 2020: no deaths (0), bottom third (1), middle third [bib_ref] Altruism and selfishness, Rachlin [/bib_ref] , and top third [bib_ref] Loss of communality at Buffalo Creek, Erikson [/bib_ref]. Consistent with prior work 57 , we used deaths rather than confirmed cases for the threat measure, because it is more consistent over time, as changes in confirmed cases may capture changes in testing availability, particularly at the beginning of the pandemic [bib_ref] Substantial underestimation of SARS-CoV-2 infection in the United States, Wu [/bib_ref].
To merge participant data from the dictator game and donor data from CN with our COVID-19 threat measure, we mapped participants' zip codes to the corresponding county using the U.S. Department of Housing and Urban Development (https:// www. hudus er. gov/ portal/ datas ets/ usps_ cross walk. html) data.
County-level median household income data was obtained from the U.S. Department of Agriculture's Economic Research Service (https:// www. ers. usda. gov/ data-produ cts/ county-level-data-sets/)www.nature.com/scientificreports/ Charity Navigator. To measure changes in generosity over time, we analyzed a dataset of donations made through CN. For each donation, the data specified the following: date, donation amount, charity name, a unique donor identifier (anonymized email address), and donor zip code. After excluding incomplete observations (0.9%; see SI Appendix, Text S1), we were left with 696,942 donations.
Dictator game. We recruited a representative panel of U.S. residents (see SI Appendix, Text S2) on Amazon's Mechanical Turk platform to respond to monthly survey waves from March to August 2020. The sample consisted of 1003 unique participants. The first wave included 998 participants, ranging between 605 and 755 thereafter (5 participants did not respond to the dictator game in the first wave, but did so on subsequent waves; for attrition details, see SI Appendix, . After excluding incomplete observations due to missing county information (0.01%), we were left with 4272 observations. Participants played the dictator game 31,62 on all six survey waves and were always assigned to the dictator role. Participants allocated an amount between $0 and $10 to a randomly selected participant. To incentivize responses, we informed participants that, in each wave, one randomly selected participant would receive a $10 bonus, which would be split between them and another randomly selected participant according to their decision. Our survey also included demographic questions capturing age, gender, and political party affiliation.
Regression models. For our primary analysis, we examined the relationship between COVID-19 threat and giving using multiple regression.
Charity Navigator. In our county-level specification, we aggregated the data to the month-year level by county and estimated the following model:
where y cmy is the sum of the amount donated over all individuals in county c in month m in year y, α c are countylevel fixed effects, and α my are month-year fixed effects. threat cmy is the average threat level in county c in month m in year y, where s indexes the threat level and coefficients β s measure the effect of COVID-19 threat on giving, relative to no threat. We log-transformed the amount donated because it was right-skewed. To test whether the effect of threat varied by median household income, we ran a similar specification, including interaction terms for median household income and threat cmy . Standard errors were clustered by state and the regression was weighted by county population.
In our individual-level specification, we exploited within-person variation in giving over time, estimating the following model:
where y id is the amount donated by individual i on date d, α i are individual-level fixed effects, and α d are date fixed effects. threat c i d is the threat level in county c of individual i on date d. To estimate the effect of COVID-19 threat on donations for each category of charities, we ran a similar specification, including charity-category dummy variables and interaction terms for threat c i d and each charity category. Standard errors for both regressions were clustered by individual and state.
Dictator game. For the dictator-game analysis, we used a similar approach and estimated the following model:
where y iw is the amount given by individual i on survey wave w, α i are individual-level fixed effects, and α w are survey-wave fixed effects. threat c i w is the threat level in county c of individual i on wave w, where s indexes the threat level and coefficients β s measure the effect of COVID-19 threat on giving, relative to no threat. Standard errors were clustered by individual and state.
See SI Appendix, Text S3 for additional methodological information.
## Data availability
Our materials, anonymized behavioral data, and additional analyses, including robustness checks, are available on OSF (https:// osf. io/ 2rykb/). www.nature.com/scientificreports/
[fig] Figure 1: Counties by COVID-19 threat and donation changes-March 2019 versus March 2020. Orange [blue] represents the presence [absence] of threat in March 2020. Darker [lighter] shades indicate an increase [decrease] in giving across all charity categories relative to March 2019. The map shows U.S. counties with inset maps for counties in Alaska and Hawaii.The chart on the right shows the proportion of counties in each "threat present" and "donations increased" group. [/fig]
[fig] Figure 2: Charity Navigator donations. The vertical axis captures the difference between giving under threat relative to no threat (dashed line). The horizontal axis indicates threat levels in each participant's county at the time of the donation. Points and error bars represent regression coefficient estimates and 95% confidence intervals, respectively. Note the "All charities" category includes human services charities. [/fig]
[fig] Figure 3: Dictator game allocations. The vertical axis captures the difference between giving under threat relative to no threat (dashed line). The horizontal axis indicates threat levels in each participant's county at the time of the survey. Points and error bars represent regression coefficient estimates and 95% confidence intervals, respectively. Scientific Reports | (2022) 12:4886 | https://doi.org/10.1038/s41598-022-08748-2 [/fig]
[fig] 1: threat cmy =s + α c + α my + ε cmy , log (y id ) = 3 s=1 β s 1 threat c i d =s + α i + α d + ε id , 1 threat c i w =s + α i + α w + ε iw ,Scientific Reports | (2022) 12:4886 | https://doi.org/10.1038/s41598-022-08748-2 [/fig]
[table] Table S4: While one might expect www.nature.com/scientificreports/ an increase in donations to health charities, this category includes organizations such as Planned Parenthood and Cure Alzheimer's Fund, which are not directly related to the COVID-19 pandemic. In contrast, human services charities include food banks and homeless services. Among repeat donors, donations to human services [/table]
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Current update on nanoplatforms as therapeutic and diagnostic tools: A review for the materials used as nanotheranostics and imaging modalities
NanomedicineNanotechnology Nanoparticles Radiopharmaceuticals a b s t r a c t In the last decade, the use of nanotheranostics as emerging diagnostic and therapeutic tools for various diseases, especially cancer, is held great attention. Up to date, several approaches have been employed in order to develop smart nanotheranostics, which combine bioactive targeting on specific tissues as well as diagnostic properties. The nanotheranostics can deliver therapeutic agents by concomitantly monitor the therapy response in real-time.Consequently, the possibility of over-or under-dosing is decreased. Various non-invasive imaging techniques have been used to quantitatively monitor the drug delivery processes.Radiolabeling of nanomaterials is widely used as powerful diagnostic approach on nuclear medicine imaging. In fact, various radiolabeled nanomaterials have been designed and developed for imaging tumors and other lesions due to their efficient characteristics.Inorganic nanoparticles as gold, silver, silica based nanomaterials or organic nanoparticles as polymers, carbon based nanomaterials, liposomes have been reported as multifunctional nanotheranostics. In this review, the imaging modalities according to their use in various diseases are summarized, providing special details for radiolabeling. In further, the most current nanotheranostics categorized via the used nanomaterials are also summed up. To conclude, this review can be beneficial for medical and pharmaceutical society as well as material scientists who work in the field of nanotheranostics since they can use this research as guide for producing newer and more efficient nanotheranostics.
# Introduction
Nanomedicine is an emerging field combining nanoscience, nanoengineering, and nanotechnology with life sciences, revealing interesting results for the medical community and society . There are several nanomedicine platforms; however nanotechnology-based drug delivery systems as well as imaging nano-agents are of the greatest interest [bib_ref] Smart sorting of tumor phenotype with versatile fluorescent Ag nanoclusters by sensing..., Chen [/bib_ref]. Theranostic nanomedicine involves the use of theranostics with nanosizes and multiple capabilities such as targeted delivery, sustained/controlled release, greater transport efficiency via endocytosis, stimuli responsive systems and the combination of therapeutic approaches such as multimodality diagnosis and therapy [bib_ref] Tuned near infrared fluorescent hyaluronic acid conjugates for delivery to pancreatic cancer..., Qi [/bib_ref]. The "theranostics" word is referred to systems that can be both applied as therapeutics and imaging agents [bib_ref] Nanotheranosticsapplication and further development of nanomedicine strategies for advanced theranostics, Muthu [/bib_ref]. Nanotheranostics [fig_ref] Figure 1 -: A schematic representation of nanotheranostics used for simultaneous release and imaging [/fig_ref] are strategies based on carriers of submicron or nano sizes . Such nanotheranostics can be obtained from polymeric nanoparticles (NPs) [bib_ref] Auto-fluorescent polymer nanotheranostics for self-monitoring of cancer therapy via triple-collaborative strategy, Shao [/bib_ref] , dendrimers [bib_ref] Multifunctional lactobionic acid-modified dendrimers for targeted drug delivery to liver cancer cells:..., Fu [/bib_ref] , liposomes [bib_ref] Organ biodistribution of radiolabelled δγ T cells following liposomal alendronate administration in..., Wang [/bib_ref] , carbon based nanomaterials [bib_ref] Functionalised carbon nanotubes enhance brain delivery of amyloid-targeting Pittsburgh compound B (PiB)-derived..., Costa [/bib_ref] , metal or inorganic nanocarriers [bib_ref] FGF2 engineered SPIONs attenuate tumor stroma and potentiate the effect of chemotherapy..., Mardhian [/bib_ref] and systems which integrate both of such categories i.e. polymeric coated nanocarriers . Carbon based nanomaterials, as carbon nanotubes either alone or decorated with other materials , graphene oxide [bib_ref] Highly sensitive determination of non-steroidal anti-inflammatory drug nimesulide using electrochemically reduced graphene..., Govindasamy [/bib_ref] [bib_ref] A facile graphene oxide based sensor for electrochemical detection of prostate anti-cancer..., Karthik [/bib_ref] [bib_ref] One-pot biosynthesis of reduced graphene oxide/prussian blue microcubes composite and its sensitive..., Keerthi [/bib_ref] , fullerenes, carbon quantum dots [bib_ref] Graphene Quantum dots-mediated theranostic penetrative delivery of drug and photolytics in deep..., Sung [/bib_ref] , have been employed for the detection of drugs in biological samples or their imaging ability. Prussian blue cubes are octahedral metal hexacyanoferrates which have been developed as detection tools due to their conductive and magnetic properties . An ideal nanotheranostic should circulate for a long time in the body, present sufficient release behavior, great tissue target specificity and penetration, imaging probability and high target to background ratio [bib_ref] Polymeric nanotheranostics for real-time non-invasive optical imaging of breast cancer progression and..., Ferber [/bib_ref]. Various nanotheranostics have the ability to localize diagnostic and therapeutic agents in specific sites of diseases and reduce undesired side effects. Their extended circulation time in blood is related with their nanometric size. In case of nanotheranostics applying for tumor diagnosis and therapy, the nanosized particles can easily extravagate from the blood into tumor tissues and be retained as a result of poor lymphatic drainage when compared to multifunctional small molecules or functionalized macromolecules [bib_ref] Novel core-shell magnetic nanoparticles for Taxol encapsulation in biodegradable and biocompatible block..., Filippousi [/bib_ref] [bib_ref] Polyhedral iron oxide core-shell nanoparticles in a biodegradable polymeric matrix: preparation, characterization..., Filippousi [/bib_ref] [bib_ref] Imaging-assisted anticancer nanotherapy, Dasgupta [/bib_ref].
At present, nanomedicine has provoked novel and promising applications in diagnostics and invasive therapy of various diseases. However, the development of novel tools with improved imaging characteristics, which can lead to early detection of diseases, is still of high importance. In addition, imaging agents, which can efficiently detect cancer in early stages, are beneficial for medical society. Aside from this, the nanotherapeutics are also key components for treatment of serious diseases [bib_ref] Diverse applications of nanomedicine, Pelaz [/bib_ref]. Nonetheless, currently, most of the reported NPs are evaluated according to the results of animal models, and clinical studies are rarely performed. In the past, most of the applications based on nanotheranostics involved their use in cancer [bib_ref] In vivo therapeutic evaluation of polymeric nanomedicines: effect of different targeting peptides..., Zhao [/bib_ref] but in the present, nanotheranostics for diseases such as neurological disorders [bib_ref] Nanotheranostics, a future remedy of neurological disorders, Sharma [/bib_ref] , cardiovascular diseases (CVD) [bib_ref] Nano-antagonist alleviates inflammation and allows for MRI of atherosclerosis, Mog [/bib_ref] have been also arised. Preclinically, theranostics have been gradually applied to CVD with encouraging findings [bib_ref] Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair, Paul [/bib_ref].
Nuclear medicine imaging has been widely examined as advanced diagnostic tool and includes the introduction of radionuclides into the body, detection of the emitted gamma rays and generation of images which provide detailed radionuclides distribution as well as physiological characterization of organs and tissues [bib_ref] Radiolabeling nanomaterials for multimodality imaging: new insights into nuclear medicine and cancer..., Ge [/bib_ref]. Various nanoimaging agents have been produced presenting dual behavior as both diagnostic and therapeutic tools. However, some of these nanomaterials present low distribution and cell penetration, and thus their undesired pharmacokinetics should be improved. Many novel strategies have been employed in order to improve their pharmacokinetics and biodistribution [bib_ref] Polymer-based cancer nanotheranostics: retrospectives of multi-functionalities and pharmacokinetics, Han [/bib_ref].
The past years, the applications of nanotheranostics or NP based theranostics have shown an enormous advancement. By summing up the current developments on nanotheranostics could assist medical professionals on providing further progress in this field. All the more, analyzing the current application of imaging modalities and nanotheranostics could also provoke an evolution on nanomedicine field.
## The application of imaging modalities in nanomedicine field
The last years, the integration of nanotools with life sciences led to the design of various diagnostic devices, contrast agents and drug delivery nanosystems. The multifunctional nanoscaled materials [fig_ref] Figure 2 -: A detailed schematic illustration of multifunctional coated nanotheranostics [/fig_ref] in most cases present promising biological activities, stability and improved biodistribution compared to larger monofunctional particles . Besides, it has been reported that there is a correlation between the sizes and the chemical composition of nanoparticles and their internalization on cellular or intracellular surfaces. In general, NPs with sizes under 200 nm can be attached into the cells [bib_ref] Insight into cellular uptake and intracellular trafficking of nanoparticles, Foroozandeh [/bib_ref]. Although these nanosystems could provide complementary information arisen from different imaging agents, they don't present equivalent properties. Thus, when a new nanotheranostic system is designed, the imaging part should be chosen according to the intended administration route. For example, the targeted delivery of imaging agents should be carefully examined in order to evaluate the possible effects of the specificity of targeting and potential toxic side effects caused by off-target accumulation .
There are many imaging systems used in radiopharmacy, nuclear medicine and radiology areas. The radiographs, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) are common imaging techniques in radiology. These methods allow the diagnosis of many diseases, mostly in cancer. Nonetheless, when the cancer cell becomes visible, it will contain about 1 billion cancer cells at about 1 cm 3 . Thus, the diagnosis of tumor area would be too late since the phenotypic changes begin. The detection of cancer earlier at the molecular level is very important for therapy. Because of this, the research has been focused on from radiological imaging to nuclear medicine imaging. Nuclear medicine imaging is described as the in vivo measurement of biological processes at the cellular and molecular levels, and the genetic alterations in the tissues. It also images the pathophysiologic situation noninvasively, and provides information regarding specific molecular changes, to perform early diagnosis, identify the stage of disease, fundamental information on pathological processes, and prognosis of disease and personalized medicine administration . Nanotheranostics offer multiple and desirable advantages over conventional drug products.
They can be easily prepared using standard nanotechnology procedures to provide designed functionalities and to achieve specific targeting; their surface can be conveniently modified with numerous ligands or moieties by linking, conjugation or coating .
Generally, NPs for imaging are classified in 4 major groups: magnetic [bib_ref] Co-precipitation synthesis of near-infrared iron oxide nanocrystals on magnetically targeted imaging and..., Syu [/bib_ref] , magnetofluorescent [bib_ref] Magnetofluorescent carbon dots derived from crab shell for targeted dual-modality bioimaging and..., Yao [/bib_ref] , Fluorescent [bib_ref] Thermo-responsive fluorescent nanoparticles for multimodal imaging and treatment of cancers, Pandey [/bib_ref] and radiolabeled [bib_ref] Uptake and subcellular distribution of radiolabeled polymersomes for radiotherapy, Roobol [/bib_ref]. They are being applied into various imaging modalities including optical, molecular, radiologic and nuclear medicine imaging [bib_ref] Strategies for target-specific contrast agents for magnetic resonance imaging, Vithanarachchi [/bib_ref] [bib_ref] Role of 18F-FDG PET/CT in the diagnosis of inflammatory and infectious vascular..., Chrapko [/bib_ref] [bib_ref] Multifunctional iron oxide nanoparticles for diagnostics, therapy and macromolecule delivery, Yen [/bib_ref]. Magnetic NPs are consisted of metals which have magnetic features. They can be prepared directly or by integrating into polymeric matrix. Recently, magnetic micro/nanoparticles have been widely used to facilitate location of cancer cells either with the help of hyperthermia or non. Due to their magnetic features, they are handful in the diagnosis of various diseases such as treatment of cancer and neurologic disorders. In various cases, drugs are attached to magnetic NPs creating a driving force for delivery. Additionally, magnetic NPs are used as contrast agents for cancer diagnosis, molecular imaging, hyperfusion region visualization, T cellbased radiotherapy, detection of angiogenesis, apoptosis and gene expressions . Fluorescent-magnetic NPs are multifunctional nanomedicals, composed of magnetic and fluorescent parts. The combination of these properties in single system plays a crucial role in theranostics field. They are beneficial for in vitro studies and in vivo imaging such as magnetic resonance imaging and fluorescence microscopy [bib_ref] Multifunctional Magnetic-fluorescent nanocomposites for biomedical applications, Corr [/bib_ref] [bib_ref] Graphene quantum Dot-MnO 2 nanosheet based optical sensing platform: a sensitive fluorescence..., Yan [/bib_ref] [bib_ref] Fluorescence turn-off detection of hydrogen peroxide and glucose directly using carbon nanodots..., Wei [/bib_ref] Radiopharmacy is a multidisciplinary science area and responsible for the development, formulation, preparation, quality control and administration of radiopharmaceuticals [bib_ref] Theranostics in nuclear medicine practice, Yordanova [/bib_ref]. Radiopharmaceuticals are drugs containing radionuclides and pharmaceutical parts, used for the diagnosis or treatment of many diseases [bib_ref] Theranostic radiopharmaceuticals: established agents in current use, Ballinger [/bib_ref]. The pharmaceutical part is chosen on the basis of its preferential localization in the organ which is desired to be imaged or participation its in the physiological function of the organ. After its selection, the pharmaceutical part is labeled with a suitable radionuclide and after its localization in the organ it can be detected with various imaging methods [bib_ref] Procedure guideline for the use of radiopharmaceuticals 4.0, Callahan [/bib_ref]. Various nanotheranostics based on polymeric NPs have been developed and radiolabeled with available radionuclides using various methods aiming the diagnosis and treatment of diseases [bib_ref] Nanoparticle probes for the detection of cancer biomarkers, cells, and tissues by..., Chinen [/bib_ref]. Linking molecules such as chelator agents are binding to radionuclides and NPs [fig_ref] Figure 3 -: The categories of radionuclides used as theranostics and diagnostic agents [/fig_ref] in order to improve their stability [bib_ref] Exploiting the metal-chelating properties of the drug cargo for in vivo positron..., Edmonds [/bib_ref]. The properties of short medium, and long half-lives of radionuclides, its radioactive decay and chemical structure could affect their potential application in clinical trials [fig_ref] Figure 3 -: The categories of radionuclides used as theranostics and diagnostic agents [/fig_ref]. Hence, the radiopharmaceutical should be carefully chosen. For example, radiopharmaceuticals which emit gamma rays and beta particles have theranostic features. They serve both of diagnostic and therapeutic facilities to determine the physiological or anatomic situation in tissues. The selection of radionuclide has important role for targeting nanotheranostic to desired area. If gamma, beta and alpha emitters are used to radiolabel NPs, then the NPs obtain the theranostic property [bib_ref] Application of nanoparticles in diagnostic imaging via ultrasonography, Mody [/bib_ref]. These emitters can also induce the visualization of the cellular function and the monitoring of the molecular process in the cells. Technetium-99 m is frequently applied as radionuclide to radiolabel nanosytems, presumably because of its wide availability, low cost, favorable imaging properties and a half-life (6 h) that allows imaging for up to 24 h [bib_ref] fac-99mTc/Re-tricarbonyl complexes with tridentate aminocarboxyphosphonate ligands: suitability of the phosphonate group in..., Lipowska [/bib_ref]. Indium-111 is the second most-used radionuclide, followed by radioisotopes of iodine [bib_ref] Indium-111 labeled gold nanoparticles for in vivo molecular targeting, Ng [/bib_ref]. More recently, positron-emitting radionuclides such as Flour-18, Manganez-52, Zirconium-89 [bib_ref] The influence of glycans-specific bioconjugation on the Fc γ RI binding and..., Vivier [/bib_ref] have been increasingly used, reflecting the growing interest in positron emission tomography (PET) imaging and the increasing availability of preclinical and clinical PET scanners in radiopharmacy and nuclear medicine applications [bib_ref] Nuclear imaging of liposomal drug delivery systems: a critical review of radiolabelling..., Man [/bib_ref]. Another important parameter for the selection of radiation types are the energy levels of the emitters. The βand γ -emitters are widely used, due to their long ranges and manageable energy levels. The α particle, or helium nucleus, is of very high mass and high energy, with very short range and thus it considered as unsuitable for radiation therapy due the extensive damage it causes to tissues [bib_ref] A proposed methodology to select radioisotopes for use in radionuclide therapy, Cuaron [/bib_ref].
Another significant factor for choosing the suitable radionuclide is the effective half-life, which is the net halflife considering both physical half-life and biological halflife within the patient's body or organs. It has been reported that an acceptable half-life is within 6 h and 7 d This is because a very limited physical half-life restricts the delivery . In further, a physical long-life reserves the radiation dose and patients should be isolated for longer time while surrounding people are being exposed in radiation doses for more time. If the biological half-life is too short, the radionuclide will be discharged with a significantly high activity, leading to extensive radioactive waste management. Another feature which should be considered is decay product. More specifically, an ideal radiopharmaceutical should be able to decay into a stable daughter product [bib_ref] Therapeutic radionuclides in nuclear medicine: current and future prospects, Yeong [/bib_ref]. While the radionuclides with short half-lives are preferred due to their diagnostic properties ( e.g., technetium-99 m, gallium-68 and iodine-123) and used as imaging agents, radionuclides with medium half-lives present therapeutic properties ( e.g., gallium-67, lutesium-177, yttrium-90 and iodine-131) and used in radiation therapy. The use of radionuclides containing very long half-lives in clinical research are common for the determination of absorption, distribution, metabolism and elimination parameters of new drug delivery systems .
## Nanoimaging applications for cancer
Cancer is the second leading cause of death worldwide [bib_ref] Cancer statistics, Siegel [/bib_ref] ; hence, early diagnosis and timely treatment of cancer are of great importance. The most common type of cancer is lung cancer followed by hepatic, colorectal, gastric and breast. In the last decade, various diagnostics and therapeutic tools for cancer have been emerged . Laboratory diagnosis of cancer is limited to later stages of the disease and specific malignancies [bib_ref] Cancers screening in an asymptomatic population by using multiple tumour markers, Wang [/bib_ref]. Consequently, early diagnostics markers should be encouraged. The classic approaches on cancer management involve the combination of surgery, radiotherapy, chemotherapy and hormonal therapy [bib_ref] Cancer diagnostics: the journey from histomorphology to molecular profiling, Ahmed [/bib_ref]. The available chemotherapy pathways as intravenous, oral [bib_ref] Biocompatible Zr-based nanoscale MOFs coated with modified poly( ε-caprolactone) as anticancer drug..., Filippousi [/bib_ref] and topical [bib_ref] Local drug delivery strategies for cancer treatment: gels, nanoparticles, polymeric films, rods,..., Wolinsky [/bib_ref]. The current cancer theranostics include the detection of novel biomarkers as advanced molecular diagnostics, new molecular imaging probes and techniques for early identification of cancer, molecular imaging guided cancer therapy and nanoplatforms incorporating both cancer imaging and therapeutic substances. The imaging techniques such as PET and single-photon emission computed tomography (SPECT) and fluorescence reflectance imaging (FRI) are widely used. Especially, PET and SPECT systems are nuclear medine imaging techniques and various radiolabeled nano systems have been administered through these methods. For these methodologies, the important fact is all the targeted molecules or the cells to become visible. Genetic agents such as photoproteins, PET [bib_ref] Bacteria-like mesoporous silica-coated gold nanorods for positron emission tomography and photoacoustic imaging-guided..., Xu [/bib_ref] and magnetic resonance detectable reporter genes or radiolabeled particles [bib_ref] Positron emission tomography imaging using radiolabeled inorganic nanomaterials, Sun [/bib_ref] , fluorochrome [bib_ref] Cancer optical imaging using fluorescent nanoparticles, Coll [/bib_ref] , magnetic molecules as labeled antibodies, small molecules or biorthogonal agents [bib_ref] Mesoporous silica nanoparticle pretargeting for pet imaging based on a rapid bioorthogonal..., Lee [/bib_ref] are widely used.
The nanotheranostics for cancer can be categorized as conventional and biomimetics [bib_ref] Recent advances of biomimetic nano-systems in the diagnosis and treatment of tumor, Li [/bib_ref]. Conventional nanocarriers, such as liposomes, micelles, nanogels, and NPs present great potentional as anticancer strategies. However, their use as imaging or diagnostics is limited since they should be efficiently conjugated with fluorescent dyes or other active molecules before their application [bib_ref] Recent advances of biomimetic nano-systems in the diagnosis and treatment of tumor, Li [/bib_ref].
In addition, such systems should be also functionalized with agents such as poly(ethylene glycol) (PEG) which can increase the circulation time . Biomimetics nanosystems as nanotheranostics have gained researchers' attention since they can combine biological activities of biomimetic compounds such as proteins, phospholipids, cholesterol, cell and cell membranes, pathogens as bacteria and viruses and other pathogens, apatite and exosomes [bib_ref] Recent advances of biomimetic nano-systems in the diagnosis and treatment of tumor, Li [/bib_ref]. The current cancer nanotherapeutics rely on the improved permeability and retention (EPR) effect according to which NPs tend to accumulate in tumor tissue much more than they do in normal tissues. However, such a phenomenon is dependent on the tumor microenvironment and is not consistently observed in all tumor types, thereby limiting drug transport to the tumor site. In addition, it has been shown that EPR effect is not translated in the majority of humans cancers. Thus, cancer nanotheranostics should be studied in more clinically relevant cancer models should be used or in tumors which present a strong EPR effect in the clinic, such as Kaposi sarcoma, head and neck tumors .
Furthermore, cancer nanotheranostics are associated with stimuli such as light, temperature, magnetism, and sound. Photodynamic, photothermal, or phototriggered therapeutic systems are related with light or photo stimuli. The photodynamic therapy (PDT) [bib_ref] H2O2-responsive biodegradable nanomedicine for cancer-selective dual-modal imaging guided precise photodynamic therapy, Zeng [/bib_ref] involves the administration of a tumor localizing photosensitizing agent, which may require metabolic synthesis ( i.e., a prodrug), followed by activation of the agent by light of a specific wavelength. This therapy results in a sequence of photochemical and photobiologic processes that cause irreversible damage to tumor tissues. However, conventional PDT is not yet widely used for clinical cancer treatment [bib_ref] An Assembled nanocomplex for improving both therapeutic efficiency and treatment depth in..., Cao [/bib_ref]. Photothermal therapy (PTT) includes cancerous tissue irradiation with electromagnetic radiation, which induces thermal tissue damage. Finally, in phototriggered drug release, the active molecule modified with light responsive agent and the drug release in proper amount and time [bib_ref] Applications of light-responsive systems for cancer theranostics, Chen [/bib_ref]. For example, recently a light triggered system for tumor diagnosis, combined photodynamic and hypoxia-activated prodrug therapy based on liposomes gene probe, hypoxiaactivated prodrug Tirapazamine and photosensitizer Chlorin e6 was prepared [bib_ref] Light-triggered theranostic liposomes for tumor diagnosis and combined photodynamic and hypoxia-activated prodrug..., Zhang [/bib_ref]. Optical imaging theranostics involve phosphorescence, bioluminescence, fluorescence Raman, and photoacoustic imaging [bib_ref] Bacteria-like mesoporous silica-coated gold nanorods for positron emission tomography and photoacoustic imaging-guided..., Xu [/bib_ref]. Fluorescence imaging [bib_ref] Fluorescent silica nanoparticles for cancer imaging, Santra [/bib_ref] is a form of luminescence that results from the emittion of light of a certain wavelength after absorbing electromagnetic radiation [bib_ref] Cancer optical imaging using fluorescent nanoparticles, Coll [/bib_ref]. Through fluorescence, images of fluorescent dyes and fluorescent proteins to mark molecular mechanisms and structures can be obtained [bib_ref] Fluorescence-guided surgery, Nagaya [/bib_ref]. A nanoplatform, comprised from silicon naphthalocyanine encapsulated in PEG-b-poly( ɛ -caprolactone) NPs, was developed for fluorescence image-guided surgery. The animal studies exhibited that the activatable NPs can accumulate at the tumor site following systemic administration, releasing the silicon naphthalocyanine. It was revealed that the combinatorial phototherapy mediated by the NPs could efficiently eradicate chemoresistant ovarian cancer tumors [bib_ref] A tumor-activatable theranostic nanomedicine platform for NIR fluorescence-guided surgery and combinatorial phototherapy, Li [/bib_ref].
Magnetic nanotheranostics composed of magnetic substances (magnetite, iron oxide etc.) which after providing an external magnetic field can concentrate and retain the nanocarriers in specific cancerous area [bib_ref] Magnetic nanoparticles in cancer theranostics, Gobbo [/bib_ref]. They are used for imaging (serving as contrast agents for MRI), therapy (combined hyperthermia-chemotherapy) as well as cell separation (cell labeling/tracking and isolation using magnetic force). Another example of magnetic nanotheranostics is their ability to be heated under a high frequency magnetic field which can induce cancer cell death . The most common used magnetic diagnostic tool is MRI. It is a noninvasive tool, which produces images with high spatial and temporal resolution and used in radiology to form pictures of the anatomy and the physiological processes of the body. MRI scanners use strong magnetic fields, magnetic field gradients, and radio waves to generate images of the organs in the body. The majority of MRI scans don't need contrast agents, however, over 35% of clinical MR scans use contrast agents to improve their sensitivity and diagnostic accuracy. For instance, Prussian blue nanoprobes were designed as nanoplatforms for tumor imaging via PTT and MRI, due to their low toxicity and excellent in vivo performance [bib_ref] Zn2 + doped ultrasmall prussian blue nanotheranostic agent for breast cancer photothermal..., Shou [/bib_ref]. Another example is the development of hollow manganese/cobalt oxide NPs as cancer nanotheranostics. The developed nanoplatforms can act as glutathione-responsive contrast agents for dual T 1 /T 2 -weighted MRI [bib_ref] Biodegradable hollow manganese/cobalt oxide nanoparticles for tumor theranostics, Ren [/bib_ref].
Ultrasound nanotheranostics are another significant category used for cancer diagnosis. Ultrasound contrast agents are mostly gas-filled microbubbles with echogenicity high degree [bib_ref] Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering, Qin [/bib_ref]. Ultrasound as tumor diagnostic tools is present various advantages as real-time, portable, nonionizing and deep tissue-penetrating capability [bib_ref] Ultrasound nanotheranostics in fighting cancer: advances and prospects, Zhou [/bib_ref]. Besides, it can be also be applied in cancer therapy as high intensityfocused ultrasound [bib_ref] High-intensity focused ultrasound in the treatment of breast tumours, Peek [/bib_ref] and sonodynamic therapy [bib_ref] Recent advances of sonodynamic therapy in cancer treatment, Wan [/bib_ref]. Contrast-enhanced ultrasound could improve ultrasound backscatter, or reflection of the ultrasound waves which can produce unique sonogram with increased contrast due to the high echogenicity difference. More specifically, ultrasound can be used for imaging blood perfusion, blood flow as well as receptor density in tumors [bib_ref] High-intensity focused ultrasound: advances in technology and experimental trials support enhanced utility..., Malietzis [/bib_ref]. In summary, ultrasound molecular imaging includes photoacoustic imaging, phasechangeable imaging, multi-modality ultrasound imaging, TME-responsive ultrasound imaging, acoustic reporter genes imaging whereas ultrasound targeting therapy: sonodynamic therapy, high intensity-focused US ablation [bib_ref] Ultrasound nanotheranostics in fighting cancer: advances and prospects, Zhou [/bib_ref]. Inorganic NPs as contrast agents for ultrasound theranostics, include perfluorocarbon NPs, noble metal materials (such as Au, Ag, Pd, and Pt) carbon-based nanomaterials (such as carbon nanotubes, carbon dots, graphenes, and graphene oxides), silica-based nanomaterials . Small molecule organic dyes with high photothermal conversion efficiency, such as indocyanine green (ICG), IR780, IR825, can absorb near infra-red (NIR) light and convert into heat energy. Most of the above diagnostic modalities can be applied for the diagnosis and treatment of other diseases.
## Nanoimaging applications for pulmonary diseases
Respiratory diseases include a wide spectrum of illnesses associated with upper and down respiratory system. Nanomedicine has shown benefit for various chronic obstructive pulmonary diseases such as asthma [bib_ref] Targeted anti-inflammatory therapeutics in asthma and chronic obstructive lung disease, Durham [/bib_ref] or genetic diseases as cystic fibrosis. In addition, pulmonary tuberculosis and lung cancer can be detected and treated by using nanoplatforms. It has been reported that lung can be easily targeted especially by using inhaled aerosols given that these nanosystems can be easily transferred to the airways [bib_ref] Inhaler usability of a pressurized metered dose inhaler and a soft mist..., Ding [/bib_ref]. Imaging nanoplatforms for pulmonary diseases detection are difficult to be found. However, various systems have been developed [bib_ref] Nanomedicine for respiratory diseases, Bahadori [/bib_ref]. For example, Aillon et al. revealed that the iodined nanoclusters could also be an alternative option as contrast agents for lung visualization [bib_ref] Iodinated nanoclusters as an inhaled computed tomography contrast agent for lung visualization, Aillon [/bib_ref]. It can be said that PET and MRI are the most frequently used techniques for NPs lung imaging [bib_ref] How to detect a dwarf: in vivo imaging of nanoparticles in the..., Roller [/bib_ref].
Various are the nanodelivery systems used for treatment of pulmonary diseases. Nonetheless, combined imaging and therapeutic nanosystems are quite limited [bib_ref] Advanced therapeutic strategies for chronic lung disease using nanoparticle-based drug delivery, Yhee [/bib_ref]. Lanza et al. prepared lipase labile phospholipid prodrug forms of fumagillin or docetaxel loading to lipid-based micelles for asthma management. The anti-angiogenic efficacy in asthma of αv β3-targeted micelles -αv β3 are expressed on lung endothelial cells-was studied via MR simultaneous dual 19F/1H neovascular molecular imaging [bib_ref] Anti-angiogenic nanotherapy inhibits airway remodeling and hyper-responsiveness of dust mite triggered asthma..., Lanza [/bib_ref]. Superparamagnetic iron oxide NPs were conjugated with a biocompatible antibody in order to examine their in vivo effect after pulmonary administration. The developed system enable specific targeting and imaging of a particular macrophage subpopulation in lipopolysaccharideinduced chronic obstructive pulmonary disease mice model [bib_ref] MR imaging and targeting of a specific alveolar macrophage subpopulation in LPS-induced..., Faraj [/bib_ref].
## Nanoimaging applications for neurological disorders
Neurodegenerative diseases as Alzheimer's (AD), Parkinson's (PD) [bib_ref] Modified chitosan coated mesoporous strontium hydroxyapatite nanorods as drug carriers, Filippousi [/bib_ref] , Huntigton's (HD) [bib_ref] Selenium nanoparticles as an efficient nanomedicine for the therapy of huntington's disease, Cong [/bib_ref] or in general neurological disorders affects millions of people worldwide. Their therapy has not been achieved yet due to the insufficient active molecules which have limited therapeutic activity, their low penetration on the targeted tissue due to blood brain barrier (BBB) and the fact that their pathophysiological mechanisms are poorly understand [bib_ref] Old drugs as new treatments for neurodegenerative diseases, Durães [/bib_ref]. Thus, more and more researchers are focused on designing promising drug delivery systems or novel drug substances able to cross BBB and reach to brain . In addition, detection of these disorders mainly focused on PET, SPECT, MRI and X-ray CT [bib_ref] Nanotheranostics, a future remedy of neurological disorders, Sharma [/bib_ref]. PET owing to its great sensitivity and better resolution provides enhanced imaging ability in comparison with SPECT. MRI and CT as non-invasive imaging techniques can depict morphological alterations in brain diseased tissues. It can be said that in order to achieve better penetration of drugs in brain, the nanocarriers surface can be modified with various biologically active substances which can exclusively couple expressed receptors on the brain endothelial cell . For example, an innovative nanosystem aiming to be used as AD nanotheranostic was prepared from ceria and iron oxide nanocrystals surface coated onto the mesoporous silica NPs (MSNs) which were functionalized with amino-T807 (PET tau tracer), and methylene blue [bib_ref] Tau-targeted multifunctional nanocomposite for combinational therapy of Alzheimer's disease, Chen [/bib_ref]. Birgitte et al. also developed manganese oxide NPs functionalized with l -DOPA able to release Mn 2 + ions and l -DOPA concurrently in water due to NPs degradation. The specific nanosystem was served as MRI agent for imaging, diagnosis and therapy tool in PD [bib_ref] L-DOPA-coated manganese oxide nanoparticles as dual MRI contrast agents and drug-delivery vehicles, Mcdonagh [/bib_ref].
## Nanoimaging applications for cardiovascular disorders
CVD such as heart failure, coronary cardiac disease, myocardial infarction and inflammatory heart disease, hypertension, dyslipidemia, diabetes are the most frequent disorders.The most common problem for medications prescribed in these disorders is their low lipophilicity and thus their bioavailability . Nanotechnology based systems are among the most useful for CVD since they present control drug delivery ability for a variety of active molecules which can be directed for the management of lipid disorders, inflammation [bib_ref] Anti-inflammatory nanomedicine for cardiovascular disease, Katsuki [/bib_ref] and angiogenesis within atherosclerotic plaques, and prevention of thrombosis. Herein, various imaging modalities can be applied for detection of disorder. For example, [bib_ref] Zn2 + doped ultrasmall prussian blue nanotheranostic agent for breast cancer photothermal..., Shou [/bib_ref] Zr-based PET imaging was employed to detect macrophages in atherosclerotic plaques. Macrophages in atherosclerotic lesions actively participate in lipoprotein ingestion and accumulation rising to foam cells filled with lipid droplets. Accumulation of foam cells contributes to lipid storage and atherosclerotic plaque growth. In this study, dextran NPs were functionalized with desferoxamine to enable hybrid PET-MR imaging and it was shown prominent localization in macrophages in plaques in the aortic root of atherogenic ApoE −/ − mice. Dextran is a biocompatible and biodegradable molecule, and thus it can be applied in bioimaging applications [bib_ref] Polymeric nanoparticle PET/MR imaging allows macrophage detection in atherosclerotic plaques, Majmudar [/bib_ref]. Another group developed dextran-coated iron oxide based magneto-fluorescent nanoagent for near-infrared fluorescent imaging and generation of singlet oxygen. The application of nanotheranostics in an ApoE −/ − mouse animal model showed desirable accumulation inside the atherosclerotic lesions and caused photo-induced apoptosis of phagocytic macrophages [bib_ref] A Light-activated theranostic nanoagent for targeted macrophage ablation in inflammatory atherosclerosis, Mccarthy [/bib_ref]. Gonçalves et al. developed PEGylated gold nanoparticles with 5-Aminolevulinic acid which were found to be accumulated into atheromatous plaques in atherosclerotic rabbits. It was further revealed that the acid was transformed to the active photosensitizer porphyrin IX. Thus, the nanoplatforms might be helpful in early diagnosis of atherosclerosis [bib_ref] Aminolevulinic acid with gold nanoparticles: a novel theranostic agent for atherosclerosis, De Oliveira Gonçalves [/bib_ref].
To summarize, nanotechnology is already playing and will play major role in developing novel and promising imaging probes and therapeutic strategies for various diseases.
## 3.
Nanotheranostics categories according to their chemical nature At present, an urgent need for early detection and diagnosis of diseases is needed. The main challenges for the development of efficient nanotheranostics are the use of non toxic contrast agents which can be circulate in body for more time in order to provide fast and detailed imaging of the lesions [bib_ref] Applications of nanoparticles in biomedical imaging, Han [/bib_ref]. In this review, the nanobased theranostics are categorized according to their chemical nature as organic and inorganic. The most frequently found organic nanotheranostics are lipid, polymers, micelles or carbon materials whereas the most common inorganic materials are calcium phosphate, iron oxide, and metal-and silica-based NPs.
The nanotheranostics could be carefully modified in order to present small sizes which will prevent their rapid clearance from blood stream mononuclear by phagocyte system (MPS) and reticuloendothelial system (RES) . Except their tiny sizes, the particles surface could be also modified with aptamers, antibodies etc. to avoid innate immunosystem recognition and to secure sufficiently long circulation to reach their targets . Thus, inorganic and organic molecules could be modified or entrapped in polymer NPs cores, liposomes or dendrimers in order to be used sufficiently [bib_ref] Surface modified multifunctional and stimuli responsive nanoparticles for drug targeting: current status..., Siafaka [/bib_ref].
The clearance and toxicity of the used nanomaterials are issues, which should be carefully examined. According to numerous studies, it is believed that the toxicity and the clearance of nanomaterials are correlated with their physicochemical characteristics. For instance, injected NPs are vigorously cleared from bloodstream as a result of their sequestration by cells of the mononuclear phagocyte system preventing them reaching their target sites. Macrophages in particular are believed to be among the first and primary cell types that process NPs, mediating host inflammatory and immunological biological responses [bib_ref] Nanoparticle uptake: the phagocyte problem, Gustafson [/bib_ref]. Several studies reported that the clearance of NPs is size and dose dependent. A study reports that iron oxide NPs presenting large sizes (over 100 nm) are rapidly trapped in the liver and spleen through macrophage phagocytosis. On the other hand, iron oxide NPs with sizes under than 10 nm are possibly eliminated through renal clearance [bib_ref] Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells, Kumar [/bib_ref]. Tsoi et al. showed that hard NPs as quantum dots, gold and silica NPs cleared primarily in liver by Kupffer cells, endothelial cells, B cells and other cells [bib_ref] Mechanism of hard-nanomaterial clearance by the liver, Tsoi [/bib_ref]. Other studies associated the shape of NPs and surface charge with their in vivo fate [bib_ref] Surface charge controls the suborgan biodistributions of gold nanoparticles, Elci [/bib_ref]. On the other hand, soft NPs resist macrophage uptake indicating higher blood circulation . PEG-like and other hydrophilic polymers could reduce opsonin adsorption on NPs inducing them as unrecognized by the elimination mechanisms from the human body [bib_ref] Long-circulating heparin-functionalized magnetic nanoparticles for potential application as a protein drug delivery..., Zhang [/bib_ref].
Toxicity is also a concern of NPs intended to be used in biomedical applications. Similarly to clearance, the toxicity of NPs is correlated with the size [bib_ref] Ultrasmall gold nanoparticles as carriers for nucleus-based Gene therapy due to size-dependent..., Huo [/bib_ref] , shape [bib_ref] Shape and surface effects on the cytotoxicity of nanoparticles: gold nanospheres versus..., Favi [/bib_ref] , the dose [bib_ref] Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes..., Feng [/bib_ref] and the surface coated . For example, Feng et al. demonstrated that PEGylated iron oxide NPs (IONs) in BALB/c mice did not show any toxicity signs whereas poly(ethylene imine) (PEI) coated IONs exhibited dose-dependent lethal toxicity [bib_ref] Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes..., Feng [/bib_ref]. A research group showed that gold NPs with sizes lower than 6 nm effectively enter the cell nucleus, whereas larger NPs (10 or 16 nm) only penetrate through the cell membrane and are found only in the cytoplasm. This fact relates that small NPs, which enter the nucleus can present higher toxicity [bib_ref] Ultrasmall gold nanoparticles as carriers for nucleus-based Gene therapy due to size-dependent..., Huo [/bib_ref]. Similarly, the cytotoxicity of metallic is strictly reliant on the pathway of cellular internalization [bib_ref] Transport across the cell-membrane dictates nanoparticle fate and toxicity: a new paradigm..., Guarnieri [/bib_ref]. Furthermore, the toxicity of NPs can be associated with their chemical composition. Since many NPs can be degraded, a leakage of metal ions from the NP core can occur. Some metal ions, such as Ag and Cd, are in fact toxic causing cell damage. Others as Fe and Zn, are biologically useful which however in high concentrations could damage cellular pathways causing high toxicity [bib_ref] Intra)cellular stability of inorganic nanoparticles: effects on cytotoxicity, particle functionality, and biomedical..., Soenen [/bib_ref]. In addition, it has been claimed that the toxicity of hard NPs can be decreased [bib_ref] Intra)cellular stability of inorganic nanoparticles: effects on cytotoxicity, particle functionality, and biomedical..., Soenen [/bib_ref] via their incorporation on biodegradable polymeric capsules-particles , coating with biocompatible polymers and materials or other strategies.
## Inorganic-metal and carbon based nanoparticles
Until now, the use of inorganic NPs as drug carriers or diagnostic tools offered great results [fig_ref] Figure 4 -: Various examples of inorganic nanoparticles used as nanotheranostics [/fig_ref]. It thought that inorganic NPs were more stable than organic NPs; nonetheless, some evidences show that inorganic NPs may degrade individually in vivo , contributting in toxicity effects [bib_ref] In vivo degeneration and the fate of inorganic nanoparticles, Feliu [/bib_ref]. Hence, in many cases such inorganic NPs are coated with other biocompatible molecules . MSN present solid framework of 50-300 nm particle diameter, interior porosity (pore size 2-6 nm), large pore volume, high surface area and ordered pore networks. They are relatively nontoxic with large loading capacity, great cytocompatibility and they can be easily modified . Chen et al. prepared supramolecular photosensitizers based on MSNs as part of PDT. Tirapazamine-MSNs were further chelated with paramagnetic Gd 3 + (Gadolinium). It was revealed that the nanosystem was able to be uptaken by CD44 receptor overexpressed in tumor cells whereas tumor growth was inhibited due to the combination of PDT and bioreductive chemotherapy under NIR fluorescence/MR imaging guidance. Bioreductive chemotherapy relies on the reductive activation of drugs by enzymes such as quinone oxidoreductase and P450 reductase as well as the identification of tumors abundant in such enzymes and differentiation in oxygen and pH levels within normal and tumor tissues. Subsequently, the developed system provides a new approach on nanotheranostics [bib_ref] Mesoporous silica-based versatile theranostic nanoplatform constructed by layer-by-layer assembly for excellent photodynamic/chemo..., Chen [/bib_ref]. Another example using MSNs and quantum dots was published from Muhammad et al. Camptothecin, was loaded in quantum dots conjugated with MSNs. In further, fluorescent doxorubicin was coupled to quantum dots surface which also led to fluorescent quantum dots. The novel nanosystem showed great in vitro anticancer activity while confocal microscopy revealed the efficient imaging and release of both active molecules [bib_ref] Responsive delivery of drug cocktail via mesoporous silica nanolamps, Muhammad [/bib_ref]. A very interesting study which involved functional nanocarriers of gold cluster bovine serum albumin nanogates which were engineered on MSNs, was prepared by Croissant et al. Authors loaded two anticancer agents: gemcitabine which was entrapped on positively-charged ammonium-functionalized MSN and doxorubicin which was conjugated in the negatively-charged of gold cluster bovine serum albumin nanogates and further electrostaticallyattached onto ammonium-functionalized MSN. The dual delivery system was applied for targeted red nuclear staining and in vivo tumor imaging revealing its capability to act as multifunctional cancer nanotheranostic [bib_ref] Protein-gold clusters-capped mesoporous silica nanoparticles for high drug loading, autonomous gemcitabine/doxorubicin co-delivery,..., Croissant [/bib_ref]. Calcium phosphate NPs are based on nanocrystalline hydroxyapatite [bib_ref] Modified chitosan coated mesoporous strontium hydroxyapatite nanorods as drug carriers, Filippousi [/bib_ref] , but there is a growing interest in amorphous calcium phosphate. Indeed, calcium phosphate has been investigated for its optical ability [bib_ref] Crystallization of citrate-stabilized amorphous calcium phosphate to nanocrystalline apatite: a surface-mediated transformation, Chatzipanagis [/bib_ref]. Last years, metal NPs have been reported of the most promising candidates on biomedical field. Metal NPs can both serve as diagnostic and drug delivery tools because of their intriguing abilities such as small size, high reactive surface area, targetability to cells, functionalization capability etc. Metal or metaloid NPs as gold , silica [bib_ref] Porous silicon in drug delivery devices and materials, Anglin [/bib_ref] and silicon oxide , silver [bib_ref] Cytotoxicity of phloroglucinol engineered silver (Ag) nanoparticles against MCF-7 breast cancer cell..., Kumar [/bib_ref] , titanium oxide [bib_ref] Recent advances in nanotheranostics for triple negative breast cancer treatment, Thakur [/bib_ref] and iron oxide [bib_ref] Superparamagnetic iron oxide nanoparticulate system: synthesis, targeting, drug delivery and therapy in..., Palanisamy [/bib_ref] are commonly used in nanotheranostics.
Magnetic NPs are based on metals (Iron-Fe, Cobalt-Co, Nickel-Ni), metal oxides (FeO, Fe 2 O 3 , Fe 3 O 4 [bib_ref] Construction of multifunctional Fe 3 O 4 -MTX@HBc nanoparticles for MR imaging..., Zhang [/bib_ref] , alloys (FePt, FePd) and ferrites (CoFe 2 O 4 , CuFe 2 O 4 ). Superparamagnetic iron oxide NPs (SPIONs) are used as contrast imaging agent in magnetic particle imaging (MPI), due to their small size, good biocompatibility and the ability of surface engineering. MPI is a novel imaging modality that accomplishes a direct measurement of the magnetization of ferromagnetic NPs to calculate their local concentration. Also, they can act as therapeutic agents in hyperthermia. There are many types of MNPs products that have been developed for biomedical applications: Resovist R (Fujifilm RI Pharma), EndoremTM (Guerbet S. A.), Feraheme R (AMAG Pharmaceuticals), Nanotherm R (MagForce Nanotechnologies) etc. Although some of are already commercially available on the market for clinical use, unfortunately, the majority of them is still under development.
Spherical gold NPs are of the most frequent gold nanoplatforms in drug delivery. Gold nanorods are used in photothermal and NIR application. Gold nanoantennas loaded with Cetuximab were characterized as an efficacious nanoprobe for in vivo tumor identification via Raman spectroscopy. Raman spectroscopy relies on the modification of frequency of light when it is inelastically scattered by molecules or atoms resulting in fingerprint information on molecular structure or environment. The nanosystem showed capability of targeting specified cancer biomarkers such as epidermal growth factor receptors. The developed nanosystem exhibits great raman signal in cancerous cells or mice tumors. According to the authors, the nanoantennas easily bind to epidermal growth factor receptors, blocking the epidermal growth factor protein from reaching the cancer cells and inhibit the signaling cascade, consequently stop proliferation and survival of targeted cells [bib_ref] Antibody-drug gold nanoantennas with Raman spectroscopic fingerprints for in vivo tumour theranostics, Conde [/bib_ref].
Carbon based nanomaterials such as fullerenes, carbon NPs, carbon nanotubes, graphene, and nano-diamonds have shown a tremendous development as biomedical applications especially in nanotheranostics [bib_ref] Nano-carbons as theranostics, Liu [/bib_ref]. Carbon nanotubes can be either single-walled (SWCNTs) or multiwalled (MWCNTs), and they are highly ordered, pseudo one-dimensional carbon allotropes. SWCNTs composed of rolled-up single layer of graphite tube of 0.3-2 nm diameter, while MWCNTs are multiple concentric cylindrical shells of graphite sheets. Furthermore, CNTs can penetrate into various cells and deliver drugs or other compounds to specific target tissue . Fullerenes have been explored as tumor theranostics since they exhibit potential in cancer therapeutic approaches such as PDT, PTT, radiotherapy and chemotherapy, but they have also been used as novel contrast agents in MRI [bib_ref] Applications of functionalized fullerenes in tumor theranostics, Chen [/bib_ref]. Huang et al. prepared fluorescent fullerene (C60) grafted with carboxyl groups. The developed NPs showed great water dispersibility, biocompatibility as well as fluorescence activity. Furthermore, researchers loaded cisplatin drug which released in controlled manner. The novel nanoplatform due to its properties of bioimaging and controlled drug delivery can act as promising nanotheranostic. However, in vivo studies should be further carried out in order to confirm the results [bib_ref] Facile fabrication of carboxyl groups modified fluorescent C 60 through a one-step..., Huang [/bib_ref]. Nanodiamonds are carbon NPs with a truncated octahedral architecture with 2 to 8 nm diameter. They demonstrate chemical stability, and extremely high hardness, stiffness and strength as well as small size, large surface area, and high adsorption capacity [bib_ref] The biocompatibility of nanodiamonds and their application in drug delivery systems, Zhu [/bib_ref]. Su et al. developed fluorescent nanodiamonds in order to quantitatively track the human placenta choriodecidual membrane-derived mesenchymal stem cells in miniature pigs through magnetic modulation [bib_ref] Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature..., Su [/bib_ref]. Graphene and graphene oxide (GO) are nanosheets of two-dimensional single monoatomic layers of sp 2 hybridized carbon atoms. They are an improtant category of theranostics due to their easy synthesis and their interesting structure which can be easily conjugated with active molecules or polymers [bib_ref] Graphene-based nanomaterials for drug delivery and tissue engineering, Goenka [/bib_ref]. Nonetheless, their in vivo tracking is quite a challenge. Recently, a dual-element labeling method using lanthanum and cerium tagged on poly(vinylpyrrolidone) (PVP) modified GO, developed from Liang et al. The authors used the laser ablation inductively coupled plasma mass spectrometry combined with Trasmission Electron Microscope observation. It was revealed that the modified GO nanosheets, after intravenous injection, entered into lung, liver, spleen and the kidney, whereas they were slowly eliminated from the accumulated organs and some excreted via urine. A previous study also exhibited that 125 I-labeled nanographene sheets after intravenous administration mainly accumulate in the reticuloendothelial system including liver and spleen and can be gradually cleared by both renal and fecal excretion [bib_ref] In vivo pharmacokinetics, long-term biodistribution, and toxicology of PEGylated graphene in mice, Yang [/bib_ref].
## Polymer, liposomes and dendrimers as nanotheranostics
Various polymeric, liposomal and dendritic formulations have been applied as nanotheranostics . As it was mentioned previously, they can coat the inorganic particles for improving their properties, avoiding their clearance and reducing their toxicity. Nonetheless, they can be also used as primary materials decorating with fluorescent molecules and drugs .
## Polymeric nanoparticles
Polymers are among the most easy-handled and economical carriers due to their important characteristics such as biocompatibility, biodegradability and stability against degradation [bib_ref] Surface modified multifunctional and stimuli responsive nanoparticles for drug targeting: current status..., Siafaka [/bib_ref]. Both synthetic and natural macromolecules have been utilized as nanotheranostics. However, in most cases they should be firstly modified in order to possess imaging ability and therapeutic activity [bib_ref] Polymer-based cancer nanotheranostics: retrospectives of multi-functionalities and pharmacokinetics, Han [/bib_ref]. Chitosan is a fundamental linear polysaccharide with numerous properties as low toxicity, cost-effectiveness, antimicrobial activity, antioxidant properties, etc. . In nanotheranostics, chitosan can be used as coating agent or polymeric core matrix. A novel multifunctional theranostic nanoplatform was fabricated via in situ growth of ultrasmall silver(I) selenide (Ag 2 Se) nanodots on the surface of chitosan coated-sodium yttrium tetrafluoride: ytterbium/erbium@sodium lutetium tetrafluoride: neodymium/ytterbium@ sodium lutetium tetrafluoride (NaYF 4 :Yb/Er@NaLuF 4 :Nd/Yb@NaLuF 4 ) upconversion NPs. The theranostic agents were able of tetra-modal imagingguided photothermal therapy of cancer for rapid and accurate delineation and elimination of tumors. The prepared nanosystems showed excellent luminescent properties, high X-ray attenuation coefficient and strong NIR absorbance. Sachdev et al. developed chitosan-based hydrogel loaded with highly fluorescent carbon dots and anticancer drug, 5-fluorouracil. It was found out that the system was able to show cellular uptake as well as therapeutic effects. In addition, in vitro studies exhibited apoptosis in A549 cells. To sum up, green fluorescence of carbon dots could be used to detect apoptosis instigated by 5-fluorouracil, eliminating the need for multiplex dyes [bib_ref] Carbon dots incorporated polymeric hydrogels as multifunctional platform for imaging and induction..., Sachdev [/bib_ref].
Chitosan due to its carboxyl and amino groups can be easily modified with various molecules showing remarkable properties. PEG and palmitoylated PEG-grafted chitosan were applied as polymeric shell for magnetic nanoparticles (Fe 1-x Mn x Fe 2 O 4 ). Methotrexate was encapsulated into chitosan coated NPs as anticancer drug. The prepared pH sensitive system was able to target in vitro tumor tissues, but still in vivo studies should also be performed [bib_ref] Pegylated and amphiphilic Chitosan coated manganese ferrite nanoparticles for pH-sensitive delivery of..., Karimi [/bib_ref]. Another derivative of chitosan, thiol chitosan was used as coating agent of gold nanoshells loaded with paclitaxel and the anti-epidermal growth factor receptor antibody. The multifunctional agents were acted as fluorescence/photoacoustic dual-modal imaging-guided chemophotothermal synergistic therapy. The nanoplatforms exhibited great biocompatibility, biosafety, broad NIR absorbance, photostability, fast and laser irradiationcontrolled release as well as high targetability. It can be concluded that the prepared nanosystem can be used for tumor visualization and sufficient chemophotothermal combination cancer therapy under the guidance of photoacoustic imaging. [bib_ref] Anti-EGFR antibody conjugated thiol chitosan-layered gold nanoshells for dual-modal imaging-guided cancer combination..., Manivasagan [/bib_ref]. A plasma membraneactivatable polymeric core-shell nanosystem was prepared by conjugating the photosensitizer protoporphyrin IX and PEG with glycol chitosan. The prepared nanoagents showed accumulation in tumor cells and improved in vivo fluorescence at the tumor site [bib_ref] Plasma membrane activatable polymeric nanotheranostics with self-enhanced light-triggered photosensitizer cellular influx for..., Jia [/bib_ref]. Chitosan and folic acid-conjugated chitosan NPs loaded with SPIONs were prepared as nanomagnetic onco-diagnostic and targeted nanomagnetic onco-diagnostic systems. In addition, uptake studies and competitive inhibition study verified the folate receptor mediated endocytosis of targeted system by MCF-7 as a folate receptor-positive cell line. The prepared novel tumor-targeting nanotheranostic agent can be applied for simultaneous MRI imaging and treatment of folate receptorpositive cancers. Nonetheless, the nanosystem lacks of in vivo studies [bib_ref] Design, preparation, and in vitro characterization of a trimodally-targeted nanomagnetic onco-theranostic system..., Zarrin [/bib_ref]. Folate-conjugated N-palmitoyl-chitosan was formulated to novel nanobubbles combined with therapeutic ultrasound (US) to act as a safe and effective physical targeted cancer therapy. The modified nanobubbles were capable of killing cancer cells and inhibiting tumor growth. Considering their US irradiation, the innovative folate nanobubbles can be associated with promising outcomes on oncology diagnosis and therapy [bib_ref] Folate-conjugated nanobubbles selectively target and kill cancer cells via ultrasound-triggered intracellular explosion, Shen [/bib_ref]. A nanoplatform for therapy and imaging of non-small cell lung cancer was fabricated by Zhang et al. The nanotheranostic agent composed of ICG entrapped into MSNs coupled with ZnO quantum dots. The nanosystem further coated with erlotinib-modified chitosan. The nanosystem exhibited a pH/redox dual-responsive release of ICG for precise fluorescent imaging. In vivo studies demonstrated that the nanotheranostic can provoke anticancer effect and can be an alternative option for imaging and therapy of non-small cell lung cancer [bib_ref] Dual-responsive nanosystem for precise molecular subtyping and resistant reversal of EGFR targeted..., Zhang [/bib_ref]. Theranostic polyfunctional gold-iron oxide NPs (polyGIONs) surface coated β-cyclodextrin-chitosan and loaded with therapeutic miRNAs and the chemotherapy drug temozolomide were able to be accumulated and released in mice glioblastoma. The prepared nanosystem was administrated via intranasal route. The stable nanosystem exhibited in vivo optical fluorescence and MRI capability and thus can be applied as nanotheranostics [bib_ref] Intranasal delivery of targeted polyfunctional gold-iron oxide nanoparticles loaded with therapeutic microRNAs..., Sukumar [/bib_ref].
Hyaluronic acid (HA) or hyaluronan is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial and neural tissues. It possesses low toxicity and has been widely utilized in pharmaceutical technology. In the literature, pure HA based nanotheranostics are limited found. Nonetheless, derivatives of HA or its salts are being fabricated into nanoplatforms. A current research of Zhang et al. involved the use of gadolinium modified mesoporous silica (GD-MS) as MRI agent. Authors in order to further improve system targeting ability, they further grafted HA on GD-MS. The grafted system was further modified with iopamidol or doxorubicin so as to prepare both diagnostic and therapeutic nanomolecules against lymph cancer [bib_ref] A versatile theranostic nanoplatform based on mesoporous silica, Zhang [/bib_ref]. Zhu et al. produced bioresponsive and fluorescent HA-iodixanol nanogels aiming to be applied as targeted X-ray computed tomography imaging and chemotherapeutic agent. The anticancer drug paclitaxel was loaded into the nanogels so as to target MCF-7 human breast tumors. A high cell uptake of the nanogel and tumor inhibition was demonstrated. Moreover, an improved CT imaging was observed for MCF-7 breast tumors in nude mice while fluorescence showed that nanogels were distributed thoughout the whole tumor indicating deep tumor penetration [bib_ref] Bioresponsive and fluorescent hyaluronic acid-iodixanol nanogels for targeted X-ray computed tomography imaging..., Zhu [/bib_ref]. A very recent study of Yu et al. included the use of deoxycholic acid-HA-methotrexate as carriers of ICG and doxorubicin. The developed NPs demonstrated intracellular doxorubicin uptake on CD44/folate receptors. Authors believe that the nanotheranostic present superior abilities as imaging-guided chemo-photothermal combination therapy [bib_ref] Imaging-guided synergistic targeting-promoted photo-chemotherapy against cancers by methotrexate-conjugated hyaluronic acid nanoparticles, Yu [/bib_ref]. In another study, nanoprobes from copolymers based on oxidized sodium hyaluronate and aggregation-induced emissionactive dye were formulated in fluorescent NPs. It was revealed that the polymeric NPs are well dispersed in water with good biocompatibility as well as confocal imaging capability. Thus, they can be applied as alternative nanotheranostic system [bib_ref] Fabrication, self-assembly and biomedical applications of luminescent sodium hyaluronate with aggregation-induced emission..., Wei [/bib_ref].
Cellulose is natural linear polysaccharide which is widely applied in pharmaceutical applications. Cellulose can be easily modified with active group providing derivatives with various functionalities. Carboxymethylcellulose (CMC) is among the most utilized cellulose derivatives. Leonel et al. produced core-shell nanofluids comprised from magnetic iron oxide and cobalt-doped magnetic IONs which further functionalized with CMC. The nanoconjugates showed hyperthermia ability due to iron oxide and thus can be used as anticancer nanotheranostics generated heat by magnetic hyperthermia of MION nanoconjugates [bib_ref] Synthesis and characterization of iron oxide nanoparticles/carboxymethyl cellulose core-shell nanohybrids for killing..., Leonel [/bib_ref]. In a different study, ZnS fluorescent quantum dots were modified with CMC and formulated nanocolloidal system which was further conjugated with doxorubicin. Nanocolloids with average size of 3.6 nm revealed photoluminescence emission property and biocompatibility. Thus the developed system can act as fluorescent nanoprobes and drug nanocarriers with ability to inhibit [bib_ref] Dual-functional supramolecular nanohybrids of quantum dot/biopolymer/chemotherapeutic drug for bioimaging and killing brain..., Mansur [/bib_ref]. SPIONs were coated with sodium CMC and cross-linked with epichlorohydrin to improve the stability of coating. Rhodamine B was loaded as model molecule. Although the study was in preliminary stages, the characterization revealed that the nanosystem can be applied in drug magnetic targeting applications [bib_ref] Rhodamine-loaded, cross-linked, carboxymethyl cellulose sodium-coated super-paramagnetic iron oxide nanoparticles: development and in..., Mallick [/bib_ref].
Other natural polymers were also acted as coating agents. Alginate is natural polymer which also used in drug delivery application due to its low toxicity [bib_ref] An alternative approach to wound healing field; new composite films from natural..., Üstünda G Okur [/bib_ref]. Doxorubicin loaded modified alginate with folate-terminated PEG and rhodamine B nanogels were produced by Pei et al. Folate conjugation led to improved targeted on cancer cells while pH-responsive release behavior was also achieved. Cytotoxicity and cellular uptake studies revealed that the anticancer drug was vastly distributed in cancer cells provoking their death. Rhodamine provides fluorescent properties to the nanogels and consequently the nanosystem can be applied as real-time and noninvasive theranostic [bib_ref] Alginate-based cancer-associated, stimuli-driven and turn-on theranostic prodrug nanogel for cancer detection and..., Pei [/bib_ref]. Nanogels with magnetic and dual responsive properties were developed by coupling SPIONs with a disulfide-modified alginate. The nanogels were simultaneously loaded with the anticancer drug doxorubicin. It was revealed that the dual nanosystem demonstrated magnetic-targeted ability, high drug loading content, co-triggered release behavior, high toxicity to tumor cells, low side effects to normal cells and MRI function [bib_ref] Novel dual responsive alginate-based magnetic nanogels for onco-theranostics, Peng [/bib_ref]. Mohapatra et al. modified SPIONs with Carboxymethyl Assam bora rice starch-in order to evaluate its potential as magnetic drug targeting moiety. Scientists further loaded the system with the anticancer doxorubicin. It was demonstrated that the magnetic nanosystem exhibited high uptake and inhibition against HER-2 and folate receptorα receptors over expressed in cancer cells [bib_ref] Doxorubicin loaded carboxymethyl Assam bora rice starch coated superparamagnetic iron oxide nanoparticles..., Mohapatra [/bib_ref]. An interesting study involved the fabrication of H 2 O 2 -activatable CO 2 bubble generating ICG-loaded boronated maltodextrin NPs for imaging and therapy of peripheral arterial disease. The developed nanobubbles demonstrated improved fluorescence, US and photoacoustic signals as well as significant antiinflammatory and proangiogenic effects in H 2 O 2 -stimulated vascular endothelial cells. Researchers revealed that the nanosystem can provide a novel insight for imaging and treatment of peripheral arterial disease [bib_ref] Stimulus-activatable echogenic maltodextrin nanoparticles as nanotheranostic agents for peripheral arterial disease, Jung [/bib_ref].
Cyclodextrins are cyclic oligosaccharides which comprised from glucopyranoside units linked with (1-4) bonds and present a conical shape with an empty cavity which can host small molecules like drugs in proportion with molecules size [bib_ref] Two different approaches for oral administration of voriconazole loaded formulations: electrospun fibers..., Siafaka [/bib_ref]. Multifunctional nanoconjugates of magnetic Fe 3 O 4 NPs, β-cyclodextrin and poly(N-isopropylacrylamide) were applied as drug carriers. Doxurobicin or curcumin was encapsulated and released under acidic pH conditions and elevated temperature whereas folic acid was further coupled in to the nanoconjugates to induce folate receptor targeting. Fluorophores were conjugated onto NPs to provide system fluorescence imaging ability. Moreover, nanoconjugated were able to accumulate on cancer tissues with magnetic hyperthermia. In vivo studies exhibited that after administration of nanoconjugates tumor growth was inhibited [bib_ref] β-cyclodextrin based dual-responsive multifunctional nanotheranostics for cancer cell targeting and dual drug..., Das [/bib_ref]. Xu et al. fabricated fluorescent organic NPs coupling red fluorescence and aggregation-induced emission dye through interactions between β-cyclodextrin and adamantine terminating aggregation-induced emission dye. The developed nanofeatures revealed excellent cytocompatibility and bioimaging ability [bib_ref] Fabrication of AIE-active fluorescent organic nanoparticles through one-pot supramolecular polymerization and their..., Xu [/bib_ref].
Polypyrrole is heterocyclic conductive polymer which is used in biomedical devices or general biomedical field. Yang et al. prepared a novel nanotheranostic system based on polypyrrole which was coupled with Gd modified bovine serum albumin. The nanotheranostic presented high stability, great photothermal property and improved uptake on cancer cells. MRI revealed that the biocompatible nanoplatforms were distributed in cancer cells. In vivo studies with mice showed that polypyrrole system after exposed to 808 nm laser can hinder the tumor growth due to photothermal ablation. From the above, all the results demonstrated the well-designed nanotheranostics can utilized as tumor diagnostic and photothermal therapy [bib_ref] One-pot synthesis of albumin-gadolinium stabilized polypyrrole nanotheranostic agent for magnetic resonance imaging..., Yang [/bib_ref]. In another work, tantalum oxide NPs were impregnated into polypyrrole for dual imaging guided photothermal removal of tumor. Small sized NPs of an average diameter at 45 nm were capable of reaching tumor site after systemic administration. It was revealed that the nanoplatform could improve X-ray CT and photoacoustic imaging in vivo . It should be noted that after intratumoral injection tumor was completely inhibited showing that the system can be efficiently act as targeted diagnostic and therapeutic system [bib_ref] Encapsulating tantalum oxide into polypyrrole nanoparticles for X-ray CT/photoacoustic bimodal imaging-guided photothermal..., Jin [/bib_ref]. Except cancer applications, polypyrrole-PEI nanocomplexes were studied by Bournouf et al. as fibrinolytic therapeutics for venous or arterial thrombotic syndromes. Authors provide novel nanocomplexes exposed to near-infrared radiation as an alternative option of recent thrombolytic agents. It was demonstrated that the nanocomplexes were able of generating local hyperthermia upon NIR treatment, which appeared to produce reactive oxygen species (ROS). In vivo studies involving rats demonstrated biocompatibility, photothermal behavior and biodistribution [bib_ref] A bioinspired hyperthermic macrophage-based polypyrrole-polyethylenimine (Ppy-PEI) nanocomplex carrier to prevent and disrupt..., Burnouf [/bib_ref].
PEG is the most frequent used hydrophilic moiety in biomedical application. PEG coating or PEGylation method onto various macromolecules is a common approach aiming to improve blood circulation of drug loaded NPs. Moreover, PEGylation can reduce toxicity of the nanosystem enhanced drug protection from degradation and numerous other properties [bib_ref] Synthesis of folate-pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing..., Siafaka [/bib_ref]. Bovine serum albumin coated PEGylated magnetic NPs were loaded with vascular endothelial growth factor and doxorubicin as a novel cancer nanotheranostic system for improved targeting in murine breast adenocarcinoma 4T1 cell line. It was demonstrated that the NPs were capable of improving survival rate of mice bearing tumors a well as MRI imaging providing simultaneous cancer therapy and diagnostics [bib_ref] Multimodal doxorubicin loaded magnetic nanoparticles for VEGF targeted theranostics of breast cancer, Semkina [/bib_ref]. An amphiphilic semiconducting polymer based on PEG-grafted poly(cyclopentadithiophene-alt-benzothiadiazole) (PEG-PCB) was developed by Jiang et al. The nanoplatform can be a diagnostic component for NIR fluorescence and photoacoustic imaging as well as therapeutic agent for photothermal cancer therapy [bib_ref] Amphiphilic semiconducting polymer as multifunctional nanocarrier for fluorescence/photoacoustic imaging guided chemo-photothermal therapy, Jiang [/bib_ref].
Another work included the preparation of a hierarchical tumor acidity-responsive magnetic nanobomb composed from chlorin e6 (Ce6)-functionalized polypeptide ligand, methoxy poly (ethyleneglycol)-block-poly (dopamineethylenediamine-2,3-dimethylmaleic anhydride)-Lglutamate-Ce6 and SPIONs. The nanobombs were able to circulated in blood system for hours and provoked tumor inhibition due to their high cancer cell uptake. Both in vitro and in vivo methods demonstrated efficient tumor distribution and superior PDT [bib_ref] Hierarchical tumor acidity-responsive self-assembled magnetic nanotheranostics for bimodal bioimaging and photodynamic therapy, Yang [/bib_ref]. Nanomicelles based on PEG-bpoly(L-leucine) entrapped doxorubicin, chemosensitizing agent XMD8-92, and superparamagnetic iron oxide NPs exhibited targeting ability, MRI imaging, controlled release behavior, improved cytotoxicity on MDR cells and sufficient tumor inhibition [bib_ref] Environmentally responsive dual-targeting nanotheranostics for overcoming cancer multidrug resistance, Yang [/bib_ref]. PEGylated GO-manganese ferrite NPs loaded with doxorubicin were developed by Qian et al. as nanotheranostic system. PEG moiety was applied for enhanced biocompatibility. The nanosystem presented MRI imaging ability whereas the system was further conjugated with radioisotope. The nanocomposites were able to reach tumor revealing high accumulation. Under the guidance of MRI/SPECT imaging, in vivo combined radioisotope therapy and chemotherapy was carried out an ihnibition of tumor growth after intravenous injection [bib_ref] Multifunctional nano-graphene based nanocomposites for multimodal imaging guided combined radioisotope therapy and..., Qian [/bib_ref]. Multi-functional core-shell Fe 3 O 4 -Au NPs, conjugated with doxorubicin, methoxy PEG (mPEG), and folic acid-linked PEG were synthesized for cancer theranostic applications. The system showed saturation magnetization, improved release property and enhanced cellular distribution on cancer cells. Moreover, the NPs demonstrated high cytotoxicity under laser irradiation which led to PTT [bib_ref] Multi-functional core-shell Fe 3 O 4 @Au nanoparticles for cancer diagnosis and..., Rajkumar [/bib_ref]. PEG was also coated magnetic gold NPs loaded with doxorubicin as anticancer nanotheranostics. In vitro and in vivo methods confirmed that the nanoplatforms were biocompatible, can release drug in controlled type, induce PTT via heat by NIR laser absorption. In addition, the multifunctional NPs can also act as MRI contrast agents [bib_ref] Multifunctional magnetic-gold nanoparticles for efficient combined targeted drug delivery and interstitial photothermal..., Elbialy [/bib_ref]. Tong et al. developed PEI-PEGreduced GO as biocompatible alternative for photothermal and chemotherapy against hepatocarcinoma. Doxorubicin was chosen as the anticancer agent. It can be concluded that the nanomaterials could effectively deliver and release drug in SMMC-7721 cells provoking cells death [bib_ref] PEGylated mBPEI-rGO nanocomposites facilitate hepotocarcinoma treatment combining photothermal therapy and chemotherapy, Tong [/bib_ref]. PEGylated black phosphorus NPs with great biocompatibility and water-solubility were prepared aiming to reduce cancer cells growth. The NPs can convert NIR light into heat, and exhibit excellent photostability. In vivo studies demonstrated the NPs accumulation in mice tumors whereas irradiation can be used for photothermal ablation of tumors [bib_ref] One-pot solventless preparation of PEGylated black phosphorus nanoparticles for photoacoustic imaging and..., Sun [/bib_ref]. A photosensitizerassembled graphene/gold nanostar hybrid was formulated for combined cancer synergistic PDT and PTT as well as effective photothermal imaging. As it was expected, the stable PEG composite showed anticancer efficacy, tumor accumulation and inhibition and improved optical imaging [bib_ref] Single wavelength light-mediated, synergistic bimodal cancer photoablation and amplified photothermal performance by..., Wu [/bib_ref]. Magnetic graphene nanohybrids based on GO-modified PEG and γ -Fe 2 O 3 were prepared by Chen et al. as an encouraging option for synergistic anticancer therapy and imaging. Doxorubicin was chosen as anticancer drug. It was exhibited that the nanosystem can be mapped by MRI, photothermal and fluorescence imaging. In addition, the anticancer efficacy was revealed by both in vitro and in vivo methods which showed tumor ablation after hyperthermia and NIR light exposure [bib_ref] Laser-assisted in situ synthesis of graphene-based magnetic-responsive hybrids for multimodal imaging-guided chemo/photothermal..., Chen [/bib_ref]. Multifunctional nanotheranostics incorporating IR-780 dye, a NIR imaging probe as well as the anticancer compound known as α-tocopheryl succinate were developed by Palao-Suay et al. The core shell NPs were fabricated using PEG and poly(methacrylic α-tocopheryl) succinate. The system presented photo-inducing and fluorescence ability due to the presence of the dye. In vitro studies showed that the theranostic NPs can be effectively localized in cells revealing promising characteristics [bib_ref] Photothermal and photodynamic activity of polymeric nanoparticles based on α-tocopheryl succinate-RAFT block..., Palao-Suay [/bib_ref]. Finally, He et al. developed a peptide-based nanotheranostic based on p53-activating peptide termed PMI, functionalized PEG and fluorescent lanthanide oxyfluoride nanocrystals. It was revealed that the nanoplatform exhibit improved tumor targeting and imaging properties. In vivo studies involving a mouse model with human colon cancer showed that the nanosystem is efficacious. More specifically, the nanorods can inhibit tumor growth and be easily mapped [bib_ref] A lanthanide-peptide-derived bacterium-like nanotheranostic with high tumor-targeting, -imaging and -killing properties, He [/bib_ref]. You et al. prepared multifunctional nanosystems with sufficient cellular uptake, specific target and controlled release efficacy. The nanosystem was consisting of folate and cyclic arginineglycine-aspartic-peptide modified NPs based on NIR light and glutathione dual stimuli-responsive release system loaded with cisplatin and ICG. The core polymer was a copolymer between poly( ε-captolactone) (PCL) and carboxylated PEG. The nanospheres were able of accumulation to tumor sites showing decreased cancer cells viability and thus it can be an alternative nanotheranostic in nanomedicine field [bib_ref] Synthesis and biological evaluation of redox/NIR dual stimulus-responsive polymeric nanoparticles for targeted..., You [/bib_ref].
PCL is hydrophobic polymer which is used in biomedical field especially in controlled release applications [bib_ref] Controlled release formulations of risperidone antipsychotic drug in novel aliphatic polyester carriers:..., Siafaka [/bib_ref]. As nanotheranostics, PCL is used as coating or core material. For instance, PCL-AuNC/Fe(OH) 3 -PAA Janus NPs were fabricated by Zhang et al. and loaded with doxorubicin and docetaxel. The nanoplatform demonstrated greater therapeutic efficacy due to synchronous release of two drugs as well as the excellent computed X-ray tomography/magnetic resonance (CT/MR) imaging capabilities. In vivo studies using mice further depicted tumor inhibition revealing that the aforementioned system can be effective as combined cancer therapy [bib_ref] Dual drug delivery and sequential release by amphiphilic Janus nanoparticles for liver..., Zhang [/bib_ref].
Poly(lactic) acid (PLA) is an aliphatic polyester with high cytocompatibility which can be easily modified or formulated in various nanoforms [bib_ref] Novel electrospun nanofibrous matrices prepared from poly(lactic acid)/poly(butylene adipate) blends for controlled..., Siafaka [/bib_ref]. In case of nanotheranostics, NPs of PLA were loaded with doxorubicin and further surface coated with Mn-porphyrin as potential MRI agent and pH-responsive drug delivery system. Firstly, the system exhibited that the drug was released at greater extent in acidic pH. Moreover, nanoformulations showed efficient tumor inhibition in HeLa cells and HT-29 cells. All the more, the system demonstrated that it can act as MRI contrast agent for sufficient cancer diagnosis and therapy [bib_ref] Covalent attachment of Mn-porphyrin onto doxorubicin-loaded poly(lactic acid) nanoparticles for potential magnetic..., Jing [/bib_ref].
Poly(lactic-co-glycolic acid (PLGA) is the copolymer between PLA and poly(glycolic acid) and presents high biocompatibility and extremely promising properties. It is widely used in pharmaceutical industry and technology as nanocarrier. As nanotheranostic, PLGA-based NPs impregnated with paclitaxel and superparamagnetic iron oxides to act as both therapeutic and imaging tool. The visualization of NPs accumulation was conducted using Electron Spin Resonance spectroscopy and MRI and it was depicted that the nanoplatforms exhibited improved NPs distribution, MRI contrast ability and most importantly anticancer activity [bib_ref] Comparison of active, passive and magnetic targeting to tumors of multifunctional paclitaxel/SPIO-loaded..., Schleich [/bib_ref]. A mPEG-PLGA-poly-l -lysine triblock copolymer was designed as photothermally triggered immunotherapeutic system loaded with SPIONs and cytosine-phosphate-guanine oligodeoxynucleotides. The under magnetic-responsive immunostimulatory nanoagents acted both as contrast agent for PA/MRI bimodal imaging and magnetic-targeting therapeutic agent. It was revealed that the NPs were accumulated in tumors and inhibit metastatic tumors simultaneously with high specificity, easy maneuverability and favorable biocompatibility [bib_ref] Magnetic-responsive and targeted cancer nanotheranostics by PA/MR bimodal imaging-guided photothermally triggered immunotherapy, Guo [/bib_ref].
Another system involved quantum dots as imaging molecule and EpCAM aptamer as target ligand which conjugated to nutlin-3a loaded PLGA NPs. The nanoplatform exhibited intriguing properties such as cell targeting and biomaging and it can be served as nanotheranostic approach on cancer therapy [bib_ref] Multifunctional nanoparticle-EpCAM aptamer bioconjugates: a paradigm for targeted drug delivery and imaging..., Das [/bib_ref]. Castellani et al. studied CdSe/ZnS QD-loaded PLGA NPs as targeted system for metastatic liver cancer as hyperthermia triggered system. Localized MW irradiation which led to mild hyperthermia played a significant role to NPs accumulation in tumor [bib_ref] Tumor-facing hepatocytes significantly contribute to mild hyperthermia-induced targeting of rat liver metastasis..., Castellani [/bib_ref]. NPs of modified PEG-PLGA with iron-gallic acid as MRI guided chemo-photothermal synergistic therapy of tumors. The drug Gallic acid was able to be released in acidic pH as tumor environment inducing apoptosis. The study revealed that the nanonetwork due to its extraordinary photostability and photothermal therapy capacity is quite promising as nanotheranostic [bib_ref] A pH-sensitive coordination polymer network-based nanoplatform for magnetic resonance imaging-guided cancer chemo-photothermal..., Zhang [/bib_ref]. Furthermore, paclitaxel was loaded into nanocapsules comprised from PLGA-PEG and perfluorooctyl bromide (PFOB) as nanotheranostic agents. The prepared nanocapsules showed anticancer efficacy when studied in vitro on CT-26 colon cancer cells whereas in vivo studies revealed passive accumulation of drug in CT-26 tumors in mice and tumor inhibition. Imaging capability was induced due to the presence of PFOB. Consequently, the system has great potential as cancer theranostic agents [bib_ref] Paclitaxel-loaded PEGylated nanocapsules of perfluorooctyl bromide as theranostic agents, Boissenot [/bib_ref].
PEI is a branched cationic polymer which can be modified and act as polymer nanotheranostic. In a recent research, Shao et al. fabricated PEI-PLA NPs which simultaneously load hydrophobic antiangiogenesis agent combretastatin A4, NIR dye IR825 and heat shock protein 70 (HSP70) inhibitor (siRNA against HSP70). The nanosystems studied in vivo in a xenograft mouse tumor model, demonstrating improved photocytotoxicity and tumor inhibition and synergistic anticancer efficacy with NIR laser irradiation. The copolymer acted as drug carrier as well as self-monitor to real-time tracking of NPs biodistribution and tumor accumulation via fluorescence imaging. Finally, due to the above and fact that the nanoplatform can also be applied as photoacoustic agent for in vivo photoacoustic imaging, the nanosystem offer encouraging results as current multifunctional cancer nanotheranostic [bib_ref] Auto-fluorescent polymer nanotheranostics for self-monitoring of cancer therapy via triple-collaborative strategy, Shao [/bib_ref]. Deng et al. also used PEI as modifying agent of black phosphorus nanomaterials along with dextran. The nanoplatforms were further functionalized with folic acid and cyanine 7 depicting great stability and cell viability, near infrared optical properties for targeted imaging of tumors through photoacoustic imaging and NIR fluorescence imaging. In addition, the nanosystem is efficient as photothermal cancer therapeutic [bib_ref] Functionalization of small black phosphorus nanoparticles for targeted imaging and photothermal therapy..., Deng [/bib_ref].
Poly(N-isopropyl acrylamide) (PNIPAM) is a pH-and temperature-responsive polymer used in variou medicine applications. Roy et al. fabricated stimuli-responsive PNIPAMco-tyrosine modified gadolinium doped IONs as cancer theranostic agent. Methotrexate was loading in great extent and released with a stimuli dependent manner. In further, the NPs showed MRI activity as well as desirable in vitro hyperthermia response [bib_ref] Stimuli-responsive poly(N-isopropyl acrylamide)-co-tyrosine@gadolinium: iron oxide nanoparticle-based nanotheranostic for cancer diagnosis and treatment, Roy [/bib_ref]. Poly(vanillin oxalate) (PVO) is antioxidant polymer which was formulated into NPs generating CO 2 through H 2 O 2 -triggered oxidation of peroxalate esters and release vanillin, which exerts antioxidant and anti-inflammatory activities. PVO NPs exhibited improved ultrasound signal in the site of hepatic I/R injury and also effectively suppressed the liver damages by inhibiting inflammation and apoptosis. Consequently, the system can act as ultrasound contrast agents and therapeutic tool in H 2 O 2 -associated diseases [bib_ref] H 2 O 2 -triggered bubble generating antioxidant polymeric nanoparticles as ischemia/reperfusion..., Kang [/bib_ref].
Other polymers have also been employed as nanotheranostics. More specifically, poly (N-(2-hydroxypropyl) methacrylamide) nanocarriers of paclitaxel were further coupled with self-quenched Cy5 (SQ-Cy5). Activatable fluorescent probes as SQ-Cy5 employ a fluorescent signal which is silenced/"OFF" under physiological conditions, and is turned-ON at the designated site. Polymeric nanotheranostics could present an 'always ON' signal. Förster resonance energy transfer is the most common and efficient turn-on mechanism. The system was able to release the drug due to enzymic degradation in cathepsin B-overexpressing breast cancer cells. The drug releases took place simultaneously with the activation of the fluorophore to its Turn-ON state. The copolymer NPs showed better distribution and drug release in comparison with the pure drug and the probe. In addition, the marketed taxol administration system utilize the toxic chremophor EL substance for the solubilization of taxol. The developed system is water soluble and thus it can be administered in aqueous solution overcoming the use of chremophor [bib_ref] Polymeric nanotheranostics for real-time non-invasive optical imaging of breast cancer progression and..., Ferber [/bib_ref]. Similarly, tumor-targeted photodynamic therapy using polymeric photosensitizers of poly (N-(2-hydroxypropyl) methacrylamide) conjugated pyropheophorbide-a was found to be a significant strategy for cancer treatment. As the majority of the nanotheranostics, the copolymeric NPs can act as encouraging therapeutic strategy in oncology field. In further, nanomicelles showed high tumor accumulation, antitumor effect under irradiation using normal xenon light source of endoscope, and clear tumor imaging profiles even in the metastatic lung cancer [bib_ref] -hydroxypropyl) methacrylamide polymer conjugated pyropheophorbide-a, a promising tumor-targeted theranostic probe for photodynamic..., Fang [/bib_ref]. Shi et al. fabricated fluorescent organic NPs based on dopamine containing copolymers (poly(AC-co-PEGMA)). PEI was also mixed and the NPs demonstrated great water dispersibility, strong green fluorescence and desirable biocompatibility [bib_ref] Facile preparation of water soluble and biocompatible fluorescent organic nanoparticles through the..., Shi [/bib_ref]. Polyacrylamide hydrogel NPs were surface modified and conjugated with oxygen indicator and PEG groups as photoacoustic oxygen imaging nanosystem for tumor targeting and detection. The prepared nanosystem provides an in vivo non-invasive imaging and assesement method of hypoxic tumor microenvironments. This method is crucial for the evaluation of cancer progression, metastasis and treatment [bib_ref] In vivo photoacoustic lifetime based oxygen imaging with tumor targeted G2 polyacrylamide..., Jo [/bib_ref].
PVP has been also used as pharmaceutical excipient. Herein, authors present ternary copper-based chalcogenide nanotheranostics which combine high photothermal conversion efficiency and a simultaneous ROS generation effect. Except these facts, the nanoplatforms also revealed remarkable contrast enhancement and thus they can act as multifunctional nanotheranostic agents for photoacoustic imaging, photothermal/photodynamic cancer therapy [bib_ref] Multifunctional NIR-responsive poly(vinylpyrrolidone)-Cu-Sb-S nanotheranostic agent for photoacoustic imaging and photothermal/photodynamic therapy, Hou [/bib_ref]. Pluronics are copolymers of polyethylene-and polypropylene oxide widely used in pharmaceutical industry. In this work, authors fabricated Pluronic coated gold NPs loaded with IR780 iodide dye as combined PDT and PTT activity with surfaceenhanced resonance Raman scattering imaging facility. The nanosystem exhibited improved water-solubility, stability and NPs accumulation in Plu-IR780 by murine colon carcinoma cells (C-26). Finally, the NPs indicated that simultaneous PDT and PTT activity can be achieved [bib_ref] IR780-dye loaded gold nanoparticles as new near infrared activatable nanotheranostic agents for..., Nagy-Simon [/bib_ref]. Another work involved the preparation of folic acid armed polymeric core-shell iron oxide NPs as a new type of nanotheranostic agent. The NPs were further coated hyperbranched polyglycerol aiming to improve their biocompatibility. Folic acid was used to target folate receptors overexpressing on cancer cells. The results showed that NPs can be applied as cancer nanotheranstics [bib_ref] Folic acid armed Fe 3 O 4 -HPG nanoparticles as a safe..., Mostaghasi [/bib_ref].
## Liposomes
Liposomes are structured biocompatible and biodegradable lipid carriers. Some liposomes have been approved from FDA [bib_ref] Recent Developments of liposomes as nanocarriers for theranostic applications, Xing [/bib_ref]. Liposomes comprised from one or more layers of natural or synthetic lipids and an aqueous core. They have been utilized as carriers for many active molecules either hydrophilic or lipophilic . Radio-labeled liposomes with radionuclides such as 67 Ga, [bib_ref] Nanomedicine in central nervous system (CNS) disorders: a present and future prospective, Soni [/bib_ref] In and 99m Tc present diagnostic, monitoring and therapeutic functions. Besides their imaging ability due to the radio-labeling, such liposomes are an excellent tool for choosing the best therapeutic action in individual patients . A research group radio-labeled iminothiolane-Tc-tricarbonyl complex a model liposome system. The system mimics the Epaxal R and Inflexal V R which are FDA approved liposomal drugs [bib_ref] Radiolabeling and quantitative in vivo SPECT/CT imaging study of liposomes using the..., Varga [/bib_ref].
In most cases, liposomes are funtionalized or coated with active molecules such as PEG, vitamins which can induce their biocompatibility. For example, vitamin E TPGS-coated liposomes are widely prepared [bib_ref] Theranostic liposomes for cancer diagnosis and treatment: current development and pre-clinical success, Muthu [/bib_ref]. In further, PEG-coated and folate-PEG-coated long-circulating and pH-sensitive liposomes loaded with [bib_ref] Antibody-drug gold nanoantennas with Raman spectroscopic fingerprints for in vivo tumour theranostics, Conde [/bib_ref] Gd and poly-l -lysine were studied as cancer nanotheranostics. The prepared liposomes provide increased animal survival and high tumor uptake [bib_ref] Scintigraphic imaging and increment in mice survival using theranostic liposomes based on..., Soares [/bib_ref]. Liposomes and PEGylated liposomes were functionalized with gadolinium (III) diethylenetriamine pentaacetic acid salt which acted as MRI contrast and zinc phthalocyanine as a model photosensitizer. The results showed that the liposomal formulations can serve as imagine agents [bib_ref] Theranostic liposomes as a bimodal carrier for magnetic resonance imaging contrast agent..., Skupin-Mrugalska [/bib_ref]. A novel system comprised from liposomes containing NIR carbon dots and the anticancer drug, cinobufagin, was developed and evaluated as potential anticancer nanotheranostics.
Bioimaging of the prepared system was significantly high whereas liposomes could be uptaken by cells and delivered to the tumor site. Moreover, a prolonged release behavior and high anticancer activity was recorded [bib_ref] Near-infrared fluorescent carbon dots encapsulated liposomes as multifunctional nano-carrier and tracer of..., Ren [/bib_ref]. The theranostic liposomes were functionalized with arginineglycine-aspartic acid-Tocopheryl succinate and loaded with docetaxel and quantum dots by Sonali et al. A prolonged drug release and biocompatibility was achieved revealing that the nanosystem can serve as brain nanotheranostic [bib_ref] RGD-TPGS decorated theranostic liposomes for brain targeted delivery, Singh [/bib_ref]. Another promising study involves the application of ultrasmall iridium nanocrystals into stealth liposomal features. The developed system showed efficient photothermal conversion ability, since the iridium nanocrystals present effective NIR responsive catalytic activity towards H 2 O 2 decomposition. In addition, the system exhibited improved blood circulation which enhances sufficient retention in mice tumors. Thus, it could be used in enhancing cancer radiotherapy [bib_ref] Iridium nanocrystals encapsulated liposomes as near-infrared light controllable nanozymes for enhanced cancer..., Feng [/bib_ref]. anticancer nanotheranostics by using the cytocombatible NIR dye, ICG. They entrapped PFOB in nanoliposomes which revealed in vivo CT contrast imaging. Moreover, the formulation after intravenous administration hindered completely the MDA-MB-231 tumor growth due to excellent oxygen carrying ability of PFOB, which effectively attenuated tumor hypoxia, improved the efficiency of collisional energy transfer between ICG and oxygen and reduced the expression of heat shock protein [bib_ref] Perfluorooctyl bromide & indocyanine green co-loaded nanoliposomes for enhanced multimodal imaging-guided phototherapy, Sheng [/bib_ref]. Stimuliresponsive liposomes were also produced featuring diagnosis and treatment features. In fact, ROS-responsive liposome were designed showing light scatter and fluorescence intensity. Authrs inserted lipid oxidation sensor, C11-BODIPY (581/591), to liposomal bilayer to provide ratiometric fluorescent nanoprobe for ROS detection. Afterwards, Mitoxantrone-chemotherapeutic substance, was loaded into C11-BODIPY (581/591) functionalized liposome. This novel system exhibited prolonged release, improved anticancer activity and imaging ability [bib_ref] Single-particle characterization of theranostic liposomes with stimulus sensing and controlled drug release..., Chen [/bib_ref]. Finally, multifunctional RNA-loaded magnetic liposomes were prepared as an early biomarker of treatment response. The iron oxide loaded RNA-liposomes deliver RNA to dendritic cells, activate those dendritic cells, and enable prediction of tumor regression with MRI [bib_ref] Dendritic cell-activating magnetic nanoparticles enable early prediction of antitumor response with magnetic..., Grippin [/bib_ref].
## Dendrimers
Dendrimers are the most significant nanostructured materials due to their external groups, which can be modified with antibodies, peptides or proteins. Dendrimers which own their name to the Greek word "dendro-tree", show a structure of tree-like arms or branches .
An interesting study reports the use of multifunctional polymeric dedrimers comprised from a copolymer of Boltorn H40, PCL and P(oligo(ethylene glycol) monomethyl ether methacrylate-co-3-azidopropyl methacrylate) as cancer targeted drug delivery and MRI contrast. In fact it was further modified with alkynyl-functionalized cancer cell-targeting moieties, alkynyl-folate, and T1-type MRI contrast agents, alkynyl-DOTA-Gd (DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakisacetic acid). Moreover, the dendritic polymers were loaded with paclitaxel. It was revealed that the drug was released in controlled manner while in vivo MRI imaging in rats showed desirable distribution of unimolecular micelles within rat liver and kidney, prominent positive contrast enhancement, and relatively long duration of blood circulation. Thus, they can be used as alternative nanotheranostics [bib_ref] Amphiphilic multiarm star block copolymer-based multifunctional unimolecular micelles for cancer targeted drug..., Li [/bib_ref]. In another study authors employed dendrimers as in vivo anti-lymphoma efficient system which also can act as diagnostic tool due to the fluorescent dye conjugation in the polymer core. Doxorubicin was used as anticancer drug, and the dendrimers were studied in murine models of malignant lymphomas including one cell line-derived xenograft and two patient-derived lymphoma xenografts (VFN-D1 and VFN-M2) [bib_ref] Effective doxorubicin-based nano-therapeutics for simultaneous malignant lymphoma treatment and lymphoma growth imaging, Etrych [/bib_ref]. Polydopamine (PDA) coated magnetite NPs were loaded in dendrimers and they were characterized as chemo-and photothermal therapeutics and imaging agents by MRI and CT-PTT of the liver cancer cells. In vivo studies exhibited that the dual therapeutics can provoke apoptosis and thus is a promising and smart system [bib_ref] Dendrimer based theranostic nanostructures for combined chemo-and photothermal therapy of liver cancer..., J Ędrzak [/bib_ref].
Similarly, doxorubicin and indodicarbocyanine fluorescent dye was attached to dendritic polyglycerol nanocarriers. The results exhibited that the dendritic carriers were monitored in real time in intact cancer cells revealing its potential [bib_ref] Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy..., Krüger [/bib_ref]. Poly(amidoamine) (PAMAM) are the most frequently used polymers as dendrimers. Carbon quantum dots from sweet lemon peel conjugated with PAMAM dendrimers to act as nanotheranostics to triple negative breast cancer. A peptide, known as arginine-glycine-aspartic acid (RGD) peptide was further conjugated to the system to target integrin which is over expressed in the specific cancer. It was found out that the system is an alternative theranostic option since it can be sufficiently bind into cancer cells [bib_ref] Dendrimer functionalized carbon quantum dot for selective detection of breast cancer and..., Ghosh [/bib_ref]. Another study included Gd-doped ferrite NPs which were further encapsulated into PAMAM dendrimers whereas doxorubicin was also added to enhance their anticancer activity. Almost 78% of doxorubicin released in the presence of a low-frequency alternating magnetic field and mildly acidic pH environment [bib_ref] Encapsulation of gadolinium ferrite nanoparticle in generation 4.5 poly(amidoamine) dendrimer for cancer..., Mekonnen [/bib_ref]. In a different study folate-PEG was attached in PAMAM dendrimers aimed to target folate receptors in cancer cells. This core was conjugated with Cadmium selenide/Zinc sulfide (CdSe/ZnS) quantum dots which are widely used as diagnostic tool. It was demonstrated that the folate coated dendritic system show high cell uptake, binding in the cell surface and doxorubicin released into the tumor cells. The developed nanosystem can act as bioimaging and therapeutic tool [bib_ref] Synthesis and grafting of folate-PEG-PAMAM conjugates onto quantum dots for selective targeting..., Zhao [/bib_ref].
Multifunctional PAMAM dendrimers loaded with gold NPS which were coupled with α-tocopheryl succinate were developed as cancer targeted nanotheranostic agent for CT imaging and therapy. In addition, PAMAM dendrimers were modified with fluorescein isothiocyanate, PEG-modified α-TOS, as well as PEGylated folic acid which served as templates to synthesize Au nanoplatforms. It was found out the nanoplatfomrs exhibited sufficient targetability to cancer cells overexpressing FA receptors as well as were able to target CT imaging of the cancer cells in vitro and the xenografted tumor model in vivo [bib_ref] Targeted cancer theranostics using alpha-tocopheryl succinate-conjugated multifunctional dendrimer-entrapped gold nanoparticles, Zhu [/bib_ref]. Same team studied multifunctional dendrimer-entrapped gold NPs with alpha-tocopheryl succinate and arginine-glycine-aspartic acid peptide for targeted chemotherapy and CT imaging of cancer cells. Authors also used fluorescein isothiocyanate, RGD peptide coupled with PEG and PEG-linked α-TOS. The prepared nanotheranostics were stable in several conditions and can target to cancer cells overexpressing αv β3 integrin. Moreover, they show improved growth inhibition of the cancer cells. Authors conclude that the system can be promisingly applied as nanoprobe [bib_ref] Dendrimer-entrapped gold nanoparticles modified with RGD peptide and alpha-tocopheryl succinate enable targeted..., Zhu [/bib_ref].
## Conclusions and authors' opinion
Nanotheranostic field is an emerging research field which however has not still meet the clinical standards. This fact is arising from the complexity and the synergistic mechanisms of the used nanotheranostics. For instance, some of the systems although show great diagnostic efficacy, they lack of therapeutic ability. Other systems have shown important therapy index with low imaging ability. Nonetheless, the scientific community has conducted great efforts to translate these new nanotheranostics into clinical trials by examining various nanomaterials or modification techniques and evaluating their in vivo performance. As it is detailed in this work, most of the current studies evaluate the diagnostic and therapeutic application of nanotheranostics using in vivo animal models, showing promising results. Nonetheless, if the nanotheranostics applied on human individuals, their application mostly fails. This is correlated with the fact that other mechanisms lead the diffusion of nanotheranostics in animals and others in human. In the past, EPR effect believed as one of the leading phenomena affecting the diffusion of NPs in tumors. Hence, many researchers evaluate the passive targeting of NPs in animals. However, EPR effect is not prominent in the majority of human tumors. Thus, efficient animal models should be utilized when studying the nanotheranostics.
Another issue of nanotheranostics is their possible toxicity and safety when used in humans. Among the applied nanotheranostics, carbon nanotubes and metallic NPs have raised a concern of safety due to their slow degradation and in vivo fate. Consequently, various attempts have been performed as surface coating with biocompatible macromolecules as PEG, PLA and chitosan to enhance their suitability for future clinical use. Another idea is the use of nanotheranostics synthesized by biodegradable or biocompatible materials which have been clinically approved. Besides, several biocompatible polymers, liposomes or dendrimers have been clinically approved for other pharmaceutical applications. Hence, the modification or conjugation of already approved therapeutic formulations or materials with functional ligands which will improve their diagnostic index could be essential.
Compared to the past, more sophisticated nanotheranostics have been developed worldwide. However, a huge advancement should be done before their clinical application. As it is addressed, in this review the current nanotheranostics have been evaluated mostly for their in vitro performance. In other works, the in vivo imaging evaluation of nanotheranostics might be performed but their in vivo drug delivery was not attenuated. In further, the toxic profile of many of the developed nanotheranostics is absent. Therefore, the promising properties of nanotheranostics, which are discussed in this review, should be advanced more before their translational in clinical medicine. To conclude, although a great effort is still needed, the future of nanotheranostics utility in clinical practice is near.
## Conflicts of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. references
[fig] Figure 1 -: A schematic representation of nanotheranostics used for simultaneous release and imaging. The functional groups of nanotheranostics can be drugs, DNA, RNA, imaging agents while their core material are polymers, inorganic and carbon based nanocarriers, dendrimers as well as liposomes. [/fig]
[fig] Figure 2 -: A detailed schematic illustration of multifunctional coated nanotheranostics. The imaging agents can be categorized on fluorescent or visible-infrared moieties, radionuclides whereas therapeutic target groups could be both chemical and biological agents. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) [/fig]
[fig] Figure 3 -: The categories of radionuclides used as theranostics and diagnostic agents. According to their emmision type they can act as therapeutic or diagnostic agents. * E max = maximum energy, half-life = the time length it takes for half of the radioactive atoms of a specific radionuclide to decay. [/fig]
[fig] Figure 4 -: Various examples of inorganic nanoparticles used as nanotheranostics. Description: Au = Gold, Ag = Silver, MSN = Mesoporous Silica Nanoparticles, Fe 3 O 4 = Iron oxide, PB = Prussian blue. [/fig]
[fig] Figure 5 -Figure 6 -: Carbon based nanomaterials used as detection diagnostic nanotools. Various structures of nanotheranostics based on polymeric NPs, liposomes and dendrimers. [/fig]
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Inverse-probability weighting and multiple imputation for evaluating selection bias in the estimation of childhood obesity prevalence using data from electronic health records
Background and objectives: Height and weight data from electronic health records are increasingly being used to estimate the prevalence of childhood obesity. Here, we aim to assess the selection bias due to missing weight and height data from electronic health records in children older than five. Methods: Cohort study of 10,811 children born in Navarra (Spain) between 2002 and 2003, who were still living in this region by December 2016. We examined the differences between measured and non-measured children older than 5 years considering weight-associated variables (sex, rural or urban residence, family income and weight status at 2-5 yrs). These variables were used to calculate stabilized weights for inverse-probability weighting and to conduct multiple imputation for the missing data. We calculated complete data prevalence and adjusted prevalence considering the missing data using inverse-probability weighting and multiple imputation for ages 6 to 14 and group ages 6 to 9 and 10 to 14.Results: For 6-9 years, complete data, inverse-probability weighting and multiple imputation obesity age-adjusted prevalence were 13.18% (95% CI: 12.54-13.85), 13.22% (95% CI: 12.57-13.89) and 13.02% (95% CI: 12.38-13.66) and for 10-14 years 8.61% (95% CI: 8.06-9.18), 8.62% (95% CI: 8.06-9.20) and 8.24% (95% CI: 7.70-8.78), respectively.
# Background
According to the World Health Organization, childhood obesity is a serious public health challenge of the twentyfirst century. In 2010, it was estimated that 42 million children under 5 years of age were overweight worldwide. Obesity is associated with conditions such as adverse cardiovascular and metabolic outcomes, mental health and psychological issues, and/or respiratory and orthopedic problems. Furthermore, childhood obesity increases the likelihood of obesity in adulthood. Public health programs are currently focused on monitoring the trend in prevalence of overweight by sex and age, as well as assessing geographic ethnic, or socioeconomic inequalities to help improve the policies and interventions. Worldwide, monitoring has been carried out through national surveys, e.g., self-reported information provided by the parents or by direct measurements of weight and height, as in the case of the National Health and Nutrition Examination Surveys in the USA, or the WHO European Childhood Obesity Surveillance Initiative. Standardized measurements of weight and height and the establishment of nationally representative samples are some of the strengths of these actions.
Over the last years, measurements of weight and height from electronic health records (EHR) databases have been used in Canadian,British,North American,Swedish,and Spanish studiesto estimate trends in childhood obesity prevalence. The use of EHR data for surveillance does not require bespoke data collection or patient recruitment,it provides information for small areas and all age groups at any time and lower cost in comparison to surveys. However, to obtain reliable results from these data, the estimations derived from EHRs should be free of potential bias.
The information available in EHRs comes from children who attend and are measured at primary care centers during well-child or office visits for any reason. Estimations of the prevalence of weight status using incomplete EHR data could be biased if there are systematic differences in characteristics associated with weight status (e.g., income, sex, age, etc.) between measured and non-measured children. It is necessary to identify the factors leading to missingness (lack of weight and height measurements in our case) and apply strategies, such as inverse-probability weighting (IPW) or multiple imputation (MI) to obtain corrected estimations. When using IPW, complete cases are weighted by the inverse of their probability of being a complete case. A pseudo-population is generated in which each individual receives a weight representing the inverse of the conditional probability of being measured (conditional to the actual values of the confounding variables). The validity of this method relies on a correctly specified model including all possible variables associated with missingness. In the same context, MI produce unbiased estimates for the parameters of interest through a simulation-based procedure that replaces each missing value with a set of plausible values, thus creating complete data sets. The results are combined in a final estimate (average) that incorporates the variability of the data and some additional variability acknowledging any uncertainty on the missing values.
The purpose of this study is to examine the selection bias associated with missing weight and height data from a single EHR and compare the prevalence of obesity derived from complete data and estimators obtained using IPW and MI.
# Methods
## Study population
This study was carried out in Navarra, a region in northern Spain with 640,000 inhabitants and around 6000 live births every year. The Navarra Health Service provides free healthcare access to most of the region's population. EHRs have been used since 2000 and include reports from primary care, hospital admissions, regional vaccinations, and laboratory test results. A single EHR is used in public primary care centers where a team formed by a pediatrician, a pediatric nurse, and occasionally a social worker perform well-child visits through the Navarra Child Health Program; the first visit is at home and from then on at the primary care centers. Vaccines are administered mostly in primary care centers until the age of six and then at school. The Child Health Program includes 15 health exams: nine during the first 3 years of life, and one at three, four, six, eight, 11, and 14 years. Weight and height, are measured during the visits using standardized methods and, in most cases, these measurements, together with the date at which they were performed, are stored as structured data in the EHRs making data mining easier. A software based on OMI-AP is used for managing the primary care EHR,which is organized in a structured list (bio-psycho-social problems, reason for consultation, etc.) following the International Classification of Primary Care, Second Edition (ICPC-2).
## Assessment of selection bias
We first determined the percentage of children with healthcare cards of the Navarra Health Service who had height and weight measurements in their first 14 years of life. By December 31, 2016, there were 95,321 children under 15 years of age in the primary EHR database from a total of 100,301 inhabitants of that age. Weight and height measurements data stored in EHR by the pediatric team during the visits were used to estimate the percentages by age of children with at least one measurement within one-year period (from January to December of 2016) and four-year period (2013-2016). The lowest percentages of measurements in one-year periods were seen for ages when well-child visits were not scheduled, and 13 years). When considering longer time-frames (4 years), the percentages of measured children were around 97-99% for children under 5 years. Thus, weight status estimates obtained using the data of these children are generalizable to the whole population of the same age. For valid overweight and obesity prevalence estimations in children older than 5 years we had to make sure that relevant characteristics related to weight status categories were not different for measured and not measured children. In case they were different, we had to correct the possible selection bias. Since there was no other sources to validate EHR height and weight data (e.g., a Nutrition Examination Survey), we analyzed if the information of the measured children older than 5 years was biased in relation to a group of variables, specifically sex, family income, urban/rural residence, and weight status at ages 2-5 years, for whom this information was available for almost all children included in the study.
## Outcomge variable
When IPW was used, measured/non-measured was the outcome variable. Having height and weight data for the same time point for ages 6 to 14 years and for 6-9 and 10-14 age groups was considered as being measured. Missing data for height and/or weight for the same time point was considered as being not-measured.
## Explanatory variables
Sex (boy/girl), rural/urban residence (urban residence included cities with ≥35,000 inhabitants; rural residence included towns with < 35,000 inhabitants), annual family income (ordinal categories from 1 to 4 [1: basic income (families living on minimum subsistence income), 2: < 18,000 €, 3: 18,000 to < 100,000 €, and 4: ≥ 100,000 €),and weight status at 2-5 years (underweight, normal weight, at risk of overweight and overweight/obese). Latest body mass index (BMI) at 2-5 years was calculated as the weight in kilograms divided by height in meters squared (kg/m 2 ). BMI z-scores were determined using the WHO standards . We categorized the weight status as follows: underweight (< − 2 SD), normal weight (≥ − 2 to ≤1 SD), at risk of overweight (> 1 to ≤2 SD), overweight/obese (> 2 SD). For older children, the cutoff for underweight was < − 2 SD), normal weight ≥ − 2 to ≤1 SD, overweight + 1 SD and obesity + 2 SD.
Children for whom there were no measurements at ages 2-5 years and those with missing values of variables used as potential predictors of missing data or implausible zscores of BMI values (≤ 5 SD and ≥ 5 SD) were excluded from the study. Thus, 10,811 children born between 2002 and 2003 that were living in Navarra by December 2016 (aged 13-14 years at that time) were included.
# Statistical analysis
We used IPW to determine whether missing data introduce bias when estimating weight status prevalence from EHRs. We fit a logistic regression model to find which variables were associated with "no weight and height measurements" for each age (between 6 and 14 years) and for two age intervals: 6-9 and 10-14 years. We included the following variables in the model: sex, weight status at 2-5 years, rural/urban residence, and annual household income.
To adjust for selection bias, a set of stabilized weights were computerized, with the probability of having complete data (measured at all ages and in the two age groups: 6-9 and 10-14 years) in the numerator and other variables associated with missing or complete data. The numerator was calculated directly from the data as the probability of being measured at the analyzed age. The denominator was computerized using logistic regression with complete data (yes/no) as the outcome and factors associated to missing data as independent variables. Robust variance estimate was used to calculate IPW. Mean standard deviation of weights was 0.09. Weighted proportions were calculated using the [pweight] statement.
## Sw ¼
f measuredà ð Þ f measuredÃjsex; weight status at 2−5yr; household income; rural=urban residence ð Þ *for each year from 6 to 14 years or for the 6-9 and 10-14 periods.
In addition to the IPW, we also used multiple imputation with the 6-9 and 10-14 age groups. An iterative Markov chain Monte Carlo method (STATA "mi" command) was applied. We generated 10 imputations for each missing measurement. The imputation models included sex, weight status at 2-5 years, rural/urban residence, and annual household income as predictors.
We calculated the prevalence for complete data, and IPW and MI estimated prevalence for each weight status at different ages and age groups.
All analyses were conducted with STATA, version 12.0 (Stata Corp).
# Results
Ninety-five percent and 88.9% of measured 2-to-5-year old children had one or more measurements at 6-9 and 10-14 years, respectively. Measurement status based on the characteristics of the children for age groups 6-9 and 10-14 years are shown in, as well as the results of the logistic regression analysis. At 6-9 years, being a girl (OR = 1.18 (95% CI: 0.99-1.42), being at risk of overweight (OR = 1.29 (95% CI: 1.02-1.63), and having an annual household income of 18,000 and < 100,000 € (OR = 1.70 (95% CI: 1.41-2.06) were associated with higher probability of being measured; while at ages 10-14 years the variables were being a girl (OR = 1.15 (95% CI:1.02-1.29) and having an annual household income of 18,000 to < 100,000 € (OR = 1.38 (95% CI: 1.22-1.57). On the other hand, belonging to a family with basic income (OR = 0.40 (95% CI: 0.31-0.54) and living in an urban area (OR = 0.79 (95% CI: 0.66-0.94) were associated with lower probability of being measured at the age of 6-9 years; being underweight (OR = 0.53 (95% CI:0.30-0.92) and belonging to a family with basic household income (OR = 0.57 (95% CI: 0.45-0.72) decreased the probability of being measured at the age of 10-14 years. Multivariate logistic regression model results for every age are shown in Additional file 1: Tables S1-S9. In ages where well-child visits were scheduled, the variable that was consistently and positively associated with having height and weight measurements was an annual household income from 18,000 to < 100,000 €. At ages at which well-child check-ups were not scheduled (7, 9, 10, 12, and 13) being overweight/obese at 2-5 years was positively associated with the existence of measurements in the EHR. and Additional file 1:show the prevalence of underweight, normal weight, overweight and obesity estimated for complete data in EHRs and adjusted for missing data using IPW for ages 6 to 14. and show the prevalence of weight status for age groups 6-9 and 10-14 estimated for complete data and adjusted for missing data using MI and IPW. As shown in the 95% CI between complete data and IPW prevalence completely overlap in all the estimations. At ages at which well-child visits were not scheduled in the Childhood Health Program (7, 9, 10, 12 and 13 years), the prevalence for complete data in EHRs overestimates the prevalence of obesity, although the differences were not statistically significant. When prevalence were calculated for longer age intervals (6-9 and 10-14 years), complete data, IPW and MI weight status estimators did not differ significantly. At 6-9 years complete data, IPW and MI obesity age-adjusted prevalence were 13.18% (95% CI: 12.54-13.85), 13.22% (95% CI: 12.57-13.89) and 13.02% (95% CI: 12.38-13.66) and at 10-14 years 8.61% (95% CI: 8.06-9.18), 8.62% (95% CI: 8.06-9.20) and 8.24% (95% CI: 7.70-8.78), respectively.
# Discussion
Our results show that weight and height data stored in primary care EHRs are highly valuable for childhood obesity surveillance in our population despite measurement under-representation for certain subpopulations. Non-measured children are more likely to belong to a family with basic income and being underweight at early age. On the other hand, being a girl, being at risk overweight in early age and having an annual household income ≥18,000 € increases the probability of being measured. Finally, missingness does not introduce significant selection bias for estimating weight status prevalence when the data is grouped by age range or at ages 6, 8, and 14 years at which the Childhood Health Program recommended measuring height and weight.
Thorough measurements of children's weight and height decreases with age, particularly ages at which there are no scheduled visits as established by the Navarra Childhood Health Program. The higher number of visits scheduled during the first years of the child's life and the fact that vaccines are administered in primary care centers until the age of six may explain the larger percentage of children under 3 years for whom weight and height measurements are available in comparison with older ages. The decrease in the number of health exams scheduled after the age of Weight status for ages 6 to 14 estimated for complete data in EHRs and adjusted for missing data using IPW three and the fact that vaccines are administered mostly at schools influences the observed decline at higher ages. Studies in which the prevalence of childhood obesity using EHRs have been assessed conclude that the use of this tool for obesity surveillance in children might be feasible but suggest that further studies are needed to determine the validity and the reliability of EHR data,particularly because at older ages there is a dramatic decrease of available measurements. In our population, a major concern was determining the representativeness of the weight status data extracted from the EHRs in the case of older children. We hypothesized that at older ages different characteristics such as sex, socioeconomic status, place of residence, and even the weight status could affect the probability of being measured. Consequently, weight status prevalence estimations obtained from complete data cannot necessarily be generalized to the population. We observed that children from poorer families and being boys, both factors associated with higher prevalence of obesity, were underrepresented in age ranges 6-9 and 10-14 years, which can result in an underestimation of obesity prevalence. On the other hand, two to five-year children being at risk of overweight or living in rural areas, which also have higher prevalence of obesity, are overrepresented in the EHRs. The fact that different selection bias are acting in opposite directions explains result similarities in complete data, IPW and MI Weight status for 6-9 and 10-14 years age groups estimated for complete data in EHRs and adjusted for missing data using IPW and MI Weight status for 6-9 and 10-14 years age groups estimated for complete data in EHRs and adjusted for missing data using IPW and MI. IPW: Inverse-probability weighting; MI: Multiple imputation prevalence estimators. It should also be noted that the highest differences between complete data and IPW overweight and obesity prevalence are seen at ages at which there were no scheduled Childhood Health Program visits (i.e., seven, nine, 10, 12, and 13 years). Furthermore, children at these ages with overweight/obesity are overrepresented probably because the pediatric team carries out additional measurements. When greater age ranges are used for weight status estimations, a large percentage of children are included in the calculations, and complete data and IPW and MI estimators that account for bias due to missing data were similar. For overweight and obesity prevalence estimations by age group (i.e., 6-9 and 10-14 years), measurement percentages (95.1 and 88.9%, respectively) are greater than those achieved by the National Study of Prevalence of Overweight and Obesity in Spanish Children in 2011 (75.5%). Within this context, a Swedish study aiming to find the variables associated to the low representation of children in a health survey observed that those of single parent families, foreign background, and poorer education and income were underrepresented in the study. All variables were associated with a risk of overweight and obesity, therefore, an adjustment of complete data estimations was necessary.
There is no increase in the probability of being measured at the age range 6-9 years of children with overweight or obesity at early years unlike those being at risk of overweight at 2-5 years. This can be partly explained by the fact that these children could be treated in specialized care, specifically in pediatric endocrinology. It can also indicate that parents of children with obesity miss well-care visits to avoid receiving advice on their children's weight, as some studies have pointed out. It has been shown that many healthcare providers hold strong negative attitudes and stereotyped beliefs toward people with obesity. Furthermore, considerable evidence suggests that such attitudes influence the perception of the individual and may lead to avoidance of care. Other characteristics, such as children of emigrants -not considered in this study-and are related with the prevalence of obesity, may help explain this finding.
In a work that is similar to the one presented here, Funk et al.conducted a study to assess the feasibility of using BMI from EHRs to estimate overweight and obesity prevalence and compare it to data from the National Health Survey and used IPW to adjust the prevalence. The authors found resemblances in the estimation of crude and adjusted prevalence to those of the National Health Survey, concluding that EHRs might be the ideal tool to identify and target patients with obesity and implement public health interventions. Moreover, a study conducted in the UK provides proof-of-concept for the use of primary care EHRs to assess children's obesity in England, suggesting it is a valuable resource for monitoring obesity trends.
A cross-sectional survey conducted in the Basque Country in 2015, a region bordering with Navarra, directly measured weight and height and found that overweight and obesity prevalence in children between 6 and 9 years seems to be quite similar (22.90 (95% CI: 19.8-26.0) and 11.27 (95% CI: 8.8-13.5), respectively) to those obtained in Navarra from the EHRs (23.70 (95% CI: 22.89-24.53) and (13.22 (95% CI: 12.57-13.89)). These data are in line with the pattern observed in the 2006-2007 National Health Survey for these two regions.
Unlike in other countries, primary care EHRs in Navarra include well-child visit measurements that contribute significantly to reduce biases and increase the reliability of weight status prevalence estimations. Another strength is the widespread use of EHRs by pediatric teams. Finally, the fact that most of the infant population is included in this single EHR ensures the generalizability to the total population.
There are some potential limitations regarding EHR data. On the one hand, the introduced information involves a number of different healthcare professionals, and therefore, there may be some variability regarding the measurement method. Another limitation is that the professionals manually enter weight and height values and this is less reliable than the data entered for research, where there is double data entry and is subjected to validity checks. To avoid implausible values, z-scores values ≤5 SD and ≥ 5 SD were excluded. A strength of EHR data is the large number of children included; this allows obtaining estimations for large and small areas, as well as for all age groups. In Spain, few national surveys provide estimations for children between 6 and 9 years; these national studies allow data comparisons against other European studies, but not estimations for certain Spanish regions due to the low number of subjects.
Some authors believe that the best place for measuring height and weight is in primary care settings during wellchild visits, ensuring the appropriate equipment is used, continuous training of the staff, and the use of measurement protocols. Furthermore, these settings minimize unintended negative consequences associated to monitoring growth in other environments (e.g., schools), such as stigmatization.
Another limitation of this study is that certain variables potentially associated to a lack of measurements, such as ethnicity or mono-parental status, among others, were not available.
# Conclusions
Missing height and weight data in the EHR did not introduce significant selection bias in weight status estimations for the 6-9 and 10-14 year age groups. Readily available EHR data could be a valid tool for surveillance of weight status in our population. Therefore, in our population, EHRs are a potentially cost-effective, emerging tool for public health surveillance.
## Additional file
Additional file 1:. Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 6 years according to baseline characteristics.. Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 7 years according to baseline characteristics.. Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 8 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 9 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 10 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 11 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 12 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 13 years according to baseline characteristics. . Odds Ratio and 95% confidence interval (OR 95%CI) of complete data at 14 years according to baseline characteristics.. Complete data and IPW adjusted prevalence and 95% confidence interval for every age between 6 to 14 years.
Abbreviations BMI: Body mass index; CI: Confidence intervals; EHRs: Electronic health records; ICPC-2: International Classification of Primary Care, 2nd Edition; IPW: Inverse-probability weighting; MI: Multiple Imputation; SD: Standard deviation; WHO: World Health Organization |
Women receiving massive transfusion due to postpartum hemorrhage: A comparison over time between two nationwide cohort studies
| on behalf of the TeMpOH-1 study group*This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
## | introduc ti on
Postpartum hemorrhage (PPH) is a serious obstetric complication and a major contributor to maternal morbidity and mortality worldwide. [bib_ref] Global, regional, and national levels and causes of maternal mortality during 1990-2013:..., Kassebaum [/bib_ref] Its incidence seems to be increasing in high-income countries accompanied by increasing rates of severe adverse outcomes. [bib_ref] Trends in postpartum hemorrhage in high resource countries: a review and recommendations..., Knight [/bib_ref] In obstetrics, massive transfusion (defined as 8 or more units of packed red blood cells transfused) after birth is associated with high rates of morbidity and hysterectomy. [bib_ref] management and outcome of women requiring massive transfusion after childbirth in the..., Ramler [/bib_ref] [bib_ref] The epidemiology and outcomes of women with postpartum haemorrhage requiring massive transfusion..., Green [/bib_ref] Incidence of massive transfusion due to PPH was notably high in the Netherlands per 100 000 births). [bib_ref] management and outcome of women requiring massive transfusion after childbirth in the..., Ramler [/bib_ref] [bib_ref] The epidemiology and outcomes of women with postpartum haemorrhage requiring massive transfusion..., Green [/bib_ref] [bib_ref] Massive blood transfusion during hospitalization for delivery, Mhyre [/bib_ref] A nationwide study based on the national perinatal database in the Netherlands showed an increased incidence of PPH (defined as ≥ 1000 mL blood loss following the first 24 h after birth) between 2004 and 2013 (from 4.1% to 6.4% of women giving birth), but a decreased incidence of any number of obstetric-related transfusion of packed red blood cells (from 23% to 3.9% of all women with PPH). [bib_ref] Increasing incidence of postpartum hemorrhage: the Dutch piece of the puzzle, Van Stralen [/bib_ref] It is unknown whether the number of women receiving massive transfusion due to PPH followed this same decreasing pattern. Assessing such a pattern and discerning possible differences over time in incidence, causes, management and outcome of PPH leading to massive transfusion could help to evaluate maternity care. Moreover, identifying antepartum risk factors may also raise awareness for women at high risk of receiving massive transfusion after birth.
The aim of this study was to describe incidence, causes, management and outcome of women who received massive transfusion due to PPH in the Netherlands between 2011 and 2012 and compare these with the same parameters previously described in the
## | material and me thods
## | study design
We performed a secondary analysis of women who received massive transfusion due to PPH as part of the Transfusion strategies in women during Major Obstetric Hemorrhage study (TeMpOH-1). TeMpOH-1 is a nationwide retrospective cohort study in 61 hospitals in the Netherlands (71% of all hospitals in the country at the time) that collected data from women of at least 18 years old, who received 4 units of packed red blood cells or any transfusion of fresh frozen plasma and/or platelets in addition to packed red blood cells because of obstetric hemorrhage (≥ 1000 mL blood loss during pregnancy or during the first 24 h following childbirth) between 1 January 2011 and 1 January 2013.
## | population
For the present analysis, women were selected from the TeMpOH-1 cohort who had experienced PPH and received massive transfu-
## | data collection
## | statistical analyses
All statistical analyses were performed with IBM SPSS StatisticsGiven that the PRN database only has summary denominator data, odds ratios (OR) were calculated by means of univariate logistic regression models, resulting in crude odds ratios with 95% CI.Women with missing values for a specific parameter were excluded from analyses that required that parameter. Furthermore, the number of births of the TeMpOH-1 cohort comprised women who gave birth under the guidance of obstetricians, but did not include women with low-risk pregnancies who had given birth under guidance of their midwives or family physicians (primary care), which represented about 29% of all births in the Netherlands between 2011 and 2012.To estimate a population-based incidence of massive transfusion due to PPH, the number of births in the TeMpOH-1 study was multiplied by 100/71 to represent all births, including those under guidance of primary care.
# | ethical approval
The TeMpOH-1 study was approved by the ethics committee of the Leiden University Medical Center on 31 January 2013 (P12.273) and by the institutional review board of each participating hospital. The study was registered in the Netherlands Trial Register (NTR4079). Need to obtain informed consent was waived by the ethics committee.
## | re sults
## | characteristics of women and bleeding
Women who received massive transfusion due to PPH had a median (IQR) age of 32 years (29-37 years), BMI of 23 kg/m 2 (21-26 kg/ m 2 ) and a gestational age of 39 weeks (37-40 weeks). The characteristics of women, pregnancy and birth are presented in and juxtaposed to the characteristics of women who experienced PPH and received massive transfusion between 2004 and 2006 in the Netherlands and to the Dutch general pregnant population in 2012. [bib_ref] management and outcome of women requiring massive transfusion after childbirth in the..., Ramler [/bib_ref] [bib_ref] Mode of delivery after previous cesarean section in the Netherlands, Schoorel [/bib_ref] [bib_ref] Pre-eclampsia increases the risk of postpartum haemorrhage: a nationwide cohort study in..., Von Schmidt Auf Altenstadt [/bib_ref] The median (IQR) estimated blood loss was 6000 mL (4500-8000 mL). . Uterine atony also remained a leading cause when causes were grouped by the total units of packed red blood cells transfused:
## | risk factors and causes of hemorrhage
"moderate" (8-12 units, n = 113), "high" (13-20 units, n = 49) and "immense" (≥ 20 units, n = 14).
## | management of pph
## | adverse maternal outcome
Of all women, 146 (83%) were admitted to an intensive care unit and 53 (30%) underwent hysterectomy as a last resort to stop bleeding.
Four women died (three due to exsanguination caused by uterine atony and one due to liver capsule rupture accompanied by uterine atony), of whom two died after hysterectomy. Case fatality rate of PPH with massive transfusion was one in 44 women (2.27%) and
case fatality rate of women who underwent peripartum hysterectomy due to major PPH was two in 53 women (3.77%).
## | d iscuss i on
The incidence of massive transfusion due to PPH decreased in the it must be noted that these numbers can be influenced by clinical decision making. There is still no definite consensus in the literature as to which definition of massive transfusion should be applied. [bib_ref] Toward a better definition of massive transfusion: focus on the interval of..., Sharpe [/bib_ref] [bib_ref] The definition of massive transfusion in trauma: a critical variable in examining..., Mitra [/bib_ref] [bib_ref] Update on massive transfusion, Pham [/bib_ref] Furthermore, number of births used as denominator for calculation of incidence of massive transfusion due to PPH was estimated by multiplying the number of hospital births in the TeMpOH-1 study by the proportion of births under guidance of primary care. However, since coverage of the PRN registry was about 99% of all births in the Netherlands and all women with hemorrhage receiving massive transfusion will eventually have been referred to a maternity unit, we believe that this estimate of incidence is reliable. 8
The decreased incidence of PPH leading to massive transfusion along with an observed increase in median blood loss reflects the more restrictive blood transfusion policy in the Netherlands over time. [bib_ref] Increasing incidence of postpartum hemorrhage: the Dutch piece of the puzzle, Van Stralen [/bib_ref] This gradual change followed, among other things, after the introduction of the "4-5-6 rule" (depending on the presence of comorbidity, the threshold for transfusion of packed cells varies be- [bib_ref] management and outcome of women requiring massive transfusion after childbirth in the..., Ramler [/bib_ref] This is known to be associated with increased risks of placenta previa, abnormally invasive placenta and uterine rupture during subsequent pregnancies. [bib_ref] Maternal morbidity associated with multiple repeat cesarean deliveries, Silver [/bib_ref] [bib_ref] Etiology and risk factors for placenta previa: an overview and meta-analysis of..., Faiz [/bib_ref] [bib_ref] Impact of multiple cesarean deliveries on maternal morbidity: a systematic review, Marshall [/bib_ref] Furthermore, studies showed that the risk of peripartum hysterectomy increased with the number of previous cesarean births and in the presence of an abnormally invasive placenta. [bib_ref] Prevalence, indications, risk indicators, and outcomes of emergency peripartum hysterectomy worldwide: a..., Van Den Akker [/bib_ref] [bib_ref] Emergency postpartum hysterectomy for uncontrolled postpartum bleeding: a systematic review, Rossi [/bib_ref] [bib_ref] Cesarean section and the risk of emergency peripartum hysterectomy in high-income countries:..., De La Cruz [/bib_ref] Other countries have reported increasing previous cesarean birth rates with an increasing trend in the incidence of placenta previa, abnormally invasive placenta and uterine rupture, which could suggest that more countries experience the high burden of hysterectomy due to PPH with massive transfusion. [bib_ref] Uterine rupture: trends over 40 years, Stray-Pedersen [/bib_ref] [bib_ref] Trends and recurrence of placenta praevia: a population-based study, Roberts [/bib_ref] [bib_ref] Abnormally invasive placenta: changing trends in diagnosis and management, Guleria [/bib_ref] Nevertheless, the hysterectomy rate in the the Netherlands re- Gynaecologists specifically recommends to "resort to hysterectomy sooner rather than later" without explicitly mentioning the option of trying embolization first. [bib_ref] Prevention and management of postpartum haemorrhage: Green-Top Guideline No. 52, Mavrides [/bib_ref] This difference could lead to a more restrictive policy of peripartum hysterectomy in the Netherlands.
Furthermore, the incidence of PPH also depends on prevalence of risk factors in the population. Our findings confirm previous observations that increased maternal age (≥ 35 years), previous cesarean birth, multiple pregnancy, pre-eclampsia, labor induction, and instrumental or cesarean birth are associated with major PPH. [bib_ref] Massive blood transfusion during hospitalization for delivery, Mhyre [/bib_ref] [bib_ref] Pre-eclampsia increases the risk of postpartum haemorrhage: a nationwide cohort study in..., Von Schmidt Auf Altenstadt [/bib_ref] [bib_ref] Incidence, risk factors, and temporal trends in severe postpartum hemorrhage, Kramer [/bib_ref] [bib_ref] Postpartum hemorrhage with transfusion: trends, near misses, risk factors and management at..., Marocchini [/bib_ref] These antepartum risk factors were only present in a certain number
## | con clus ion
Incidence of PPH leading to massive transfusion decreased in the Netherlands, but continues to be an important cause of maternal morbidity and mortality. Improved continuous registration of major obstetric hemorrhage with confidential enquiries to identify substandard factors could lead towards better maternal care and may prevent morbidity and mortality from major PPH in the Netherlands.
International comparison of our findings could provide high-quality evidence for the best management practices of major PPH.
[fig] 3: Causes of PPH in women who received massive transfusion were further analyzed by mode of birth and the number of packed red blood cells transfused, using the same cut-off points described by Green et al 4 in the UK: "moderate" (8-12 units of packed red blood cells), "high" (1-20 units of packed red blood cells) and "immense" (> 20 units of packed red blood cells). Adverse maternal outcome was defined as the need for hysterectomy, admission to an intensive care unit and/ or maternal death. [/fig]
[fig] TheF: TeMpOH-1 source population comprised 270 101 births (including births under guidance of primary care) in the Netherlands during the 2-year inclusion period. A total of 176 women experienced PPH and received transfusion of 8 or more units of packed red blood cells, making the incidence of massive transfusion due to PPH in the Netherlands 65 per 100 000 births (95% CI 56-I G U R E 1 Incidence of women requiring massive transfusion due to postpartum hemorrhage. *Defined massive transfusion as ≥10 packed red cells and included all pregnancy-realted hemorrhage 5 [Color figure can be viewed at wileyonlinelibrary.com] [/fig]
|
Malignant renal schwannoma in a cat
A nine-year-old male European shorthair cat with rapidly enlarging mass at the left kidney doubted to be malignant was presented. The purpose of this study is to present the clinical, radiological and pathological findings of a primary renal tumor in the cat. Grossly, the mass mostly encapsulated the kidney. Histologically, excisional biopsy showed worrying histological features. A sarcoma-like tumor composed mainly of neoplastic spindle-shaped cells. Neoplastic nodules of aggregations of fusiform cells arranged in multidirectional bundles. Immunohistochemically, several immunohistochemical satins (melan-A, S-100, vimentin, actin, desmin, cytokeratin, neurofilament, melan-A, NSE, synaptophysin, chromogranin, Glial Fibrillary Acidic Protein GFAP, Collagen IV and CD99) were used to differentially diagnose the mass. The stained neoplastic sections positively tested to S-100, but negative to the other aforementioned immunohistochemical stains. Immunohistochemistry with S-100 antibody staining showed an unusually strong positive reaction throughout the tumor cells. Based on our comparative diagnosis relative to other tumors, in addition to the progressive clinical signs, histopathological and immunohistochemical results, this case was presumptively diagnosis as a malignant schwannoma. According to our investigation of the relevant literature, this study of malignant renal Schwannoma (malignant peripheral nerve sheath tumor) is a highly rare case not previously characterized in a cat.
# Introduction
There are a few reports of schwannomas in cats: in the oral cavity , in the head [bib_ref] Malignant peripheral nerve sheath tumor in a cat, Watrous [/bib_ref] , in the forelimb [bib_ref] Of all the nerve! A subcutaneous forelimb mass on a cat, Tremblay [/bib_ref] , and in the eye . Primary renal tumors are uncommon in domestic animals, so that only 12% of renal tumors are primary, while 88% are secondary. In general, 99% of renal tumors are malignant, 77% of which are epithelial and 23% mesenchymal in origin. Renal tumors of neural origin are extremely rare [bib_ref] Tumors of the urinary system, Meuten [/bib_ref]. According to our survey, only one case of renal tumor from neural origin has been reported in a cat [bib_ref] Nerve sheath tumours in the dog and cat, Jones [/bib_ref]. Schwannoma is a tumor that arises from the Schwann's cells that are present among the cell types that form the nerve sheath. Malignant Schwannoma is part of a larger group of rare malignant peripheral nerve sheath tumors (MPNSTs) which are also called soft tissue sarcomas or neurofibrosarcomas.
Case details A 9-year-old male European shorthair male, castrated, cat was referred for clinical investigation. Initial diagnosis showed that the cat suffered from loss of appetite and low water intake with abdominal distension.
Radiography and ultrasonography investigations revealed a mass approximately 15 cm in diameter within the left kidney. The cat was admitted for nephrectomy and the excised specimen was submitted for histopathology investigation at the Institute for Veterinary Pathology, Justus-Liebig-University, Giessen, Germany. No information on the cat after performing surgery. The kidney together with the tumor mass were fixed in neutral buffered 10% formalin solution. A specimen from the tissue was processed, embedded in paraffin, sectioned at 4 m thickness and stained with Haematoxylin and Eosin (H&E). Other sections were stained with Periodic Acid Schiff Stain (PAS), Azan trichrome stain and Warthin-Starry (WS) stain. Immunohistochemistry was performed using polyclonal rabbit anti-S-100 antibody (Z0311 DAKO Co.). Anti-S-100 has strong tendency to positively react with most melanocytic tumors, Schwannomas, ependymomas, astrogliomas and glioblastomas, and sometimes with salivary gland tumors. Along with anti-S-100, and for differential diagnosis, the sample was stained with other antibodies for Neurofilaments, Glial fibrillary acidic protein (GFAP), Melan-A, NSE, Synaptophysin, Chromogranin (using the ABC method) and CD99 (monoclonal antibody 12E7 raised against the MIC2 protein).
http: , Vol. 7(3): 214-220 ________________________________________________________________________________________________________
## 215
The gross examination, of formalin fixed kidney, showed a singular, roughly spherical encapsulated tumor mass in the left kidney. The mass was about 5.5 x 4.5 x 4 cm superficially nodular, greyish-white in color, and greasy in texture. The tumor was detected at one pole of the kidney. The tumor is well demarcated and distinguished from the normal renal tissue; it encapsulates the outside of the kidney and infiltrates inside the renal pelvis but it does not expand to the renal parenchyma [fig_ref] Figure 1: Growth of tumor mass around and inside the hemisected kidney [/fig_ref]. The left adrenal gland could not be located. Right kidney was normal. Based on mass location and size, this tumor might be a primary tumor. Excised specimen was sent for histopathological examination which demonstrates thick capsules with loose tissues in some parts. Most of the neoplastic cells were arranged in dense bundles with increased cellularity like Antoni A areas of a classic schwannoma, and some in loosely arranged streams of cells like Antoni B [fig_ref] Figure 2: Hematoxylin and eosin stained kidney tissue section reveals Neoplastic cells arranged in... [/fig_ref]. In other parts of the tumor, single-cell groups were distributed as a fibro-vascular stroma which appeared to be mostly myxomatous. The neoplastic cells appeared spindle-like or oval in shape with indistinct borders. The cytoplasm was eosinophilic with a moderate amount of PAS-positive fine granules. The nuclei, which appeared at the center as oval, elongated or spindle-like in shape, were hyperchromatic and finely stippled. The nucleoli were mostly singular and basophilic. Mitotic activity ranged from 3 to 5 per high power field (HPF). WS stain showed no evidence of external lamina to confirm the diagnosis of Schwannoma. The Azan stain showed a low to moderate amount of collagen fibres between the tumor cells. Renal tissue invasion, necrosis and hemorrhaging were evidence of malignancy. There were heterologous elements in form of cartilaginous islets [fig_ref] Figure 3: Hematoxylin and eosin stained kidney tissue section shows spindle and oval Neoplastic... [/fig_ref]. Additionally, a mild multifocal lymphocytic interstitial nephritis was noted. Immunostaining showed that all tumor cells were strongly positive for anti-S-100 polyclonal antibodies [fig_ref] Figure 4: Immunohistochemistry [/fig_ref] and negative for Melan-A, Vimentin, actin, desmin, cytokeratin, neurofilament, melan-A, NSE, synaptophysin, chromogranin, Glial Fibrillary Acidic Protein GFAP, Collagen IV and CD99.
# Discussion
In such cases, morphological appearance might point at carcinoma as the primary suspect. Histological examination as well as immunohistochemistry, however, opposed this indication. Negative reaction with cytokeratin in particular excluded epithelial origin for this tumor. On the other hand, S-100 protein is normally expressed in cells originating from the neural crest (Schwann's cells, and melanocytes), as well as myoepithelial cells, chondrocytes, macrophages, adipocytes, keratinocytes, Langerhans cells, [bib_ref] CD1a and S100 antigen expression in skin Langerhans cells in patients with..., Wilson [/bib_ref] , dendritic cells, and some breast epithelial cells [bib_ref] Differentiation of Langerhans cells from interdigitating cells using CD1a and S-100 protein..., Shinzato [/bib_ref]. According to the literature, the expression of S-100 proteins varies between tumor types: 100% of Schwannomas and 100% of neurofibromas (with considerably lower stain intensity in neurofibromas than in schwannomas) 70-90% of melanomas and 50% of malignant peripheral nerve sheath tumors, paraganglioma stromal cells, histiocytoma and clear cell sarcomas [bib_ref] Differential expression of S100 protein subtypes in malignant melanoma, and benign and..., Nonaka [/bib_ref]. Immunohistochemistry showed negative reaction with antibodies to Melan-A, which is normally expressed by melanocytes. The negative expression of Melan-A excluded melanoma as a candidate and limited the diagnosis to a tumor of neural origin. In using anti-S-100 as an antibody indicator of neural origin, it is important to note that antibodies to S-100 family proteins react with many neural tumors outside the central nervous system, such as:
1-Schwannomas [bib_ref] The enigmatic origin of olfactory schwannoma, Sabel [/bib_ref] [bib_ref] Different patterns of DNA copy number changes in gastrointestinal stromal tumors, lelomyomas,..., Sarlomo-Rikala [/bib_ref]. 2-Neurofibroma [bib_ref] Occult neurofibroma and increased S100 protein in the skin of patients with..., Karvonen [/bib_ref]. 3-Perineurioma: Fletcher (2007) stated that a small minority of perineuriomas show focal S-100 positivity. However, [bib_ref] Perineurial cell tumor, Weidenheim [/bib_ref] , [bib_ref] World Health Organization classification of tumors, Kleihues [/bib_ref] , [bib_ref] Perineurioma: a clinicopathological study of eight cases, Rankine [/bib_ref] and reported that perineuriomas were negative for S-100 proteins. 4-Melanocytic tumors [bib_ref] S100 protein: a marker for human malignant melanomas?, Gaynor [/bib_ref].
## 5-adrenal oncocytic pheochromocytoma:
Fletcher (2007) stated that only sustentacular cells within pheochromocytomas showed positive reaction with antibodies to S-100 proteins. However, [bib_ref] Oncocytic adrenocortical neoplasms: a report of seven cases and review of the..., Lin [/bib_ref] and [bib_ref] Adrenal oncocytic neoplasm: a systematic review, Mearini [/bib_ref] reported that Pheochromocytomas showed negative reaction with antibodies to S-100 proteins. 6-Liposarcomas [bib_ref] Liposarcoma arising in uterine lipoleiomyoma: a report of 3 cases and review..., Mcdonald [/bib_ref]. 7-Synovial sarcomas [bib_ref] Comparison of immunohistochemical labelling of melanocyte differentiation antibodies melan-A, tyrosinase and HMB..., Olsen [/bib_ref]. 8-Chondrosarcomas (rarely, and express cytokeratin) had positive reaction with antibodies to S-100 according to [bib_ref] Brachyury, SOX-9, and podoplanin, new markers in the skull base chordoma vs..., Oakley [/bib_ref] , but negative reaction according to [bib_ref] Mesenchymal chondrosarcoma. An immunohistochemical study, Swanson [/bib_ref]. 9-Ossifying fibromyxoid tumor of soft parts [bib_ref] Ossifying fibromyxoid tumor of soft part--a clinicopathologic and immunohistochemical study of 104..., Miettinen [/bib_ref]. According to the WHO classification of neural origin tumors, Schwannomas diffusely, strongly and uniformly react with antibodies to S-100 protein, vimentin, and GFAP. In this particular case, S-100 staining was strongly and diffusely positive but vimentin and GFAP staining were negative. Ultrastructure of external lamina usually persists, in an attenuated and interrupted form, adjacent to cells of malignant Schwannomas. External lamina tends to be absent in Schwannomas . In this tumor, the external lamina was not clear and contained sporadic remnants. As a result of the mutual expression of S-100 proteins by a variety of neural tumors to different degrees, and due to the absence of conclusive evidence, differential diagnosis was conducted to investigate and compare potential candidates with Schwannoma mainly based on the expression of S-100 and distinctive histopathological features: Neurofibromas exhibit less reactivity (40%) with S-100 antibody in comparison with Schwannomas, which react 100% with S-100 antibody. Neurofibromas were categorized into neuro and fibroblastic sub-tumor regions. Neuro components express only 40% of the tissue stained with S-100 antibody whereas the fibroblastic component does not express S-100 and reacts with antibodies to vimentin . In our study, cells were 100% S-100 positive. Microscopically, neurofibroma cells are elongated and arranged in interlacing bundles. Based on differential histological test and S-100 immune reaction, neurofibroma was a weak candidate for this case study. Perineurioma is an uncommon nerve sheath tumor expressing epithelial membrane antigen (EMA). Therefore, most Perineuriomas stain positively for EMA, Claudin-1, vimentin and collagen [bib_ref] World Health Organization classification of tumors, Kleihues [/bib_ref] but negatively for S-100 protein and cytokeratin [bib_ref] Soft-tissue perineurioma: evidence for an abnormality of chromosome 22, criteria for diagnosis,..., Giannini [/bib_ref] [bib_ref] Reticular perineurioma: a distinctive variant of soft tissue perineurioma, Van Roggen [/bib_ref]. Although some other previously mentioned research studies stated that perineurioma cells do not express S-100 proteins, Fletcher (2007) reported that a small minority of cases may show positive reaction with S-100. Histologically, perineurioma has been categorized into two types: intraneural and soft tissue perineuroma. Perineurioma cells appear well-differentiated and spindle-like in shape with stretched nuclei and eosinophilic cytoplasm. Perineuriomas are characterized by parallel cell arrangement to form concentric whorls known as onion bulbs [bib_ref] Hybrid schwannoma/perineurioma: clinicopathologic analysis of 42 distinctive benign nerve sheath tumors, Hornick [/bib_ref] , Vol. 7(3): 214-220 ________________________________________________________________________________________________________ This feature does not appear in the histological examination for this case. Both differences in histological appearance and the controversial reaction with S-100 antibody gave considerable reason for eliminating Perineurioma tumor in this case. Malignant melanoma was initially a strong suspect due to the positivity of the tumor for S-100. For specificity, we applied immune staining using an antibody to Melan-A, which is a melanoma-specific marker [bib_ref] A comparative review of melanocytic neoplasms, Smith [/bib_ref] for which more than 67% of feline melanomas are cytoplasmatic positive [bib_ref] Melan A and S100 protein immunohistochemistry in feline melanomas: 48 cases, Ramos-Vara [/bib_ref]. Our case was negative for expression of Melan-A. Ossifying fibromyxoid tumor of soft parts (OFMT) is an extremely rare tumor of bone and soft tissue [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic, proteomic and genomic study, Graham [/bib_ref]. Although our survey of the literature showed no previous report of OFMT in domestic animals, previous studies in human revealed different expression levels of S-100 by ossifying fibromyxoid tumor cells: 73% [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic, proteomic and genomic study, Graham [/bib_ref] , 60% [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic study of 70 cases..., Folpe [/bib_ref] and 53% [bib_ref] Recurrent rearrangement of the PHF1 gene in ossifying fibromyxoid tumors, Gebre-Medhin [/bib_ref]. These expression levels were considerably lower in comparison with our study, in which the degree of expression of S-100 was unusually high, with almost 100%. Furthermore, our IHC results showed no expression of desmin which had been reported 38% in [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic, proteomic and genomic study, Graham [/bib_ref] , 13% [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic study of 70 cases..., Folpe [/bib_ref] and 82% in OFMT [bib_ref] Recurrent rearrangement of the PHF1 gene in ossifying fibromyxoid tumors, Gebre-Medhin [/bib_ref]. Histologically, OFMT presents as a multi lobular growth consisting of nests of spherical-to-ovoid cells embedded in fibromyxoid stroma [bib_ref] Recurrent rearrangement of the PHF1 gene in ossifying fibromyxoid tumors, Gebre-Medhin [/bib_ref]. It is also characterized by atypical necrosis and mitosis with high cellular activity rates >2 MF/50 HPF [bib_ref] Ossifying fibromyxoid tumor of soft parts: a clinicopathologic study of 70 cases..., Folpe [/bib_ref]. According to the known histological features of OFMT and the IHC results (S-100 and desmin) obtained in this study, OFMT was likely not the correct tumor in this case. However, theoretically, OFMT could not be completely excluded from the comparison with Schwann's cell tumors. Adrenal oncocytic pheochromocytoma positively expresses chromogranin, synaptophysin, neuronspecific enolase, neurofilament, serotonin, bombesin, ACTH, vimentin, desmin, S-100 protein, and cytokeratins including AE1/3, CAM 5.2, cytokeratin 7, and cytokeratin 20. [bib_ref] Adrenal oncocytic pheochromocytoma, Li [/bib_ref]. Gross appearance examination of our case showed that the tumor originated from the kidney pelvis whereas Pheochromocytoma originates from the medulla of the adrenal gland. In addition, according to our observations, Oncocytic pheochromocytoma usually stains with yellow in formalin fixation. This phenomenon was not observed in our case. Liposarcomas express S-100 in fat cells and lipoblasts. PAS positive elements might be seen as well in some adipocytes and lipoblasts, which positively react with anti-vimentin but vary in intensity and might not react in poorly differentiated lesions due to lack of expression. Transition from low to high grade nonlipogenic morphology can occur within a welldifferentiated liposarcoma. Heterologous elements occur in 5-10% of cases with myogenic, osteo/chondrosarcomatous, or rarely angiosarcomatous elements. Myxoid liposarcoma [bib_ref] Myxoid tumours of soft tissue, Van Roggen [/bib_ref] and spindle cell liposarcoma were both reported as S-100 positive. In our case, no adipose cell differentiation was observed, and the diagnosis of liposarcoma can be easily excluded based on the histological appearance. Diagnosis of schwannomas is based on presence of some features, including: (1) Antoni Type A or B histologic feature . (2) Round cells with few cytoplasmic components, enclosed by a basal lamina . (3) Positive immunohistochemical staining to S-100 of neuronal origin [bib_ref] S-100 protein in methyl-and ethylnitrosourea induced tumors of the rat nervous system, Wechsler [/bib_ref] [bib_ref] Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other..., Vinores [/bib_ref]. Generally, discrimination between benign and malignant schwannoma is sometimes challenging; since both kinds have comparable morphological features . Malignant schwannoma is usually densely packed arranged in patterns with high mitotic range [bib_ref] Malignant epithelioid schwannoma affecting the trigeminal nerve of a dog, Pumarola [/bib_ref]. Whereas, benign schwannoma usually is small in size, grows superficially with low mitotic activities [bib_ref] Benign epithelioid schwannoma, Kindblom [/bib_ref]. Although positive immunoreactivity for S-100 is an indication of benign and most malignant Schwannomas [bib_ref] Histopathologic and immunohistochemical study of malignant tumors of peripheral nerve sheath (malignant..., Matsunou [/bib_ref] , cells in this tumor showed malignant characteristics in histological examination, e.g. Antoni A and B patterns and neoplastic cells with different sizes and shapes.
Conclusion From these comparisons, which were conducted based on the strong expression of S-100 and from the exclusion of the aforementioned tumors, the findings of our investigation matched malignant schwannoma as the most likely diagnosis in this case. Unlike these malignant tumors, cellular schwannoma reveals a strong and diffuse reactivity for S-100. [fig_ref] Figure 2: Hematoxylin and eosin stained kidney tissue section reveals Neoplastic cells arranged in... [/fig_ref] clearly shows two different groups of cells (Antoni patterns A and B); a looser area with low density of cells and interwoven dense cells with clear myxoid changes. Antoni patterns, in this case, may be a good indicator for malignant schwannoma versus benign schwannoma. In addition, and in comparison with similar studies [bib_ref] Malignant, solitary, nasopharyngeal schwannoma in a cow, Mandrioli [/bib_ref] , histomorphological and immunohistochemical means helped in reaching that malignant schwannoma is the final diagnosis in our case.
[fig] Figure 1: Growth of tumor mass around and inside the hemisected kidney; tumor mass, in light brownish color, is encapsulating most of the kidney (C), infiltrating inside the pelvis (B) without reaching the yellowish medulla and cortex (A). [/fig]
[fig] Figure 2: Hematoxylin and eosin stained kidney tissue section reveals Neoplastic cells arranged in bundles and in streams (100x). The black arrow points at a dense area (Antoni A) and the blue arrow points at a looser area (Antoni B). [/fig]
[fig] Figure 3: Hematoxylin and eosin stained kidney tissue section shows spindle and oval Neoplastic cells with cartilaginous islets at the middle (400x). [/fig]
[fig] Figure 4: Immunohistochemistry (IHC) shows tumor cells are diffusely immunoreactive for S-100 antibody (dark brown). (40x). The dark brown area is an indication for a positive reaction for S-100 antibody (A), where (B) indicates the negative reaction. [/fig]
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Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in patients with immunocompromising conditions: a review of available evidence
Immunocompromising conditions increase the risk of invasive pneumococcal disease (IPD). Vaccine uptake in patients with these conditions may be low in part because of concerns about decreased immunogenicity and safety in these high-risk groups. We conducted a literature search to identify publications describing antibody responses to 13-valent pneumococcal conjugate vaccine (PCV13) in immunocompromised individuals recommended for PCV13 vaccination by the US Advisory Committee on Immunization Practices (ACIP). This review summarizes immunogenicity data from 30 publications regarding the use of PCV13 comprising 2406 individuals considered at high risk for IPD by the ACIP. Although antibody responses to PCV13 in individuals with immunocompromising and high-risk conditions were variable and generally lower compared with healthy controls, the vaccine was immunogenic and was largely well tolerated. Based on these findings, concerns regarding immunogenicity and safety of PCV13 are not supported and should not be barriers to vaccination in high-risk populations.ARTICLE HISTORY
# Introduction
Streptococcus pneumoniae is a major cause of serious diseases, including meningitis and pneumonia. [bib_ref] Streptococcus pneumoniae colonisation: the key to pneumococcal disease, Bogaert [/bib_ref] Individuals with weakened immune systems are at higher risk for pneumococcal infection, associated hospitalizations, and related mortality compared with healthy individuals. [bib_ref] Risk of underlying chronic medical conditions for invasive pneumococcal disease in adults, Baxter [/bib_ref] [bib_ref] Risk of pneumococcal disease in children with chronic medical conditions in the..., Pelton [/bib_ref] [bib_ref] Rates of pneumococcal disease in adults with chronic medical conditions, Shea [/bib_ref] [bib_ref] Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs, Shigayeva [/bib_ref] [bib_ref] The effect of underlying clinical conditions on the risk of developing invasive..., Van Hoek [/bib_ref] Some estimates show over one quarter of all cases of invasive pneumococcal disease (IPD) occur in immunocompromised individuals, with the highest risk occurring among individuals with hematological malignancies. [bib_ref] Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs, Shigayeva [/bib_ref] Studies have shown that individuals with multiple myeloma (MM) have the highest risk for IPD compared with individuals with other immunocompromising conditions, with the risk reported to be up to 176 times greater in patients with MM compared with immunocompetent individuals. [bib_ref] Risk of underlying chronic medical conditions for invasive pneumococcal disease in adults, Baxter [/bib_ref] [bib_ref] Risk of pneumococcal disease in children with chronic medical conditions in the..., Pelton [/bib_ref] [bib_ref] Rates of pneumococcal disease in adults with chronic medical conditions, Shea [/bib_ref] [bib_ref] Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs, Shigayeva [/bib_ref] Individuals with sickle cell disease and chronic renal failure on dialysis have also been shown to be at 25 and 19 times greater risk of IPD than immunocompetent individuals, respectively. [bib_ref] Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs, Shigayeva [/bib_ref] Another study estimated that adults with HIV infection had an IPD incidence 49 times higher than individuals without HIV; moreover, infection reoccurrence is up to 9 times more frequent in HIV-infected individuals. [bib_ref] Active bacterial core surveillance program of the emerging infections program network. The..., Kyaw [/bib_ref] [bib_ref] The evidence for using conjugate vaccines to protect HIV-infected children against pneumococcal..., Bliss [/bib_ref] Two vaccines are available in the United States to prevent pneumococcal disease. The 13-valent pneumococcal conjugate vaccine (PCV13; Prevnar 13®/Prevenar 13®, Pfizer Inc, Philadelphia, PA) contains pneumococcal capsular polysaccharides 1, 3, 4, 5, 6A, 6B, 7F, 9V, individually conjugated to a nontoxic diphtheria protein CRM 197.Chemical linkage to a carrier protein stimulates a T cell−dependent response. [bib_ref] Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines, Pollard [/bib_ref] The 23-valent pneumococcal polysaccharide vaccine (PPSV23; Pneumovax® 23, Merck and Co., Inc., Whitehouse Station, NJ) contains 12 of the serotypes included in PCV13 (all but 6A) plus serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F; capsular polysaccharides in this vaccine are unconjugated and stimulate a T cell−independent response that does not induce immunological memory. [bib_ref] A review of pneumococcal vaccines: current polysaccharide vaccine recommendations and future protein..., Daniels [/bib_ref] [bib_ref] Immunogenicity and immunological memory induced by the 13-valent pneumococcal conjugate followed by..., Farmaki [/bib_ref] Since June 2012, the US Advisory Committee on Immunization Practices (ACIP) has been recommending the use of PCV13 in addition to PPSV23 in individuals ≥19 y of age with underlying conditions perceived as a high risk for IPD, including functional or anatomic asplenia, cerebrospinal fluid (CSF) leaks, cochlear implants, and specified immunocompromising conditions [fig_ref] Table 1: Underlying medical conditions for which both PCV13 and PPSV23 are currently recommended... [/fig_ref]. In February 2013, the routine use of PCV13 for individuals with the same high-risk conditions was also recommended in children aged 6 to 18 y. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine among..., Bennett [/bib_ref] Despite the increased risk of IPD among immunocompromised individuals, immunization rates in this high-risk population remain low. Available surveillance reports show that only 23% of adults (19-64 y of age) at increased risk, including immunocompromised individuals, ever received a pneumococcal vaccine. [bib_ref] Surveillance of vaccination coverage among adult populations -United States, Williams [/bib_ref] Possible reasons include lack of health-care provider knowledge about vaccine recommendations and appropriate timing of vaccination relative to disease treatment; uncertainty about potential vaccine benefit in an immunocompromised host; and safety concerns, such as allograft rejection. [bib_ref] Inadequate knowledge of immunization guidelines: a missed opportunity for preventing infection in..., Yeung [/bib_ref] [bib_ref] Vaccination guidelines after hematopoietic stem cell transplantation: practitioners' knowledge, attitudes, and gap..., Ariza-Heredia [/bib_ref] [bib_ref] Outcomes associated with influenza vaccination in the first year after kidney transplantation, Hurst [/bib_ref] [bib_ref] A comprehensive review of immunization practices in solid organ transplant and hematopoietic..., Chong [/bib_ref] Concerns regarding low vaccine effectiveness may stem from the stated possibility of impaired ability of immunocompromised individuals to mount an antibody response to vaccination and limited awareness of the amount of immunogenicity data for PCV13 in diverse patients with immunocompromising conditions.The objective of this review is to summarize current data on PCV13 vaccination in patients with immunocompromising and other high-risk conditions included in the ACIP PCV13 recommendation. Search terms included all relevant terminology for the 7-valent pneumococcal conjugate vaccine (PCV7) and PCV13 (e.g., "prevnar 7," "prevenar 7," "pneumococcal 7-valent," "pneumococcal heptavalent," or "CRM197," "diphtheria CRM 197 protein," "prevnar 13," "pneumococcal 13-valent," "pneumococcal tridecavalent," respectively) and the following high-risk conditions included in the ACIP recommendation for PCV13 vaccination: sickle cell disease (SCD), other hemoglobinopathy, congenital asplenia-SCD, acquired asplenia (splenectomy), HIV, chronic renal failure, nephrotic syndrome, leukemia, lymphoma, Hodgkin disease, generalized malignancy, iatrogenic immunosuppression, solid organ transplant, and MM. No limitations were placed on study design or setting. Search results were manually evaluated for relevance to the topic of PCV13 effectiveness in patients with these conditions. For search results for underlying conditions that returned PCV13 and PCV7 articles, only PCV13 articles were selected; for search results that returned no PCV13 articles, PCV7 articles were selected. Search results of abstracts later published as full articles were included as full articles.
# Methods
## Searches in
# Results
A total of 184 articles were identified by the literature search and reviewed for eligibility; of these, 30 publications describing studies of PCV13 use in individuals with conditions included by ACIP vaccination recommendation were eligible for inclusion. In total, 2406 individuals with immunocompromising conditions were included in this review, including those with congenital or acquired asplenia/SCD (four articles; n = 277), HIV infection (eight articles; n = 1025), hematologic disorders (eight articles; n = 534), autoimmune/rheumatologic disorders (seven articles; n = 487), solid organ transplant (two articles; n = 66), and chronic renal failure (one article; n = 17). No studies of PCV13 use were identified in individuals with cochlear implants, nephrotic syndrome, congenital or acquired immunodeficiencies (other than HIV infection), CSF leaks, Hodgkin disease, or generalized malignancy. Although two of the identified studies evaluated PCV7 use in children with idiopathic nephrotic syndrome or cochlear implants, these were not included here to limit the focus to PCV13. [bib_ref] Safety and immunogenicity of booster immunization with 7-valent pneumococcal conjugate vaccine in..., Liakou [/bib_ref] [bib_ref] Immunogenicity of pneumococcal vaccination of patients with cochlear implants, Rose [/bib_ref] Studies used a combination of assays, primarily (i) opsonophagocytic (OPA) assayswhich are used to measure the functional activity of antibody titers in vivoand/or (ii) immunoglobulin G (IgG) assays, which measure the concentrations of serotype-specific antibodies circulating in sera. These assays are used to assess protective immunity following vaccination. [bib_ref] Assignment of weight-based antibody units for 13 serotypes to a human antipneumococcal..., Quataert [/bib_ref] [bib_ref] Correlation of opsonophagocytosis and passive protection assays using human anticapsular antibodies in..., Johnson [/bib_ref] The endpoints for immune response are presented for study populations as OPA geometric mean titers (GMTs) and/or IgG geometric mean concentrations (GMCs). In infants, the World Health Organization (WHO) recognizes a population-based threshold of ≥0.35 µg/mL for serotypespecific IgG following pneumococcal conjugate vaccine administration for use in the evaluation of new PCVs.There is no defined protective threshold in adults, for whom functional antibody assessment via OPA is favored. [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] Included studies used a variety of endpoints to assess vaccine responses and were grouped according to medical condition; these are summarized in [fig_ref] Table 2: Overview of studies. [/fig_ref] and described below.
## Congenital or acquired asplenia/sickle cell disease
Two studies investigated the immunogenicity of one dose of PCV13 in asplenic adults with thalassemia major and a history of pneumococcal vaccination, including receipt of one to four doses of PPSV23. [bib_ref] Antigen-specific B-cell response to 13-valent pneumococcal conjugate vaccine in asplenic individuals with..., Papadatou [/bib_ref] [bib_ref] Conjugate and 23-valent pneumococcal polysaccharide booster vaccination in asplenic patients with thalassemia..., Rezai [/bib_ref] In both studies, IgG GMCs were significantly increased following PCV13 vaccination. In the first study by Papadatou and colleagues, 39 patients aged 19 to 48 y received PPSV23 at 1 to 11 y before PCV13 and PCV7 at 7 y before PCV13. [bib_ref] Antigen-specific B-cell response to 13-valent pneumococcal conjugate vaccine in asplenic individuals with..., Papadatou [/bib_ref] Previous PPSV23 vaccination had a dose-and time-dependent effect on PCV13 immunogenicity and immunologic memory. Specifically, a positive correlation was observed between both IgG and IgM memory B cells and IgG antibodies elicited by PCV13 and the time elapsed since last PPSV23 vaccination; a negative correlation was observed between memory B cell and antibody levels and the number of PPSV23 doses. In the second study by Rezai and colleagues, PCV13 and PPSV23 were administered 8 weeks apart in 47 individuals aged 20 to 44 y who had received PPSV23 >5 y previously. [bib_ref] Conjugate and 23-valent pneumococcal polysaccharide booster vaccination in asplenic patients with thalassemia..., Rezai [/bib_ref] IgG GMCs elicited by PCV13 were significantly greater when PCV13 was administered before versus after PPSV23. These data are supportive of the general recommendations for administering PCV13 before PPSV23. [bib_ref] Intervals between PCV13 and PPSV23 vaccines: recommendations of the advisory committee on..., Kobayashi [/bib_ref] In another study by Nived and colleagues, PCV13 immunogenicity in 33 splenectomized adults was investigated; the most common reasons for splenectomy were trauma or abdominal surgery due to a benign process. Increased IgG GMCs for serotypes 1, 3, 4, 5, 7F, 18C, 19A, 19F, and 23F were observed following PCV13 vaccination in asplenic individuals who had received previous vaccination with one, two, or three doses of PPSV23 (92% of participants had not received PPSV23 within the previous 12 months), suggesting that PCV13 is immunogenic for these serotypes when used as a booster dose in asplenic patients previously vaccinated with PPSV23. [bib_ref] Vaccination status and immune response to 13-valent pneumococcal conjugate vaccine in asplenic..., Nived [/bib_ref] A study by De Montalembert and colleagues investigated the immunogenicity and safety of two doses of PCV13 administered approximately 6 months apart in 158 children (mean age, 13.3 y) with SCD who had received one or more doses of PPSV23 ≥6 months earlier. Statistically significant increases in IgG GMCs and OPA GMTs were observed after a single dose, with similar GMCs observed after doses 1 and 2. One year after the second PCV13 vaccination, OPA GMTs were significantly higher than pre-PCV13 levels for all 13 vaccine serotypes, and IgG GMCs were significantly higher for all 13 serotypes except for serotypes 3 and 5. No relationship between the time interval between PPSV23 and PCV13 administration and antibody responses to PCV13 was identified. Overall, PCV13 was well tolerated.
## Hiv infection
Eight studies examined antibody responses after pneumococcal vaccination in HIV-infected individuals. Two phase 3 clinical studies assessed the immunogenicity and safety of a three-dose PCV13 series, and four studies compared prime-boost PCV13 or PCV13/PPSV23 vaccination with PPSV23-only vaccination. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] [bib_ref] Immunological efficacy of pneumococcal vaccine strategies in HIV-infected adults: a randomized clinical..., Sadlier [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine versus the 23-valent..., Lombardi [/bib_ref] [bib_ref] Quantitative and functional antibody responses to the 13-valent conjugate and/or 23-valent purified..., Ohtola [/bib_ref] [bib_ref] Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine..., Ohtola [/bib_ref] One study evaluated PCV13 responses at various intervals after PPSV23 vaccination, and another evaluated responses to PCV13 followed by PPSV23 vaccination. 13-29
## Pcv13 three-dose regimen
Both open-label, single-arm, phase 3 studies of PCV13 immunogenicity and safety in HIV-infected individuals evaluated serotype-specific IgG and OPA responses 1 month after vaccination. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] In both studies, participants received stable highly active antiretroviral therapy (HAART) for at least 6 weeks prior to the first vaccination or did not receive antiretroviral therapy. In the first study by Bhorat and colleagues, 301 vaccine-naive PCV13 recipients (mean age, 25.8 y) received at least one of three PCV13 doses 1 month apart followed by one PPSV23 dose. After one dose, PCV13 elicited significantly increased antibody responses for PCV13 serotypes compared with prevaccination; however, for most serotypes, antibody responses increased modestly with subsequent doses of PCV13 and after PPSV23. Safety events were generally mild and did not increase with the number of PCV13 doses. [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] In the second study by Glesby and colleagues, 329 subjects aged 19 to 73 y previously vaccinated with one or more doses of PPSV23 ≥6 months earlier received at least one of three doses of PCV13 administered 6 months apart. The first PCV13 dose elicited a significant antibody response to all vaccine serotypes with only modest increases after subsequent doses. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] Rates of injection-site redness and swelling as well as fatigue, headache, and vomiting after dose 3 were slightly higher in subjects who had received two or more previous PPSV23 doses compared with those who had received one dose. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] In both studies, the second and third doses only provided incremental antibody increases compared with the response after the initial dose of PCV13. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] These findings support the current ACIP recommendation of a single dose of PCV13 in HIV-positive adults. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for..., Bennett [/bib_ref]
## Pcv13 prime-boost versus ppsv23-only regimen
Lombardi and colleagues compared a PCV13/PCV13 primeboost schedule (8-week interval) with a single PPSV23 dose in 100 pneumococcal vaccine-naive HIV-infected adults aged 18 to 65 y, nearly all (99%) of whom received stable antiretroviral therapy; both vaccine groups showed similar GMCs for IgGs against the common antigens at 48 weeks postvaccination. There was no significant increase in IgG levels for eight of the 13 serotypes (serotypes 1, 4, 5, 6B, after the second dose of PCV13. The percentage of participants achieving threshold IgG antibody concentrations was similar overall, but notably the percentage of responses to serotype 3 was significantly lower for thresholds of ≥1 µg/mL (at week 8) and ≥0.35 µg/mL (at week 48) for the PCV13 group. The IgG seroconversion rate was significantly higher only for serotype 6B at 24 weeks for the PCV13 group. Both vaccines were safe and well tolerated. [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine versus the 23-valent..., Lombardi [/bib_ref] A single-center study by Sadlier and colleagues compared the immunogenicity of a PCV13/PPSV23 prime-boost schedule (4-week interval) with that of PPSV23 alone in 60 pneumococcal vaccine-naive HIV-infected adults (mean age, 37 y), about half of whom were on HAART. Results showed substantially greater IgG and OPA responses in the primeboost group for five serotypes (1, 3, 4, 19F, 23F) and three serotypes (14, 23F, 6A) at week 28, respectively, compared with those receiving PPSV23 only. Two-fold IgG responses and 4-fold GMT increases were also more frequently observed in the prime-boost group compared with the PPSV23-only group (70% vs 52%, p < .01 and 48% vs 36%, p < .01, respectively). [bib_ref] Immunological efficacy of pneumococcal vaccine strategies in HIV-infected adults: a randomized clinical..., Sadlier [/bib_ref] An additional study by Ohtola and colleagues compared a PCV13/PPSV23 prime-boost schedule (8-week interval) with a single dose of PPSV23 in 51 HIV-infected adults aged 49 to 64 y (84% had PPSV23 >5 y prior) on antiretroviral therapy for ≥1 y; the PCV13/PPSV23 group was also compared with a PCV13/PPSV23 HIV-negative control group. [bib_ref] Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine..., Ohtola [/bib_ref] Among HIV-infected individuals, IgG and OPA responses to serotypes 14 and 23F were measured, showing similar increases after vaccination with PCV13/PPSV23 compared with PPSV23 alone, with no significant difference between the groups. Compared with HIV-negative individuals, HIVinfected individuals had reduced IgG responses to serotype 14 and reduced OPA responses to serotype 23F. [bib_ref] Quantitative and functional antibody responses to the 13-valent conjugate and/or 23-valent purified..., Ohtola [/bib_ref] The authors also assessed B-cell responses through experimental assays, with results suggesting that PCV13 may not enhance responses to subsequent PPSV23 vaccination in older HIVinfected adults. [bib_ref] Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine..., Ohtola [/bib_ref] Serotype-specific B-cell responses were measured 1 week postvaccination after administration of PCV13 followed 8 weeks later by PPSV23 or PPSV23 alone in HIVinfected adults (aged 51-59 y) on antiretroviral therapy for ≥1 y. Serotype-specific B cell percentages were significantly increased for only serotype 23F after PCV13 and for both serotype 23F and 14 after PPSV23 in the PCV13/PPSV23 group compared to prevaccination levels; significant increases occurred for both serotypes in the PPSV23-only group. Transmembrane activator and calcium-modulating cyclophilin ligand interactor-positive B-cell percentages were significantly lower in the PCV13/PPSV23 HIV-infected group compared with the PPSV23-only HIV-infected group. [bib_ref] Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine..., Ohtola [/bib_ref]
## Pcv13 after ppsv23
A study by Rossheim and colleagues in 96 HIV-infected adults aged 20 to 65 y receiving combination antiretroviral therapy evaluated the effect of time since PPSV23 receipt on antibody responses to subsequent PCV13 vaccination. [bib_ref] Association of time since pneumococcal polysaccharide vaccine receipt and CD4 count with..., Rossheim [/bib_ref] At 1 month after vaccination with PCV13, the foldrise in IgG GMCs for the three tested serotypes (3, 7F, 19A) was higher for individuals who received PPSV23 >3 y earlier compared with those who received PPSV23 between 1 and 3 y earlier, although only serotype 7F reached significance on univariate analysis. The authors suggested that receipt of PCV13 >1 y after PPSV23 may improve serologic responses to both vaccines in HIVinfected adults. [bib_ref] Association of time since pneumococcal polysaccharide vaccine receipt and CD4 count with..., Rossheim [/bib_ref]
## Ppsv23 after pcv13
Farmaki and colleagues evaluated the immunogenicity of one PCV13 dose followed by one PPSV23 dose 1 y later in 40 HIV-infected adults (median ± SD age, 50.6 ± 8.6 y) receiving antiretroviral therapy. [bib_ref] Immunogenicity and immunological memory induced by the 13-valent pneumococcal conjugate followed by..., Farmaki [/bib_ref] An approximately 2-fold rise in IgG antibody concentrations was elicited after PCV13 receipt across all patients for each of the two measured serotypes (serotypes 3 and 14), with a smaller increase after PPSV23. Antibody concentrations were 40% lower in the group with lower CD4+ T-cell counts (200-399 cells/µL). Immunologic memory was assessed by evaluating polysaccharide specific IgM+ memory B cells (IgM+ MBC) and isotype-switched immunoglobulin (sIg+ MBC). IgM+ MBC remained stable after PCV13 dosing; however, the IgM+ MBC pool was significantly reduced after PPSV23 dosing compared with baseline levels. In contrast, sIg+ memory B-cell counts were increased after PCV13 vaccination but had no increase after PPSV23 vaccination compared with baseline. [bib_ref] Immunogenicity and immunological memory induced by the 13-valent pneumococcal conjugate followed by..., Farmaki [/bib_ref]
## Hematologic conditions
Eight studies evaluated responses to PCV13 in patients with hematologic malignancies, including leukemia, MM, and mixed hematological disorders. 33-40
## Leukemia
In one study by Pasiarski and colleagues involving patients with leukemia, one dose of PCV13 was assessed in 24 adults aged 47 to 79 y with treatment-naive chronic lymphocytic leukemia (CLL) and 15 healthy control subjects aged 54 to 83 y. All healthy subjects and 58.3% of patients with CLL responded to vaccination (i.e., ≥2-fold increase in serotype-specific IgG concentrations 30 d post-PCV13). A significant increase in the percentage of plasmablasts 7 d postvaccination was observed in both groups, indicating a rapid response to PCV13. [bib_ref] Pneumococcal conjugate vaccine triggers a better immune response than pneumococcal polysaccharide vaccine..., Svensson [/bib_ref] prospectively compared OPA GMTs following vaccination with either PCV13 or PPSV23 in 128 patients with CLL aged 46 to 87 y, finding that PCV13 elicited higher serotype-specific GMTs compared with PPSV23 for 10 of the 12 serotypes common to both vaccines (serotypes 1, 3, 4, 5, 7F, 9V, 18C, 19A, 19F, 23F) in addition to seven or more 6A; no differences were found for serotype 6B and 14. Positive responses (OPA titer greater than or equal to the lower limit of quantitation for seven or more serotypes) were more frequent after PCV13 vs PPSV23. [bib_ref] Pneumococcal conjugate vaccine triggers a better immune response than pneumococcal polysaccharide vaccine..., Svensson [/bib_ref] An additional study by Andrick and colleagues evaluated eight patients aged 53 to 77 y with CLL, four taking ibrutinib (a Bruton tyrosine kinase inhibitor for B cell malignancies, which is associated with increased risk of infection as a treatment side effect) and four control patients not receiving ibrutinib. All control patients with CLL not taking ibrutinib met the defined antibody response to PCV13 (≥2-fold increase in postvaccination IgG concentrations for three or more serotypes), but responses were not observed in the four patients being treated with ibrutinib. [bib_ref] Lack of adequate pneumococcal vaccination response in chronic lymphocytic leukaemia patients receiving..., Andrick [/bib_ref]
## Multiple myeloma
Two small studies led by Locke and Bahuaud assessed PCV13 vaccination in patients with MM. In the first, patients with MM undergoing autologous HCT were given PCV13: two patients received three doses (pretransplant and at 7-10 and 21-24 d following transplant); three patients received two doses (pretransplant and at d 21 post-transplant); and one patient received a single dose (pretransplant). [bib_ref] Boosting humoral and cellular immunity to pneumococcus by vaccination before and just..., Locke [/bib_ref] [bib_ref] Immunogenicity and persistence of the 13-valent pneumococcal conjugate vaccine (PCV13) in patients..., Bahuaud [/bib_ref] All patients who received pre-and post-transplant PCV13 vaccinations showed significantly greater IgG antibody responses against vaccine-specific serotypes compared with nonvaccine serotypes. There was a significant increase in serotype-specific IgG levels when compared with prevaccination levels for 6 of the 11 serotypes tested (3, 4, 6B, 9V, 18C, 23). [bib_ref] Boosting humoral and cellular immunity to pneumococcus by vaccination before and just..., Locke [/bib_ref] The other study assessed a single dose of PCV13 in 20 patients aged 57 to 75 y with smoldering (i.e., early) MM. Immunogenicity was assessed for PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) at 1 month, 6 months, and 12 months post-vaccination. [bib_ref] Immunogenicity and persistence of the 13-valent pneumococcal conjugate vaccine (PCV13) in patients..., Bahuaud [/bib_ref] One month post-PCV13, 12 patients responded based on IgG levels measured in an enzyme-linked immunosorbent assay (ELISA; i.e., >2-fold increase in IgG antibody concentration and concentration ≥1 µg/mL for more than five serotypes) and eight patients responded based on OPA (titer greater than or equal to the lower limit of quantitation and 4-fold increase from baseline for more than five serotypes). When persistent immunity was assessed at 6 months postvaccination, seven patients were responders based on the ELISA (IgG) criteria and six based on OPA. This decreased to five responders based on ELISA (IgG) criteria and two responders based on OPA criteria after 12 months. [bib_ref] Immunogenicity and persistence of the 13-valent pneumococcal conjugate vaccine (PCV13) in patients..., Bahuaud [/bib_ref]
## Hematopoietic stem cell transplant recipients
An open-label study by Cordonnier and colleagues investigated the immunogenicity and safety of a four-dose PCV13/ one-dose PPSV23 vaccination schedule in 216 individuals aged 2 to 71 y with different hematologic disorders who had received allogeneic HCT 3 to 6 months previously. [bib_ref] Immunogenicity, safety, and tolerability of 13-valent pneumococcal conjugate vaccine followed by 23-valent..., Cordonnier [/bib_ref] Subjects received the first three PCV13 doses at 1-month intervals, the fourth dose 6 months later, and the single PPSV23 dose 1 month thereafter. IgG antibody concentrations and OPA titers increased significantly for all 13 vaccine serotypes from baseline to after the third dose of PCV13. Responses declined during the interval between PCV13 doses 3 and 4 and then increased from pre-to postdose 4; little change was observed after PPSV23. Local and systemic adverse events (AEs) were more frequent after PCV13 dose 4, but the overall safety profile of the four-dose PCV13 schedule was considered acceptable. [bib_ref] Immunogenicity, safety, and tolerability of 13-valent pneumococcal conjugate vaccine followed by 23-valent..., Cordonnier [/bib_ref] Shah and colleagues evaluated responses to PCV13 (or its precursor, PCV7) in 58 cord blood transplantation recipients aged 0.9 to 64 y undergoing treatment for hematologic malignancies. Among these 58 patients, 24 received PCV7, 33 received PCV13, and one patient received both vaccines. [bib_ref] Robust vaccine responses in adult and pediatric cord blood transplantation recipients treated..., Shah [/bib_ref] Following three doses of PCV13 (or PCV7 if available at the time; doses 1 month apart and administered ≥6 months postcord blood transplantation), 52% (30/58) of patients responded (i.e., >3-fold rise in IgG GMC) to all three pneumococcal serotypes tested and 33% (19/58) responded to one or two of the three serotypes tested. A total of 16% (9/58) of patients did not respond to any tested serotypes; of these, six patients were revaccinated with one or two booster doses but only five patients were evaluated. All five evaluated patients who initially did not respond to primary vaccination did so when revaccinated with one or two booster doses. Response rates did not differ between children and adults. No significant differences were noted between vaccine responses and prior chemotherapy, graft versus host disease, or low dose immunosuppression therapy. [bib_ref] Robust vaccine responses in adult and pediatric cord blood transplantation recipients treated..., Shah [/bib_ref] A study by Small and colleagues evaluated responses to three doses of PCV13 in 59 hematopoietic stem cell transplant (HCT) recipients aged 3 to 68 y, most of whom had undergone transplantation for leukemia or myelodysplastic syndrome. [bib_ref] Safety and immunogenicity of the 13-valent protein-conjugated pneumococcal vaccine (PCV13) following related..., Small [/bib_ref] Immunization was performed upon immune competency milestone achievements (i.e., >200 CD4 cells/μL and IgG levels >500 mg/dL at ≥6 weeks without gammaglobulin). Overall, 73% of patients responded to PCV13 (i.e., >3-fold rise in titer to specified pneumococcal serotypes), including 87% of patients aged <21 y and 67% of patients aged >21 y. [bib_ref] Safety and immunogenicity of the 13-valent protein-conjugated pneumococcal vaccine (PCV13) following related..., Small [/bib_ref]
## Autoimmune/rheumatologic disorders
Seven studies assessed PCV13 in patients with diverse underlying medical conditions who were taking various immunosuppressive drugs to treat immunologic disorders. [bib_ref] Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines..., Kantso [/bib_ref] [bib_ref] Evaluation of the immunogenicity of the 13-valent conjugated pneumococcal vaccine in rheumatoid..., Rakoczi [/bib_ref] [bib_ref] Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving..., Nived [/bib_ref] [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] [bib_ref] T-cell-mediated immune response to pneumococcal conjugate vaccine (PCV-13) and tetanus toxoid vaccine..., Winthrop [/bib_ref] [bib_ref] Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B..., Kapetanovic [/bib_ref] A study by Kantsø and colleagues compared antibody responses to PCV13 and PPSV23 in 151 patients (mean age, 44 y) with Crohn disease in the presence or absence of immunosuppressive drugs containing azathioprine (AZA) or mercaptopurine (immunomodulators associated with increased infection susceptibility) and tumor necrosis factor (TNF)-α antagonist therapy. [bib_ref] Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines..., Kantso [/bib_ref] One month postvaccination, serotypespecific IgG GMCs were higher for PCV13 compared with PPSV23 (significantly greater for five serotypes); overall increases were highest in untreated patients (significantly greater in PCV13 compared to PPSV23 for four serotypes). Immunity, while impaired, was induced in both immunosuppressive drug-treated patients and patients treated with immunosuppressive drugs along with TNF-α antagonists; PCV13 responses in these groups were significantly greater than PPSV23 for three and two serotypes, respectively. [bib_ref] Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines..., Kantso [/bib_ref] Two other studies also compared PCV13 antibody responses in patients with rheumatoid arthritis (RA) on immunosuppressant therapy to responses in patients with the same or a similar disease who were not taking immunosuppressants. [bib_ref] Evaluation of the immunogenicity of the 13-valent conjugated pneumococcal vaccine in rheumatoid..., Rakoczi [/bib_ref] [bib_ref] Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B..., Kapetanovic [/bib_ref] In the study by Rakoczi and colleagues, patients with RA (median age, 55.1 y) treated with etanercept (ETA; a TNF-α inhibitor associated with serious lung infections, including pneumonia) or a combination of ETA and methotrexate was compared to controls. Serotype-specific IgG antibody concentrations significantly increased (≥2-fold increases) from pre-to 1 and 2 months postvaccination in all groups, but those on drug therapy had significantly lower PCV13 antibody responses compared with untreated patients (median age, 63.9 y). [bib_ref] Evaluation of the immunogenicity of the 13-valent conjugated pneumococcal vaccine in rheumatoid..., Rakoczi [/bib_ref] [bib_ref] Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving..., Nived [/bib_ref] [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] [bib_ref] T-cell-mediated immune response to pneumococcal conjugate vaccine (PCV-13) and tetanus toxoid vaccine..., Winthrop [/bib_ref] [bib_ref] Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B..., Kapetanovic [/bib_ref] [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) in solid organ..., Sun [/bib_ref] [bib_ref] Immunogenicity of 13-valent conjugate pneumococcal vaccine in patients 50 years and older..., Mitra [/bib_ref] [bib_ref] Safety and immunogenicity of booster immunization with 7-valent pneumococcal conjugate vaccine in..., Liakou [/bib_ref] [bib_ref] Immunogenicity of pneumococcal vaccination of patients with cochlear implants, Rose [/bib_ref] [bib_ref] Assignment of weight-based antibody units for 13 serotypes to a human antipneumococcal..., Quataert [/bib_ref] [bib_ref] Correlation of opsonophagocytosis and passive protection assays using human anticapsular antibodies in..., Johnson [/bib_ref] [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] [bib_ref] Serious infections in patients receiving ibrutinib for treatment of lymphoid cancer, Varughese [/bib_ref] [bib_ref] Infections in patients with rheumatoid arthritis treated with biologic agents, Listing [/bib_ref] The other study by Kapetanovic and colleagues evaluated 10 patients with RA on methotrexate compared to 10 patients with RA not on disease-modifying antirheumatic therapy (DMARD). [bib_ref] Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B..., Kapetanovic [/bib_ref] Immunogenicity was assessed 4-6 weeks postvaccination for two vaccine serotypes (6B and 23F). Patients on methotrexate showed a significant increase in 6B IgG GMCs postvaccination, likely as a result of a dramatic response from a single patient. Patients not on therapy had a significant increase to both serotypes tested. [bib_ref] Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B..., Kapetanovic [/bib_ref] Three studies compared PCV13 responses in immunosuppressed patients with those in healthy controls and found that PCV13 antibody responses were generally higher in healthy individuals. [bib_ref] Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving..., Nived [/bib_ref] [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] Caporuscio and colleagues compared 24 patients with RA on low dose glucocorticoids, methotrexate, and/or anti-TNF therapy to three healthy controls. One month postvaccination, 61% of patients developed a positive response (2-fold increase in concentration) to the 13 serotypes. [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] Mild local and systemic AEs were more frequently reported in immunosuppressed patients with RA compared with healthy individuals (44% vs 16%, respectively). [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] Another study by Nived and colleagues evaluated IgG concentrations for serotype 6B and 23F in patients on treatment for vasculitis compared to controls. Treatment included AZA, methotrexate, cyclophospharmide, mycophenolate mofetil, prednisolone, rituximab, and TNF inhibitors in a variety of therapy combinations. Patients were stratified into two groups based on treatment. Both the patient groups and the control group had increases in IgG GMCs to the two serotypes tested (6B and 23F) postvaccination with no difference between groups in the proportion achieving a response (2-fold increase in IgG). [bib_ref] Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving..., Nived [/bib_ref] The proportion of patients with IgG concentrations ≥1.0 µg/mL also increased across both patients and controls. Both the patient groups and control group had postvaccination increases in OPA titers for serotype 23F; however, responses were lower in patients on treatment. [bib_ref] Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving..., Nived [/bib_ref] Finally, Nagel and colleagues compared immune responses of 47 patients with systemic lupus erythematosus (SLE) on therapy (mean age, 50.8 y) with those of 21 healthy controls (mean age, 43.6 y). [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] Therapy included various combinations of AZA and other DMARDs, hydroxychloroquine, prednisolone, and belimumab. IgG GMCs increased significantly for all 12 serotypes tested (1, 3, 4, 5, 6B, 7F, 9V, 14, 18 C, 19A, 19F, 23F) in patients with SLE and controls postvaccination, but the response was significantly lower in those with than in controls. There were no differences noted in the antibody response of the belimumabtreated patients compared with those who received other therapies. [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] A single-arm study by Winthrop and colleagues evaluated the geometric mean fold rise from baseline in serotypespecific OPA titers for the 13 PCV13 serotypes 4 weeks after immunization in patients with severe to moderate psoriasis receiving immunosuppressive therapy. [bib_ref] T-cell-mediated immune response to pneumococcal conjugate vaccine (PCV-13) and tetanus toxoid vaccine..., Winthrop [/bib_ref] Sixty patients aged 23 to 70 y with psoriasis treated with tofacitinib, a Janus kinase inhibitor, were included. GMTs ranged from 4.9 to 99.5 at baseline and from 66.1 to 2782.2 after 4 weeks. More than 80% of patients achieved measurable serotype-specific OPA titers for each serotype. [bib_ref] T-cell-mediated immune response to pneumococcal conjugate vaccine (PCV-13) and tetanus toxoid vaccine..., Winthrop [/bib_ref]
## Solid organ transplantation
Two publications reported outcomes of PCV13 in solid organ transplant (SOT) recipients. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) in solid organ..., Sun [/bib_ref] In one study by Dendle and colleagues, a prospective cohort of 45 clinically stable kidney transplant recipients (median time since transplant, 2.24 y) aged 47.0 to 63.9 y received a single PCV13 dose.The majority (81%) of patients were on tacrolimus + mycophenolate + prednisolone therapy. Antipneumococcal IgG antibodies were measured for the 13 vaccine serotypes pre-and 1 month postvaccination, and OPA antibody titers were measured for four serotypes postvaccination. Median increases in antipneumococcal IgG GMCs were 1.1-to 1.7-fold for all 13 serotypes, and OPA GMTs were ≥1:8 for three of the four serotypes tested (1, 4, 9V, 23F). No transplant rejections, de novo anti-HLA antibody development, or IPD episodes occurred during the 12-month postvaccination period.The second study by Sun and colleagues was described in a conference abstract and assessed the immunogenicity and safety of a single PCV13 dose in two heart transplant recipients and 19 SOT candidates. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) in solid organ..., Sun [/bib_ref] At 1 month postvaccination, 58.3% to 75.0% of 13 patients who were followed up showed anticapsular antibody responses (i.e., ≥2-fold increase compared with baseline) to the four pneumococcal serotypes tested (6B, 14, 19F, 23F). [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) in solid organ..., Sun [/bib_ref] A case report described by Gupta and Brennan discussed the occurrence of PCV13-associated immune thrombocytopenic purpura in a 77-y-old renal transplant recipient (summarized here, but not part of the 30 studies included in [fig_ref] Table 2: Overview of studies. [/fig_ref]. [bib_ref] Pneumococcal 13-valent conjugate vaccine (Prevnar 13)-associated immune thrombocytopenic purpura in a renal..., Gupta [/bib_ref] The patient presented approximately 1 month after PCV13 vaccination with bruising at insulin injection sites; following methylprednisolone, immunoglobulin, and platelet transfusion treatment, the patient's blood counts improved to baseline within a month and remained stable thereafter. [bib_ref] Pneumococcal 13-valent conjugate vaccine (Prevnar 13)-associated immune thrombocytopenic purpura in a renal..., Gupta [/bib_ref]
## Renal failure
A single study by Mitra and colleagues assessed the immunogenicity of PCV13 in 17 older adults (median age, 62.6 y) with end-stage renal disease receiving dialysis. [bib_ref] Immunogenicity of 13-valent conjugate pneumococcal vaccine in patients 50 years and older..., Mitra [/bib_ref] Among the 17 patients analyzed, 11 had received prior pneumococcal vaccination (>5 y previously). IgG GMCs increased significantly compared to baseline for all 13 serotypes at 2 months and four serotypes (5, 19F, 6B, 18C) remained higher at 12 months after vaccination. Vaccine responses (i.e., ≥2-fold increase in IgG concentration and absolute post-PCV13 IgG concentration ≥1 µg/mL) to >75% of the PCV13 serotypes were observed in nine of the 17 patients at 2 months postvaccination and in four patients at 12 months after vaccination. [bib_ref] Immunogenicity of 13-valent conjugate pneumococcal vaccine in patients 50 years and older..., Mitra [/bib_ref]
# Discussion
To our knowledge, this is the first review to focus exclusively on studies evaluating PCV13 immunogenicity in patients with immunocompromising conditions. A 2014 review summarized the efficacy and safety of available pneumococcal vaccines (PPSV23, PCV7, and PCV13) in immunocompromised patients, but more data have since become available. [bib_ref] Pneumococcal immunization in immunocompromised hosts: where do we stand?, Cordonnier [/bib_ref] The studies summarized herein included patients with diverse underlying medical conditions, covering the majority of conditions specified for coverage in the ACIP recommendations for PCV13 vaccination. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for..., Bennett [/bib_ref] Overall immunogenicity findings support that patients, despite an underlying condition or immunosuppressive treatment regimen, generally mounted antibody responses against the serotypes contained in PCV13 following a single dose. Results from multiple studies suggest that a second or third dose in HIV-infected populations may not provide additional benefit in terms of increases in antibody levels. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] In individuals who had undergone hematopoietic bone marrow transplantation, four doses of PCV13 were evaluated. [bib_ref] Immunogenicity, safety, and tolerability of 13-valent pneumococcal conjugate vaccine followed by 23-valent..., Cordonnier [/bib_ref] As expected, individuals with an underlying condition or therapy that can cause immunosuppression tended to have lower antibody responses to PCV13 compared with healthy counterparts; however, these individuals are also at high risk for pneumococcal disease. Immune responses varied by condition, serotype, and type of antibody response measured; most of these data were derived from open-label studies. These findings are consistent with the results of PCV7 use in immunocompromised individuals. [bib_ref] PCVs in individuals at increased risk of pneumococcal disease: A literature review, Fletcher [/bib_ref] The ACIP recommends that individuals with immunocompromising conditions receive PCV13 followed by PPSV23 at least 8 weeks later to offer broader protection against the high burden of IPD these individuals face. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine among..., Bennett [/bib_ref] [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for..., Bennett [/bib_ref] The rationale behind the sequence of PCV13 followed by PPSV23 is based on studies that demonstrated a better response to serotypes included in both vaccines if PCV13 was administered first. [bib_ref] Intervals between PCV13 and PPSV23 vaccines: recommendations of the advisory committee on..., Kobayashi [/bib_ref] At the time of the ACIP recommendation, data on the immunogenicity of PCV13 in immunocompromised individuals were not available. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for..., Bennett [/bib_ref] As this current review shows, there are now PCV13 immunogenicity data in this population. Overall, findings generally support the ACIP recommended PCV13/PPSV23 vaccine sequence in immunocompromised individuals and further reinforce the importance of administering the conjugate vaccine first. [bib_ref] Immunogenicity and immunological memory induced by the 13-valent pneumococcal conjugate followed by..., Farmaki [/bib_ref] [bib_ref] Conjugate and 23-valent pneumococcal polysaccharide booster vaccination in asplenic patients with thalassemia..., Rezai [/bib_ref] [bib_ref] Immunogenicity and safety of the 13-valent pneumococcal conjugate vaccine in HIV-infected individuals..., Bhorat [/bib_ref] [bib_ref] Immunological efficacy of pneumococcal vaccine strategies in HIV-infected adults: a randomized clinical..., Sadlier [/bib_ref] [bib_ref] Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine..., Ohtola [/bib_ref] [bib_ref] Immunogenicity, safety, and tolerability of 13-valent pneumococcal conjugate vaccine followed by 23-valent..., Cordonnier [/bib_ref] PCV13 was generally well tolerated, and safety events were consistent with those previously reported, such as redness, swelling, and injection-site pain.No new safety concerns emerged during the studies. [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in HIV-infected adults previously..., Glesby [/bib_ref] [bib_ref] Immunogenicity, safety, and tolerability of 13-valent pneumococcal conjugate vaccine followed by 23-valent..., Cordonnier [/bib_ref] [bib_ref] Treatment with belimumab in systemic lupus erythematosus does not impair antibody response..., Nagel [/bib_ref] [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] This review is limited by variability in designs, size, and endpoints of the currently available studies. A more complete understanding of PCV13 use and the ACIP recommended PCV13/PPSV23 sequence in immunocompromised individuals will emerge as more clinical studies are conducted and real-world usage data are examined. It should be noted that three of the studies included in this review were published as conference abstracts rather than expanded, peer-reviewed publications, limiting the data available. In other cases, patient groups were too small to draw robust conclusions, patient therapies varied within groups, and prevaccination histories were not always available. [bib_ref] Safety and immunogenicity of the 13-valent protein-conjugated pneumococcal vaccine (PCV13) following related..., Small [/bib_ref] [bib_ref] AB0391 immunogenicity of 13-valent conjugate pneumococcal vaccine in patients with rheumatoid arthritis, Caporuscio [/bib_ref] [bib_ref] Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) in solid organ..., Sun [/bib_ref] The use of immunosuppressive drugs makes it difficult in some cases to extrapolate PCV13 immunogenicity results from patients with a specific medical condition to the treatment of patients with another condition and/or treatment.
Finally, the collective interpretation of all studies included in this review should be considered with the understanding that endpoints used across studies to measure immunogenicity differed. ELISAs are widely used to measure IgG levels to evaluate vaccine immunogenicity because they are generally easier to perform and are easily scalable. [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] Some studies utilized the threshold of IgG antibody concentrations ≥0.35 µg/mL, a widely accepted population-based level for use in the evaluation of PCVs after an infant series; others utilized an IgG threshold of ≥1 µg/mL. [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] Responders were often quantified as achieving a 2-fold rise in preto postvaccination concentrations. However, no clear threshold IgG antibody concentration or foldrise has been established to indicate protection or efficacy in an individual, and IgG results may not always correlate with functional activity, particularly in adults. [bib_ref] Immunogenicity and persistence of the 13-valent pneumococcal conjugate vaccine (PCV13) in patients..., Bahuaud [/bib_ref] [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] OPA titer is a preferred measure of vaccine immunogenicity in adults because it directly measures functional activity (the ability of antibodies to opsonize and kill bacteria), and therefore adult studies employing OPA endpoints are expected to generate more reliable results. [bib_ref] Pneumococcal vaccine and opsonic pneumococcal antibody, Song [/bib_ref] However, as with the IgG, there is no OPA titer that has been established as a protective threshold. Additional variability among the conclusions of studies in this review may be related to inconsistency among the subsets of serotypes evaluated in each experiment.
Vaccine efficacy trials with PCV13 in immunocompromised patients are not currently available. A recent study demonstrated a vaccine effectiveness of 72.8% for PCV13 against hospitalized vaccine-type community-acquired pneumonia in adults over the age of 65 y. [bib_ref] Effectiveness of 13-valent pneumococcal conjugate vaccine against hospitalization for community-acquired pneumonia in..., Mclaughlin [/bib_ref] In this real-world population, approximately 46% of the patient population had an immunocompromising condition or a condition otherwise defined in the review as high risk, and the vaccine effectiveness did not change substantially when adjusting for risk group.
Randomized, controlled efficacy trials of pneumococcal conjugate vaccines to demonstrate protection against IPD are not feasible. Results from these immunogenicity studies provide valuable support for the use of PCV13 in immunocompromised individuals for whom PCV13 vaccination is recommended because of their increased risk of pneumococcal disease. [bib_ref] Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for..., Bennett [/bib_ref] Concerns regarding a poor safety profile or the lack of immunogenicity of PCV13 are not supported by data and should not create barriers to appropriate pneumococcal immunization in this patient population.
[table] Table 1: Underlying medical conditions for which both PCV13 and PPSV23 are currently recommended by ACIP for individuals ≥6 y of age.[14][15][16] [/table]
[table] Table 2: Overview of studies. [/table]
|
The impact of trophic and immunomodulatory factors on oligodendrocyte maturation: Potential treatments for encephalopathy of prematurity
Encephalopathy of prematurity (EoP) is a major cause of morbidity in preterm neonates, causing neurodevelopmental adversities that can lead to lifelong impairments.Preterm birth-related insults, such as cerebral oxygen fluctuations and perinatal inflammation, are believed to negatively impact brain development, leading to a range of brain abnormalities. Diffuse white matter injury is a major hallmark of EoP and characterized by widespread hypomyelination, the result of disturbances in oligodendrocyte lineage development. At present, there are no treatment options available, despite the enormous burden of EoP on patients, their families, and society. Over the years, research in the field of neonatal brain injury and other white matter pathologies has led to the identification of several promising trophic factors and cytokines that contribute to the survival and maturation of oligodendrocytes, and/or dampening neuroinflammation. In this review, we discuss the current literature on selected factors and their therapeutic potential to combat EoP, covering a wide range of in vitro, preclinical and clinical studies. Furthermore, we offer a future perspective on the translatability of these factors into clinical practice.
# | introduction
Worldwide, approximately 10% of live-born babies is born preterm, that is, before 37 weeks of gestation. Preterm birth can be subdivided into extremely preterm (<28 weeks), very preterm and moderate or late preterm birth (32-<37 weeks [bib_ref] National, regional, and worldwide estimates of preterm birth rates in the year..., Blencowe [/bib_ref] and is associated with multiple neurodevelopmental morbidities, ranging from motor problems and cognitive impairments to an increased risk of psychiatric disorders. The risk of neurological consequences of preterm birth is inversely correlated with gestational age, meaning that extreme preterm infants are most at risk [bib_ref] Neurobiology of injury to the developing brain, Deng [/bib_ref] [bib_ref] Neurodevelopmental disabilities and special care of 5-year-old children born before 33 weeks..., Larroque [/bib_ref] [bib_ref] Cognitive trajectories from infancy to early adulthood following birth before 26 weeks..., Linsell [/bib_ref] [bib_ref] Gestational age at delivery and special educational need: Retrospective cohort study of..., Mackay [/bib_ref] [bib_ref] Long-term medical and social consequences of preterm birth, Moster [/bib_ref]. Neurodevelopmental morbidities after preterm birth are thought to arise from encephalopathy of prematurity (EoP), an umbrella term used to describe the brain abnormalities that result from impeded brain development due to preterm birth-related complications [bib_ref] Brain injury in premature infants: A complex amalgam of destructive and developmental..., Volpe [/bib_ref]. The most prominent hallmark of EoP is white matter injury (WMI); however, neuronal and axonal deficits, such as GABAergic interneuron maldevelopment, have received growing attention over the years [bib_ref] Estrogen treatment reverses prematurity-induced disruption in cortical interneuron population, Panda [/bib_ref] [bib_ref] Interneuron development is disrupted in preterm brains with diffuse white matter injury:..., Stolp [/bib_ref] [bib_ref] The encephalopathy of prematurity-Brain injury and impaired brain development inextricably intertwined, Volpe [/bib_ref]. Preterm WMI is a collective term referring to a spectrum of pathological changes in the developing white matter and is often classified based on neuropathological findings, such as (small) necrotic lesions [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Confusions in nomenclature: "periventricular leukomalacia" and "white matter injury"-identical, distinct, or overlapping?, Volpe [/bib_ref]. As a result of advances in supportive care, diffuse WMI (dWMI), characterized by global hypomyelination without focal necrosis, is currently the most prevalent form of preterm WMI with reported prevalence rates up to 80% in all affected preterm neonates [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Brain injury in premature neonates: A primary cerebral dysmaturation disorder?, Back [/bib_ref] [bib_ref] Preterm brain injury: White matter injury, Schneider [/bib_ref]. Histopathological findings in dWMI display injured immature oligodendrocytes, along with astrocytosis and microgliosis. Consequently, a deficit of mature myelinating oligodendrocytes is observed, leading to a reduction in axonal myelination [bib_ref] Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants, Buser [/bib_ref] [bib_ref] White matter injury of prematurity: Its mechanisms and clinical features, Lee [/bib_ref] [bib_ref] Preterm brain injury: White matter injury, Schneider [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] The developing oligodendrocyte: Key cellular target in brain injury in the premature..., Volpe [/bib_ref]. Due to the key role of dWMI in EoP, this review will focus primarily on potential treatments aimed at restoration of white matter development.
Although treatment options for dWMI in preterm infants are currently limited, research in the last decade has created larger understanding of the pathophysiology underlying myelination failure, pinpointing impaired oligodendrocyte maturation as an critical target for therapeutic intervention. Experimental research has identified several growth factors and cytokines that play essential roles in healthy white matter development or that boost myelination in other white matter pathologies, such as multiple sclerosis (MS), (neonatal) stroke, and traumatic brain injury. Despite the evident differences in pathophysiology-most of the mentioned diseases are characterized by demyelination of existing white matter tracts while dWMI is associated with impaired myelin formation-, there are some overlapping characteristics, such as insufficient oligodendrocyte maturation and the occurrence of neuroinflammation. Thus, knowledge from previous research in the above-mentioned pathologies could aid in the refinement and identification of potential therapeutic strategies to restore white matter development in (extreme) preterm infants. Using a wide range of in vivo and in vitro studies, this review aims to integrate the current knowledge on a selection of trophic and immunomodulatory factors that boost oligodendrocyte maturation and white matter development, leading to the identification of potent therapeutic targets to combat preterm dWMI.
## | developmental white matter (patho)physiology
During normal human brain development, the formation of myelin sheaths by oligodendrocytes starts relatively late, at >32 weeks of gestation [bib_ref] Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human..., Back [/bib_ref]. Before oligodendrocytes are able to produce myelin, the proliferation, migration, and maturation of oligodendrocyte precursors has to be completed. This typically occurs in four stages: (1) oligodendrocyte precursor cells (OPCs) originate from differentiated neural stem cells (NSCs) in the ventral forebrain; (2) OPCs migrate throughout the brain and proliferate to increase their numbers; (3) at the site of destination, OPCs differentiate into premyelinating oligodendrocytes (pre-OLs), which are still nonmyelinating cells until (4) differentiation-repressive factors are lifted and pre-OLs differentiate into postmitotic, mature oligodendrocytes that produce myelin to enwrap axons [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. Not all OPCs reach this end stage during brain development: a homeostatic pool of OPCs populates the brain to maintain regenerative capacity after oligodendrocyte damage throughout adulthood. Under physiological circumstances, oligodendrocyte maturation is aided by microglia and astrocytes, through the release of essential nutrients, proteins, and cytokines [bib_ref] Microglia and the brain: Complementary partners in development and disease, Hammond [/bib_ref] [bib_ref] Astrocytes and microglia as major players of myelin production in normal and..., Traiffort [/bib_ref] [bib_ref] Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions, Volpe [/bib_ref]. Interestingly, interneurons, other cells at risk in EoP pathophysiology, have been reported to regulate oligodendrocyte lineage development, by emitting pro-differentiation cues through transient synaptic input and secreted factors [bib_ref] The cerebral cortex is a substrate of multiple interactions between GABAergic interneurons..., Benamer [/bib_ref] [bib_ref] GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white..., Zonouzi [/bib_ref].
The particular vulnerability of white matter in prematurely born infants results from the fact that (extreme) preterm birth coincides with the initiation of oligodendrocyte lineage development. Especially between 24 and 30 weeks of gestation, the brain contains a large population of OPCs and pre-OLs, which are particularly vulnerable to insults associated with preterm birth [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions, Volpe [/bib_ref]. Two major preterm birth-related insults known to affect maturation of pre-OLs to myelinating oligodendrocytes are inflammation and oxygen fluctuations. Moreover, encountering multiple hits is reported to aggravate neonatal brain damage and worsen neurodevelopmental outcome [bib_ref] Interaction of inflammation and hyperoxia in a rat model of neonatal white..., Brehmer [/bib_ref] [bib_ref] Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions, Volpe [/bib_ref] [bib_ref] The developing oligodendrocyte: Key cellular target in brain injury in the premature..., Volpe [/bib_ref].
Inflammation is estimated to occur in 65-79% of very low birth weight or extremely preterm infants, either caused by perinatal immune activation (such as intrauterine infection or maternal fever) or postnatal inflammation/infections (such as neonatal sepsis or necrotizing enterocolitis (NEC) of the immature bowel; [bib_ref] Inflammation and preterm birth, Cappelletti [/bib_ref] [bib_ref] The developing oligodendrocyte: Key cellular target in brain injury in the premature..., Volpe [/bib_ref]. After extremely preterm birth, oxygen fluctuations due to immature lungs and cardiovascular system (hypoxia) and/or the need for ventilation are also detrimental to the immature brain [bib_ref] Interaction of inflammation and hyperoxia in a rat model of neonatal white..., Brehmer [/bib_ref].
Inflammation induces the release of pro-inflammatory cytokines into the circulation which subsequently reach the immature brain [bib_ref] Brain-immune interactions in perinatal hypoxic-ischemic brain injury, Li [/bib_ref]. This process triggers microglia, the resident immune cells of the brain, to shift to a pro-inflammatory (M1) phenotype, stimulating additional release of pro-inflammatory cytokines by these cells in the brain parenchyma [bib_ref] Brain-immune interactions in perinatal hypoxic-ischemic brain injury, Li [/bib_ref]. The pro-inflammatory microglial shift may be sustained by multiple hits during pregnancy and following preterm birth [bib_ref] Brain-immune interactions in perinatal hypoxic-ischemic brain injury, Li [/bib_ref] [bib_ref] Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions, Volpe [/bib_ref].
Pre-OLs possess a high amount of cytokine receptors, and are therefore particularly sensitive to the release of these cytokines in the immature brain, which causes apoptosis [bib_ref] A silver lining of neuroinflammation: Beneficial effects on myelination, Goldstein [/bib_ref]. Activated microglia cause further harm to immature oligodendrocytes by reducing their trophic factor support, and releasing excessive amounts of glutamate which causes excitotoxicity [bib_ref] The potential of stem cell therapy to repair white matter injury in..., Vaes [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] The developing oligodendrocyte: Key cellular target in brain injury in the premature..., Volpe [/bib_ref]. Similarly, astrocytes respond to inflammation by increased reactivity, during which astrocytes release growth factors that stimulate OPC proliferation but impair oligodendrocyte maturation [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. As will become apparent in this review, neuroinflammation and myelination are complexly linked, since cytokines that are secreted during inflammation have a dual role in oligodendrocyte development [bib_ref] A silver lining of neuroinflammation: Beneficial effects on myelination, Goldstein [/bib_ref].
Oxygen fluctuations such as hypoxia and/or hyperoxia can directly and indirectly induce apoptosis and necrosis, and contribute to accumulation of reactive oxygen species (ROS) in the brain [bib_ref] Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α, Brill [/bib_ref] [bib_ref] Brain-immune interactions in perinatal hypoxic-ischemic brain injury, Li [/bib_ref] [bib_ref] Oligodendroglial maldevelopment in the cerebellum after postnatal hyperoxia and its prevention by..., Scheuer [/bib_ref]. Oligodendrocyte precursors are sensitive to the release of ROS from activated microglia, as they lack enzymes needed to counteract oxidative stress [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. Similarly, oligodendrocyte precursors express a large amount of glutamate receptors, which increases their susceptibility to the release of excess glutamate by microglia and the halted or reversed uptake of glutamate by astrocytes [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. However, astrocytes may also shift to a protective type A2 activation state in response to hypoxic conditions, during which they release neurotrophic factors that stimulate proliferation and survival of multiple cell types, among which oligodendrocytes and their precursors [bib_ref] Reactive astrocytes: Production, function, and therapeutic potential, Liddelow [/bib_ref].
Together, preterm-birth related insults such as inflammation and oxygen fluctuations lead to a reduced number of mature oligodendrocytes and consequent myelin insufficiency in the preterm brain. It is under current debate whether survival of oligodendrocytes precursors is impaired as a result of neuroinflammation and oxygen fluctuations, or whether these hits cause arrested maturation of pre-OLs [bib_ref] White matter injury in the preterm infant: Pathology and mechanisms, Back [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions, Volpe [/bib_ref]. In fact, both cell death and arrested maturation of oligodendrocyte precursors may contribute to dWMI, as accumulation of ROS and cytokines leads to apoptosis particularly in pre-OLs, and oxygen fluctuations induce the upregulation of trophic factors that sustain OPC proliferation and survival, keeping them in an immature state [bib_ref] Reactive astrocytes: Production, function, and therapeutic potential, Liddelow [/bib_ref] [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref].
Thus, reducing the impact of detrimental insults such as inflammation and oxygen fluctuations, plus providing trophic or immunomodulatory factors that stimulate oligodendrocyte maturation and myelination are two important strategies to reduce preterm dWMI.
## | the role of growth factors and cytokines in oligodendrocyte maturation and neuroinflammation
Although dWMI can lead to life-long neurological impairments, no therapeutic options to promote white matter development in the preterm brain are currently available. Future therapies would preferably stimulate oligodendrocyte lineage survival and maturation, increasing the proportion of mature, myelin-producing oligodendrocytes. Stimulation of differentiation could be achieved through growth factors and cytokines that directly affect the oligodendrocyte lineage, boosting maturation and/or survival of oligodendrocyte precursors, or indirectly by modulating neuroinflammation, providing a more favorable intracerebral milieu for myelination . In this review, we integrate the current knowledge on the role of promising growth factors and cytokines in healthy white matter development and oligodendrocyte maturation and neuroinflammation after injury from (a) in vitro and (b) in vivo animal models and (c) clinical studies, in order to identify potential therapeutic targets for preterm dWMI.
3.1 | Insulin-like growth factor 1 A compelling quantity of experimental studies has provided evidence for the essential role of insulin-like growth factor 1 (IGF-1) in normal fetal white matter development and in regeneration following cerebral injury [bib_ref] Expanding the mind: Insulin-like growth factor I and brain development, D'ercole [/bib_ref] [bib_ref] The role of growth factors as a therapeutic approach to demyelinating disease, Huang [/bib_ref]. The effects of IGF-1 are mediated by activation of the Type I IGF receptor (IGF1R), broadly expressed on different cell types in the central nervous system (CNS), among which all cells of the oligodendrocyte lineage [bib_ref] Expanding the mind: Insulin-like growth factor I and brain development, D'ercole [/bib_ref] [bib_ref] Insulin-like growth factor type 1 receptor signaling in the cells of oligodendrocyte..., Zeger [/bib_ref]. IGF1R activation is thought to trigger the PI3kinase-Akt Over the years, a wide range of in vitro studies have provided evidence of a direct effect of IGF-1 on proliferation and differentiation of healthy oligodendrocyte lineage cells, ultimately resulting in myelination [bib_ref] Cell-death and control of cellsurvival in the oligodendrocyte lineage, Barres [/bib_ref] [bib_ref] Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from..., Masters [/bib_ref] [bib_ref] Insulin-like growth factor I/somatomedin C: A potent inducer of oligodendrocyte development, Mcmorris [/bib_ref] [bib_ref] Insulin-like growth factor I stimulates oligodendrocyte development and myelination in rat brain..., Mozell [/bib_ref] [bib_ref] Insulinlike growth factor I increases myelination and inhibits demyelination in cultured organotypic..., Roth [/bib_ref] [bib_ref] Human oligodendrocyte precursor cells in vitro: Phenotypic analysis and differential response to..., Wilson [/bib_ref]. However, a study using OPCs derived from human fetal tissue did not find a proliferative response following IGF-1 administration, suggesting the mitogenic effect of IGF-1 may be more pronounced in rodents [bib_ref] Human oligodendrocyte precursor cells in vitro: Phenotypic analysis and differential response to..., Wilson [/bib_ref]. Other than its role in healthy oligodendrocyte development, multiple in vitro studies show that IGF-1 promotes differentiation and myelin production, and inhibits oligodendrocyte cell death when exposed to different WMI-associated stimuli (e.g., hypoxia or inflammation; [bib_ref] IGF-1 protects oligodendrocyte progenitors against TNFalpha-induced damage by activation of PI3K/Akt and..., Pang [/bib_ref] [bib_ref] Delayed IGF-1 administration rescues oligodendrocyte progenitors from glutamate-induced cell death and hypoxic-ischemic..., Wood [/bib_ref].
In vivo studies underline the prominent role of IGF-1 in white matter development, as well as in injury repair. Transgenic mice lacking IGF-1 display a dramatic reduction in size of white matter structures, myelination, and oligodendrocyte numbers [bib_ref] Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss..., Beck [/bib_ref] [bib_ref] Myelination is altered in insulin-like growth factor-I null mutant mice, Ye [/bib_ref] [bib_ref] Insulin-like growth factor type 1 receptor signaling in the cells of oligodendrocyte..., Zeger [/bib_ref]. In line with these findings, mice overexpressing IGF-1 show excessive oligodendrocyte numbers and myelin content [bib_ref] Insulin-like growth factor I increases brain growth and central nervous system myelination..., Carson [/bib_ref] [bib_ref] In vivo effects of insulin-like growth factor-I (IGF-I) on prenatal and early..., Popken [/bib_ref] [bib_ref] In vivo actions of insulin-like growth factor-I (IGF-I) on brain myelination: Studies..., Ye [/bib_ref].
Exogenous IGF-1 treatment in animal models of neonatal brain injury has shown beneficial effects on oligodendrocyte differentiation, survival, and myelination. The effectiveness of IGF-1 therapy in term hypoxic/ischemic encephalopathy (HIE) has been studied in both rodent and larger animal models using different routes of administration. [bib_ref] Intranasal administration of IGF-1 attenuates hypoxic-ischemic brain injury in neonatal rats, Lin [/bib_ref] showed a significant improvement in myelination of the subcortical white matter after intranasal IGF-1 treatment in a rat model of near-term HIE. Moreover, the authors report a mitogenic effect of IGF-1 administration based on an increase in proliferating NG2+ cells, a marker for OPCs. In a similar rat model, intracerebral IGF-1 treatment induced activation of Akt and pGSK3β, inhibiting activation of caspases, thereby reducing brain injury [bib_ref] IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and..., Brywe [/bib_ref]. These results were confirmed in sheep models of near-term HIE, using intracerebral IGF-1 administration [bib_ref] Insulin-like growth factor (IGF)-1 suppresses oligodendrocyte caspase-3 activation and increases glial proliferation..., Cao [/bib_ref] [bib_ref] Insulin-like growth factor-1 reduces postischemic white matter injury in fetal sheep, Guan [/bib_ref]. In a rodent model of severe preterm WMI, where intracerebral LPS was injected, intranasal IGF-1 therapy was shown to reduce preoligodendrocyte (O4+) and mature oligodendrocyte (CC1+) loss, which resulted in myelin recovery [bib_ref] Intranasal administration of insulin-like growth factor-1 protects against lipopolysaccharide-induced injury in the..., Cai [/bib_ref]. In these studies, the reported increases in total oligodendrocyte numbers after IGF-1 treatment were attributed to prevention of oligodendrocyte death, with a less prominent role for oligodendrocyte proliferation. Conversely, a previous study from this group showed conflicting outcomes of IGF-1 treatment, detecting both recovery and exacerbation of injury.
Coadministration of intracerebral LPS and IGF-1 in a low dose reduced oligodendrocyte loss and myelin deficits, while higher doses F I G U R E 1 The underlying pathophysiology of encephalopathy of prematurity (EoP) and proposed mechanism of therapeutic intervention. Preterm birth-related issues such as inflammation and hypoxia/hyperoxia (yellow flash) are thought to lead to oligodendrocyte (OL) death (apoptosis) and/or a maturational arrest of the oligodendrocyte lineage, through the activation of microglia (purple cell) and astrocyte (red cell) reactivity. Microglia release reactive oxygen species (ROS) and pro-inflammatory cytokines, which leads to the death of vulnerable oligodendrocyte precursors (OPCs). Reactive astrocytes respond to inflammation and/or oxygen fluctuations by reducing their uptake of glutamate and increasing the release of proliferative factors (red dots), that inhibit the differentiation of oligodendrocyte precursor cells (OPCs). The proposed therapeutic effect of growth factor-and cytokine-based treatments is indicated in dotted lines. This review summarizes growth factors (blue dots) and cytokines (green dots) that have the potential to stimulate oligodendrocyte maturation/differentiation and increase survival of these cells directly, while simultaneously inhibiting the harmful neuroimmune response caused by microglia and astrocytes. Several routes of administration are proposed, such as intranasal through a local opening in, or through the compromised blood-brain-barrier (BBB) in the acute phase after brain injury of IGF-1 led to intracerebral hemorrhage and exacerbation of brain damage [bib_ref] IGF-1 can either protect against or increase LPS-induced damage in the developing..., Pang [/bib_ref]. These damaging effects were not observed following intranasal treatment with IGF-1 nor in any of the other aforementioned studies, implying that caution is only advised when locally injecting (very high) doses of IGF-1 in an acute inflammatory environment. However, intracerebral injection is a clinically unfeasible route of administration in the instable preterm infant.
Although these models do not represent the commonly observed pattern of WMI in human preterm infants (i.e., dWMI) and/or use a rather invasive administration method of IGF-1, IGF-1 generally seems to have beneficial effects on the oligodendrocyte lineage.
Results from a handful in vitro studies indicate an immunoregulatory effect of IGF-1 directly on astrocytes and microglia, implying an indirect role of IGF-1 on pre-OL differentiation and survival by contributing to a more favorable environment by reducing microglia and astrocyte activation [bib_ref] Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through..., Dodge [/bib_ref] [bib_ref] Insulin-like growth factor-1 abrogates microglial oxidative stress and TNFalpha responses to spreading..., Grinberg [/bib_ref]. [bib_ref] Insulin-like growth factor-1 abrogates microglial oxidative stress and TNFalpha responses to spreading..., Grinberg [/bib_ref] demonstrated a reduction in microglial ROS and tumor necrosis factor α (TNF-α) production following IGF-1 supplementation in rat hippocampal slice cultures. Astrocytic ROS and TNF-α production, however, were not reduced by IGF-1. In contrast,did demonstrate a decrease in ROS levels following IGF-1 treatment in astrocytic cultures, protecting the brain against oxidative injury.
Evidence for the immunomodulatory properties of IGF-1 obtained from in vivo studies seems inconclusive. In a sheep model near-term HIE intracerebral IGF-1 administration led to increased proliferation of (reactive) astrocytes and microglia. While reactive (micro)glia are traditionally associated with dWMI pathophysiology, the authors propose that the increased numbers of reactive glial cells after IGF-1 treatment are associated with improvement of white matter repair. It is suggested that the neuroprotective properties of reactive glia might be the result of paracrine signaling [bib_ref] Insulin-like growth factor (IGF)-1 suppresses oligodendrocyte caspase-3 activation and increases glial proliferation..., Cao [/bib_ref] [bib_ref] Insulin-like growth factor-1 reduces postischemic white matter injury in fetal sheep, Guan [/bib_ref]. Similar observations of potential proregenerative glial subtypes, particularly for astrocytes, have been reported in other studies [bib_ref] Reactive astrocytes: Production, function, and therapeutic potential, Liddelow [/bib_ref] [bib_ref] Lipopolysaccharide-induced microglia activation promotes the survival of midbrain dopaminergic neurons in vitro, Zhou [/bib_ref]. The possible role of reactive glia subtypes in perinatal brain injury remains unclear, though in the majority of studies (micro)glia activation is linked to exacerbation of brain injury and a poorer outcome [bib_ref] Neuroinflammation in the developing brain: Risk factors, involvement of microglial cells, and..., Baud [/bib_ref] [bib_ref] Microglial aggregation in the dentate gyrus: A marker of mild hypoxic-ischaemic brain..., Bigio [/bib_ref] [bib_ref] Prenatal ischemia and white matter damage in rats, Olivier [/bib_ref] [bib_ref] Microglial reaction in axonal crossroads is a hallmark of noncystic periventricular white..., Verney [/bib_ref]. Intranasal IGF-1 administration in a rodent model of severe LPS-induced preterm WMI reduced microglia activation and peripheral immune cell infiltration, even though pro-inflammatory IL-1β and TNF-α concentrations remained unchanged. The authors hypothesize that the direct anti-inflammatory effect of IGF-1 is likely limited and that the observed attenuation of microglia activation and peripheral immune cell infiltration could be the result of reduced oligodendrocyte apoptosis [bib_ref] Intranasal administration of insulin-like growth factor-1 protects against lipopolysaccharide-induced injury in the..., Cai [/bib_ref]. As mentioned previously, another study by this group showed exacerbation of brain injury after local coinjection of a high dose of IGF-1 and LPS. Even though a low dose of IGF-1 did provide protection against oligodendrocyte loss and myelination deficits, it failed to attenuate LPS-induced micro-and astrogliosis and was associated with an increase in peripheral immune cell infiltration. These effects were more pronounced in the higher doses of IGF-1, leading to profuse leukocyte infiltration and subsequent exacerbation of brain injury. The authors suggest that IGF-1 likely negatively affects blood-brain-barrier (BBB) integrity while upregulating chemotactic signaling during episodes of acute inflammation [bib_ref] IGF-1 can either protect against or increase LPS-induced damage in the developing..., Pang [/bib_ref].
Glial cells are an important source of local IGF-1 production during brain development. Endogenous microglial IGF-1 secretion was shown to be hampered in vitro following glutamate treatment in a primary microglial culture obtained from hypoxic rats, mimicking glutamate excitotoxicity in WMI [bib_ref] Role of glutamate and its receptors and insulin-like growth factors in hypoxia..., Sivakumar [/bib_ref]. These findings are supported by in vivo evidence demonstrating a decrease in IGF-1 gene expression in the ipsilateral hemisphere following a hypoxic-ischemic insult in near-term rats [bib_ref] Coordinate IGF-I and IGFBP5 gene expression in perinatal rat brain after hypoxia-ischemia, Lee [/bib_ref]. Interestingly, this decrease in local IGF-1 secretion seems to be distinctive for the neonatal period, as hypoxia/ischemia in older animals leads to a local upregulation IGF-1 [bib_ref] Insulin-like growth factors in the response to cerebral ischemia, Lee [/bib_ref]. These studies emphasize the potential need for IGF-1 supplementation following perinatal hits [bib_ref] Coordinate IGF-I and IGFBP5 gene expression in perinatal rat brain after hypoxia-ischemia, Lee [/bib_ref] [bib_ref] Role of glutamate and its receptors and insulin-like growth factors in hypoxia..., Sivakumar [/bib_ref].
Interestingly, evidence from human studies indicated a reduction in circulatory IGF-1 in the first weeks following extreme preterm birth
## Box 1 jagged-1 and notch in eop
Although the Notch receptor and its ligand Jagged-1 have previously been associated with OPC proliferation thereby regulating the timing of differentiation, Jagged-1 and Notch may be more complexly linked to oligodendrocyte development. Subtle increases in Notch and Sonic hedgehog (Shh) signaling may skew the OPC fate to differentiation instead of recruitment into the homeostatic NG2+ OPC pool, as was demonstrated in the spinal cord of zebrafish larvae [bib_ref] Sequential specification of oligodendrocyte lineage cells by distinct levels of Hedgehog and..., Ravanelli [/bib_ref]. In EoP, neuroinflammation and oxygen fluctuations upregulate the expression of Jagged-1 by astrocytes, thereby activating the Notch-pathway which can contribute to the oligodendrocyte maturational arrest [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] Notch signaling: Key role in intrauterine infection/inflammation, embryonic development, and white matter..., Yuan [/bib_ref]. This was also shown in a mouse model of neonatal hyperoxia, in which WMI could be rescued by blocking the Notch-signaling pathway using a γ-secretase inhibitor . However, a recent in vivo study showed the downregulation of Notch-1 and Hes-1 proteins in the corpus callosum of rat pups subjected to fetal inflammation, which corresponded with reduced maturation of OPCs into mature oligodendrocytes and myelination [bib_ref] Inhibitory effect of LPS on the proliferation of oligodendrocyte precursor cells through..., Ying [/bib_ref]. Together, these results indicate that oligodendrocyte development is tightly regulated by the Notch-pathway, and that subtle imbalances in Notch pathway activation caused by EoP lead to failure of myelination.
due to inadequate endogenous IGF-1 production. This relative IGF-1 deficiency during a developmental time-window analogous to the third trimester of pregnancy has been associated with a poorer neurodevelopmental outcome, as well as with retinopathy of prematurity severity and respiratory complications [bib_ref] Insulin-like growth factor 1 has multisystem effects on foetal and preterm infant..., Hellstrom [/bib_ref]. These observations, along with the promising preclinical data instigated the first feasibility and pharmacokinetics studies using IGF-1 administration in neonates. Even though intravenously administered IGF-1 (including IGF binding protein 3; either from fresh frozen plasma or provided by a pharmaceutical company) was shown to successfully elevate serum IGF1/IGFBP3 concentrations in human extreme preterm infants, IGF-1 half-life was shown to be extremely short (<1 hr; [bib_ref] Longitudinal infusion of a complex of insulinlike growth factor-I and IGF-binding protein-3..., Ley [/bib_ref] [bib_ref] A pharmacokinetic and dosing study of intravenous insulin-like growth factor-I and IGFbinding..., Lofqvist [/bib_ref]. Continuous intravenous infusion during a mean of 2 weeks was deemed safe and feasible; however, one-third of all included infants did not reach target serum IGF-1 levels. Additional studies are needed to determine the optimal dosing regimen and to assess treatment efficacy [bib_ref] Continuous longitudinal infusion of rhIGF-1/rhIGFBP-3 in extremely preterm infants: Evaluation of feasibility..., Hansen-Pupp [/bib_ref]. Even though IGF-1 has been shown to cross the (intact) BBB by active transport, the proportion of the polypeptide that reaches CNS following intravenous injection is likely limited [bib_ref] Interactions of IGF-1 with the blood-brain barrier in vivo and in situ, Pan [/bib_ref] [bib_ref] Insulin-like growth factors cross the blood-brain barrier, Reinhardt [/bib_ref] [bib_ref] Delivery of insulin-like growth factor-I to the rat brain and spinal cord..., Thorne [/bib_ref]. An adult rat study comparing intravenous and intranasal administration of IGF-1 showed significantly higher CNS concentrations following intranasal treatment, with similar blood and peripheral tissue levels. Intranasally administered IGF-1 was shown to bypass the BBB, entering the CNS via the olfactory system and trigeminal nerve [bib_ref] Delivery of insulin-like growth factor-I to the rat brain and spinal cord..., Thorne [/bib_ref]. Thus, while continuous intravenous infusion of IGF-1 would pose a substantial clinical burden with a limited supply to the brain, intranasal IGF-1 administration might offer a less invasive, rapid and direct route to target the CNS [bib_ref] Intranasal administration of insulin-like growth factor-1 protects against lipopolysaccharide-induced injury in the..., Cai [/bib_ref] [bib_ref] Intranasal administration of IGF-1 attenuates hypoxic-ischemic brain injury in neonatal rats, Lin [/bib_ref] [bib_ref] Noninvasive intranasal insulin-like growth factor-I reduces infarct volume and improves neurologic function..., Liu [/bib_ref].
3.2 | Epidermal growth factor family 3.2.1 | Epidermal growth factor and transforming growth factor alpha The epidermal growth factor (EGF) family consists of several factors including EGF, heparin-binding EGF (hb-EGF) and transforming growth factor alpha (TGF-α), that are involved in the proliferation and survival of many cell types, among which cells of the oligodendrocyte lineage [bib_ref] Essential roles of heparin-binding epidermal growth factor-like growth factor in the brain, Oyagi [/bib_ref] [bib_ref] EGF enhances oligodendrogenesis from glial progenitor cells, Yang [/bib_ref]. EGF-family members and the EGF receptor (EGFR) are upregulated in the CNS during development and in response to injury, for example, after hypoxiaischemia [bib_ref] A functional role for EGFR signaling in myelination and remyelination, Aguirre [/bib_ref] [bib_ref] Transforming growth factor-α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal..., Ferrer [/bib_ref] [bib_ref] Prenatal ontogeny of the epidermal growth factor receptor and its ligand, transforming..., Kornblum [/bib_ref] [bib_ref] Essential roles of heparin-binding epidermal growth factor-like growth factor in the brain, Oyagi [/bib_ref]. The EGFR signals through multiple intracellular pathways, such as PI3K/Akt, RAS/ERK, and JAK/STAT (see Box 2; [bib_ref] Epidermal growth factor receptor: Mechanisms of activation and signalling, Jorissen [/bib_ref].
In vitro studies indicate that during normal white matter development, EGF interacts with the mitogens platelet-derived growth factor AA (PDGF-AA) and basic fibroblast growth factor (bFGF) to skew glial precursor cells toward OPC cell fate, and to enhance survival and proliferation of OPCs [bib_ref] EGF enhances oligodendrogenesis from glial progenitor cells, Yang [/bib_ref]. However, when OPCs are cultured with EGF in the absence of PDGF-AA, EGF promotes differentiation into mature (myelin basic protein (MBP)expressing) oligodendrocytes, indicating a role for EGF in both proliferation and differentiation . Since cerebral PDGF-levels decrease during third trimester development [bib_ref] Control of progenitor cell number by mitogen supply and demand, Van Heyningen [/bib_ref] , the role of EGF shifts to promote oligodendrocyte differentiation. In contrast, when PDGF is upregulated in response to, for example, pro-inflammatory cytokines [bib_ref] Astroglial control of oligodendrocyte survival mediated by PDGF and leukemia inhibitory factor-like..., Gard [/bib_ref] [bib_ref] Cytokineregulated expression of platelet-derived growth factor gene and protein in cultured human..., Silberstein [/bib_ref] , EGF may halt oligodendrocyte differentiation and promote proliferation of the extensive OPC pool that is already present in preterm brain injury, making it a less feasible therapeutic candidate to treat preterm WMI.
In vivo rodent experiments confirm the potential of EGF to stimulate both OPC proliferation and differentiation. In a transgenic mouse model, overexpression of EGFR in CNPase+ cells (i.e., pre-OLs) led to increased proliferation of OPCs, mature oligodendrocyte numbers, MBP expression, and myelinated axons [bib_ref] A functional role for EGFR signaling in myelination and remyelination, Aguirre [/bib_ref]. Conversely, hypoactive EGFR signaling in a mouse mutant reduced the number of OPCs (NG2+) and mature (CC1+) oligodendrocytes and myelination during development, supporting the in vitro evidence that EGF plays a role in both proliferation and maturation. Results from several preclinical studies indicate a protective role of EGF in animal models of white matter pathologies. EGFR-overexpressing neonatal mice were less susceptible to developing dWMI after subjection to chronic hypoxia, while an EGFR-antagonist reduced the number of OPCs, mature oligodendrocytes and production of myelin [bib_ref] Intranasal epidermal growth factor treatment rescues neonatal brain injury, Scafidi [/bib_ref]. This had previously been shown in adult EGFRoverexpressing mice recovering from focal demyelination [bib_ref] A functional role for EGFR signaling in myelination and remyelination, Aguirre [/bib_ref]. Moreover, intranasal treatment with exogenous hb-EGF following neonatal chronic hypoxia reduced apoptosis of mature oligodendrocytes, preserved axonal myelination and improved behavioral outcome, through a reduction of Notch-signaling (see Box 1; [bib_ref] Intranasal epidermal growth factor treatment rescues neonatal brain injury, Scafidi [/bib_ref]. Interestingly, in a rabbit model of neonatal intraventricular hemorrhage (IVH), EGF levels were reduced, indicating a deficit in endogenous EGF production after injury [bib_ref] Epidermal growth factor preserves myelin and promotes astrogliosis after intraventricular hemorrhage, Vinukonda [/bib_ref]. Intraventricular injection of EGF increased OPC proliferation, oligodendrocyte maturation, and astrogliosis [bib_ref] Epidermal growth factor preserves myelin and promotes astrogliosis after intraventricular hemorrhage, Vinukonda [/bib_ref].
In an in vitro model of HIE, in which OPCs were deprived of oxygen and glucose, treatment with EGF-family member TGF-α significantly reduced apoptosis of OPCs and mature oligodendrocytes through STAT3 signaling (see Box 2), but had no direct effect on oligodendrocyte differentiation or myelination . This may indicate that TGF-α might preferably be given shortly after WMI is induced to reduce apoptosis of oligodendrocyte precursors. Consistent with in vitro findings, EGF-family member TGF-α also protected OPCs and mature oligodendrocytes against apoptosis in adult ischemic stroke, while TGF-α knockout mice displayed more extensive white matter lesions compared to wild-type mice .
Together, these studies indicate the therapeutic potential of EGF-family members in protection of oligodendrocyte-lineage cells against apoptosis and stimulating OPC proliferation and differentiation after WMI.
Besides their involvement in oligodendrocyte development and survival, EGF-family members modulate neuroinflammatory processes that contribute to the pathophysiology of EoP. It has been shown in vitro that shedding of hb-EGF by astrocytes can be induced by inflammatory cytokines such as IFN-γ and TNF-α, inducing proliferation of microglia, enhancing their phagocytotic capacity and increasing monocyte migration [bib_ref] Secreted phospholipase A 2-IIA-induced a phenotype of activated microglia in BV-2 cells..., Martín [/bib_ref] [bib_ref] Roles for HB-EGF and CD9 in multiple sclerosis, Schenk [/bib_ref]. TGF-α and EGF are known to promote astrogliosis through the EGFR [bib_ref] Epidermal growth factor and fibroblast growth factor-2 have different effects on neural..., Kuhn [/bib_ref] [bib_ref] A role for transforming growth factor α as an inducer of astrogliosis, Rabchevsky [/bib_ref] [bib_ref] Striatal TGF-α: Postnatal developmental expression and evidence for a role in the..., Weickert [/bib_ref]. Expression of hb-EGF by reactive astrocytes has also been demonstrated in active MS lesions, which may trigger further inflammatory events in the lesioned area [bib_ref] Roles for HB-EGF and CD9 in multiple sclerosis, Schenk [/bib_ref]. Single treatment with an anti-EGF antibody was therefore beneficial in an experimental autoimmune encephalomyelitis (EAE) mouse model, by shifting NSC differentiation toward neurons and oligodendrocytes instead of astrocytes (Amir-Levy,. Simultaneous activation of both mitogen receptors (bFGF and PDGF) and EGFR further induces neuroinflammation and oligodendrocyte apoptosis in response to pathogens [bib_ref] Receptor tyrosine kinases play a significant role in human oligodendrocyte inflammation and..., Parthasarathy [/bib_ref] , suggesting that supplementation of EGF should not coincide with extensive FGFR and PDGFR activation.
Further research is warranted to design therapies that balance the potential beneficial effect of EGF-family members on oligodendrocyte survival and maturation while avoiding excessive astrogliosis F I G U R E 2 Proposed model of intracellular pathways that mediate the different stages of oligodendrocyte development. Astrocytes (red cell), triggered by inflammation and/or hypoxia (yellow flash), produce factors that stimulate oligodendrocyte precursor cell (OPC) proliferation, which contributes to the maturational arrest of oligodendrocytes in preterm WMI. In contrast, several factors discussed in this review either directly or indirectly stimulate oligodendrocyte maturation/differentiation, myelination, and survival, through shared intracellular pathways. IGF-1, insulin-like growth factor 1; EGF, epidermal growth factor; TGF, transforming growth factor (α and β); NRGs, neuregulins; GDNF, glial cell-line derived neurotrophic factor; NTs, neurotrophins; Gp130, glycoprotein 130; IL, interleukin (4 and 10); BMP, bone morphogenetic protein; BMPR, BMP receptor; RTK, receptor tyrosine kinase; JAK, janus kinase; STAT, signal transducer and activator of transcription proteins; JNK, c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; cAMP, cyclic adenosine monophosphate; ERK, extracellular signalregulated kinase; mTOR, mammalian target of rapamycin; FoxO1, forkhead box protein O1; Sp1, specificity protein 1; CREB, cAMP response element-binding protein, mTORC1, mTOR complex 1; GSK3β, glycogen synthase kinase 3 β; PPARγ, peroxisome proliferator-activated receptor γ BOX 2 Shared intracellular pathways targeting oligodendrocyte survival and maturation Several trophic and immunomodulatory factors discussed in this review activate shared intracellular pathways downstream of their specific receptors, such as PI3K/Akt, MAPK pathways and JAK/STAT, that are associated with oligodendrocyte survival and maturation . Next to supplementing growth factors and/or cytokines that activate these pathways, a potent treatment strategy for EoP could be to directly target these pathways on a molecular level.
The PI3K/Akt pathway drives oligodendrocyte survival, differentiation and maturation [bib_ref] Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental..., Ishii [/bib_ref] [bib_ref] Insulin-like growth factor 1: At the crossroads of brain development and aging, Wrigley [/bib_ref]. Specifically, the down- The rat sarcoma/extracellular signal-regulated kinases (RAS/ERK) MAP kinase pathway is activated after growth factors (e.g., IGF-1, EGF) bind specific tyrosine kinase receptors, and is involved in all stages of oligodendrocyte lineage progression, but most prominently during myelination [bib_ref] The roles of extracellular related-kinases 1 and 2 signaling in CNS myelination, Gonsalvez [/bib_ref]. Although in vitro evidence has revealed a role for RAS/ERK in oligodendrocyte differentiation, this has not been conclusively demonstrated in vivo (reviews by [bib_ref] The roles of extracellular related-kinases 1 and 2 signaling in CNS myelination, Gonsalvez [/bib_ref]. Moreover, a recent study by [bib_ref] Inhibition of MAPK/ERK pathway promotes oligodendrocytes generation and recovery of demyelinating diseases, Suo [/bib_ref] demonstrated that inhibition of MEK, the direct regulator of ERK1/2, promoted oligodendrocyte differentiation from NPC-derived OPCs in vitro and in vivo in an EAE model. This has led to the hypothesis that RAS/ERK is not directly involved in OPC differentiation, but does come into play during the process of myelination [bib_ref] Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental..., Ishii [/bib_ref]. The RAS/ERK pathway is hypothesized to determine proper myelin thickness relative to axon diameter in vivo through ERK1/2 activation [bib_ref] ERK1/ERK2 MAPK signaling is required to increase myelin thickness independent of oligodendrocyte..., Ishii [/bib_ref] , and in myelin maintenance throughout adulthood [bib_ref] Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental..., Ishii [/bib_ref]. Furthermore, the RAS/ERK pathway has been shown to converge with the PI3K/Akt/mTOR pathway to promote myelination during development and after demyelinating injury at the level of mTORC1, as RAS/ERK by itself could not promote sufficient myelination in mTOR-deficient mice [bib_ref] Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental..., Ishii [/bib_ref]. The potential pleiotropic role of the RAS/ERK pathway in both proliferation and myelination could help explain the involvement of some OPC mitogens, such as bFGF (see Section 3.8) during myelination in mature oligodendrocytes.
Another MAP kinase, JNK, has been described to inhibit differentiation of OPCs by promoting proliferation [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. The JNK pathway is activated after EoP-associated injury, such as neuroinflammation or oxygen fluctuations, which makes it a salient target for intervention after preterm birth [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. Therapeutic inhibition of the JNK pathway rescues myelination after an inflammatory stimulus in vitro and in a rat model of preterm dWMI, potentially through inhibiting OPC proliferation which induces differentiation (van Tilborg et al., unpublished results of our group).
Activation of the MAP kinase p38 has also been associated with oligodendrocyte development [bib_ref] Mechanisms of regulation of oligodendrocyte development by p38 mitogen-activated protein kinase, Chew [/bib_ref]. p38 inhibition leads to reduced expression of oligodendrocyte signature genes such as O1 and O4, and reduced myelin production in vitro. Furthermore, p38MAPK induces phosphorylation of the cyclic adenosine monophosphate response element-binding protein, which induces gene transcription that has been proposed to underlie stimulation of oligodendrocyte differentiation and mitogenesis, for example, after innervation of the IGFR [bib_ref] Intracellular events mediating insulin-like growth factor Iinduced oligodendrocyte development: Modulation by cyclic..., Palacios [/bib_ref] [bib_ref] Insulin-like growth factor 1: At the crossroads of brain development and aging, Wrigley [/bib_ref]. Next to its function during normal development, the p38 MAPK pathway has been associated with upregulation after inflammation. OPCs from p38a specific knockout mice failed to myelinate after differentiation in vitro, and brains of these mouse mutants showed abnormalities in myelin microstructure [bib_ref] The p38α mitogen-activated protein kinase is a key regulator of myelination and..., Chung [/bib_ref]. The p38 MAPK has also been shown to interact with other MAP kinases, such as which may exacerbate neuroinflammation, for example, by targeting the EGFR on oligodendrocytes specifically. Clinical trials involving the exogenous administration of EGF, TGF-α or hb-EGF for the treatment of preterm WMI have not been conducted yet to the best of our current knowledge (clinicaltrials.gov).
## | neuregulins
Neuregulins (NRGs) are members of the EGF-family that have been classically linked to myelination through their secretion by neuronal axons (e.g., [bib_ref] Type III neuregulin-1 promotes oligodendrocyte myelination, Taveggia [/bib_ref]. OPCs and oligodendrocytes also express NRGs, which may give them the capacity to self-regulate their development [bib_ref] Neuregulin signaling regulates neural precursor growth and the generation of oligodendrocytes in..., Calaora [/bib_ref] [bib_ref] Effect of neu differentiation factor isoforms of neonatal oligodendrocyte function, Raabe [/bib_ref]. NRGs signal through different homodimer and heterodimer of the ErbB receptor tyrosine kinases that are related to the EGFR (i.e., ErbB1) and can activate PI3K/Akt and MAPK pathways intracellularly (see Box 2; [bib_ref] GGF/neuregulin induces a phenotypic reversion of oligodendrocytes, Canoll [/bib_ref]. NRG1 is the most widely studied NRG, and has been extensively studied in the context of peripheral myelination.
It has been demonstrated in vitro that NRG1 possesses isoformdependent effects on OPCs and oligodendrocytes [bib_ref] Effect of neu differentiation factor isoforms of neonatal oligodendrocyte function, Raabe [/bib_ref].
Specifically, Type II NRG1 glial growth factor 2 (GGF2) promotes proliferation in OPCs through ErbB3, as OPCs differentiate after soluble ErbB3 is administered to neutralize GGF2 [bib_ref] Neuregulin signaling regulates neural precursor growth and the generation of oligodendrocytes in..., Calaora [/bib_ref] [bib_ref] GGF/neuregulin induces a phenotypic reversion of oligodendrocytes, Canoll [/bib_ref]. In contrast, Type I NRG1 isoforms induce differentiation and reduce apoptosis [bib_ref] Effect of neu differentiation factor isoforms of neonatal oligodendrocyte function, Raabe [/bib_ref]. ErbB4, which binds multiple NRGs, was found to be particularly involved in promoting oligodendrocyte maturation and myelination [bib_ref] Implication of γ-secretase in neuregulin-induced maturation of oligodendrocytes, Lai [/bib_ref] [bib_ref] The ErbB4 neuregulin receptor mediates suppression of oligodendrocyte maturation, Sussman [/bib_ref]. More recently, it has been discovered that Type I isoforms of NRG1 promote a shift in oligodendrocyte phenotype which increases their responsiveness to glutamate through generation of NMDA receptors [bib_ref] Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by oligodendrocytes, Lundgaard [/bib_ref]. This enables oligodendrocytes to respond to environmental triggers to enhance their myelin production. NRG1 Type III was also found to be involved specifically in myelination, as oligodendrocytes cocultured with Type III deficient DRGs formed significantly less myelin sheaths [bib_ref] Type III neuregulin-1 promotes oligodendrocyte myelination, Taveggia [/bib_ref].
In vivo evidence supports the role of NRGs in myelin formation.
Knockout of the nardilysin (Nrd1) gene, an important regulator of NRG1 shedding, resulted in hypomyelination in the CNS and peripheral nervous system, while neuronal Nrd1 overexpression induced NRG1 Types I and III availability and consequent hypermyelination, while the availability of NRG1 Type II was not investigated [bib_ref] Nardilysin regulates axonal maturation and myelination in the central and peripheral nervous..., Ohno [/bib_ref]. Similarly, NRG1 Type III knockout mice showed hypomyelination throughout development and adulthood [bib_ref] Nardilysin regulates axonal maturation and myelination in the central and peripheral nervous..., Ohno [/bib_ref] [bib_ref] Type III neuregulin-1 promotes oligodendrocyte myelination, Taveggia [/bib_ref]. In both studies, numbers of OPCs and mature oligodendrocytes were not altered between mutants and wild types, suggesting that NRG1 Types I and III are specifically involved in myelination and do not play a prominent role in oligodendrocyte lineage progression. In mutant mice with oligodendrocytespecific impairment in NRG1/ErbB signaling, oligodendrocyte morphology was altered, leading to smaller cells and less myelin production per cell [bib_ref] Loss of erbB signaling in oligodendrocytes alters myelin and dopaminergic function, a..., Roy [/bib_ref]. NRGs may therefore be a promising therapeutic agent to enhance myelination capacity of mature oligodendrocytes, perhaps in combination with a therapeutic agent that triggers oligodendrocyte survival or maturation. To our knowledge, in vivo studies in which NRGs are administered as a therapeutic in models of EoP have not yet been conducted.
Microglia have been shown to release NRG1 Types I and III and NRG3 after stimulation with LPS in vitro [bib_ref] Microglia-derived neuregulin expression in psychiatric disorders, Ikawa [/bib_ref]. In vivo, NRG expression was enhanced in microglia and astrocytes after injury [bib_ref] Microglia-derived neuregulin expression in psychiatric disorders, Ikawa [/bib_ref] [bib_ref] Regulation of neuregulin expression in the injured rat brain and cultured astrocytes, Tokita [/bib_ref]. This may be a protective response, as exogenous NRG1β treatment partly reduced microglial and astrocytic activation in mice exposed to LPS, leading to reduced inflammatory cytokine production and improved neuronal survival . However, there have also been some reports indicating antagonism of NRG1 may be beneficial in neuroinflammatory conditions, by reducing the trophic effect of NRG1 on microglia (e.g., [bib_ref] Neuregulin1 modulation of experimental autoimmune encephalomyelitis (EAE), Allender [/bib_ref]. Thus, although NRGs have been In humans, genetically caused disturbances in the NRG/ErbB balance have been associated with several neuropsychiatric disorders, such as schizophrenia, depression and bipolar disorder (see review by [bib_ref] Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases, Mei [/bib_ref] , and have been directly linked to an impaired social performance in children with ASD [bib_ref] Microglia-derived neuregulin expression in psychiatric disorders, Ikawa [/bib_ref]. Interestingly, NRG1 is endogenously upregulated in human umbilical through inhibition of c-JUN phosphorylation [bib_ref] Mechanisms of regulation of oligodendrocyte development by p38 mitogen-activated protein kinase, Chew [/bib_ref].
Numerous cytokines, including the interleukin-family of which IL-4, IL-6, IL-10, and IL-11 discussed in this review, activate the JAK/STAT-pathway downstream of their specific receptors [bib_ref] Opportunities for translation from the bench: Therapeutic intervention of the JAK/STAT pathway..., Liu [/bib_ref]. Activation of STAT1 is linked to oligodendrocyte apoptosis , while STAT3 activation promotes oligodendrocyte survival [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref]. Furthermore, STAT3 is implicated in myelinogenesis during postnatal brain development, but a mouse model with specific STAT3 ablation in oligodendrocytes showed that it is not essential for developmental myelination [bib_ref] Activation of oligodendroglial Stat3 is required for efficient remyelination, Steelman [/bib_ref]. However, when STAT3 activation was ablated in oligodendrocytes, maturation and remyelinating capacity after focal demyelination was impaired [bib_ref] Activation of oligodendroglial Stat3 is required for efficient remyelination, Steelman [/bib_ref]. STAT6 promotes oligodendrocyte differentiation through activation of PPARγ . endothelial cells after preterm birth, and a SNP increasing its availability is linked to improved neurodevelopmental outcome after preterm WMI [bib_ref] Neuregulin-1, the fetal endothelium, and brain damage in preterm newborns, Hoffmann [/bib_ref]. Considering this and its role in myelination described above, NRG1 has been put forward as a protective agent in preterm WMI (see review by [bib_ref] Neuregulin-1: A potential endogenous protector in perinatal brain white matter damage, Dammann [/bib_ref]. However, more recent evidence points to NRG1 isoform-dependent effects that must be taken into consideration. Further preclinical research regarding the effectiveness of NRGs in EoP is warranted to substantiate the initiation of clinical trials.
## | transforming growth factor beta superfamily
The transforming growth factor beta (TGF-β) superfamily consists of at least 30 cytokines, and can be subdivided into TGF-β-type and bone morphogenetic protein (BMP) type-proteins, that play a role in a wide array of physiological processes, among which oligodendrocyte development and gliosis (see Weiss & Attisano, 2013 for a review).
## | tgf-β-type proteins
There are three isoforms of TGF-β that are postulated to play different roles in the CNS during development and disease [bib_ref] The neuroprotective functions of transforming growth factor beta proteins, Dobolyi [/bib_ref] [bib_ref] Lesion-associated expression of transforming growth factor-beta-2 in the rat nervous system: Evidence..., Stoll [/bib_ref]. TGF-β1 has been the most stud- TGF-β1 has been shown to increase proliferation of OPCs and differentiation to mature oligodendrocytes in vitro [bib_ref] TGF-β pro-oligodendrogenic effects on adult SVZ progenitor cultures and its interaction with..., Pinto [/bib_ref] [bib_ref] A role for TGF-beta in oligodendrocyte differentiation, Mckinnon [/bib_ref]. The mitogenic effect of TGF-β1 on OPCs is likely to be indirect and mediated through astrocytes, which secrete Jagged-1 upon stimulation with TGF-β1 (Gómez [bib_ref] TGF-β pro-oligodendrogenic effects on adult SVZ progenitor cultures and its interaction with..., Pinto [/bib_ref] [bib_ref] TGFβ1 induces Jagged1 expression in astrocytes via ALK5 and Smad3 and regulates..., Zhang [/bib_ref]. Through activation of the Notch-1 receptor on OPCs, Notch ligands such as Jagged-1 stimulate OPC proliferation to prevent early differentiation, thereby regulating the timing of oligodendrocyte lineage progression [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref] [bib_ref] Notch receptor activation inhibits oligodendrocyte differentiation, Wang [/bib_ref]. Jagged-1 is also secreted by adult oligodendrocytes, possibly to signal to OPCs that the region is sufficiently myelinated [bib_ref] Notch receptor activation inhibits oligodendrocyte differentiation, Wang [/bib_ref]. However, the relationship between the Notch pathway and oligodendrocyte development is likely more complex, which is highlighted in Box 1. OPCs also express TGF-β receptors, and direct TGF-β1 stimulation is believed to exert a maturational effect [bib_ref] TGF-β pro-oligodendrogenic effects on adult SVZ progenitor cultures and its interaction with..., Pinto [/bib_ref] , likely after Jagged-1 expression by astrocytes is downregulated during normal development (e.g., [bib_ref] Notch receptor activation inhibits oligodendrocyte differentiation, Wang [/bib_ref].
In a series of loss-and gain-of-function experiments, [bib_ref] TGFβ signaling regulates the timing of CNS myelination by modulating oligodendrocyte progenitor..., Palazuelos [/bib_ref] demonstrated that TGF-β signaling is critical for OPC differentiation in vivo, by mediating cell cycle withdrawal through SMAD2/3/4 and downstream FoxO1 and Sp1 activation. It is therefore perhaps surprising that the TGF-β pathway was found to be upregulated in a rat model of neonatal HI brain injury at preterm-equivalent age in humans (Sun, [bib_ref] Ischemia induced neural stem cell proliferation and differentiation in neonatal rat involved..., Strelau [/bib_ref].
Although TGF-β is able to stimulate differentiation of oligodendrocytes, the upregulation of TGF-β after HI injury may have adverse effects on EoP pathology through Jagged-1 expression by astrocytes.
Therefore, studies in HI-injured neonatal rats have aimed to target the TGF-β pathway by antagonizing the ALK-5 (or TGFBR1), that mediates the effect of TGF-β on astrocytes. In a model of preterm injury, the ALK-5 antagonist SB505124 decreased microgliosis and astrogliosis, improved oligodendrogenesis and myelination, and promoted autophagy of potentially toxic debris [bib_ref] Age-dependent effects of ALK5 inhibition and mechanism of neuroprotection in neonatal hypoxic-ischemic..., Kim [/bib_ref]. However, when hypoxic-ischemic injury was induced at a later postnatal age in mice (p9 instead of p6), administration of the ALK-5 antagonist exacerbated hippocampal injury and functional outcome [bib_ref] Age-dependent effects of ALK5 inhibition and mechanism of neuroprotection in neonatal hypoxic-ischemic..., Kim [/bib_ref]. It can be speculated that astrogliosis caused by TGF-β/ALK-5 signaling after preterm birth negatively impacts oligodendrocyte maturation through Jagged-1 expression, while astrogliosis is necessary for demarcating lesions in hypoxic-ischemic injury at term [bib_ref] Age-dependent effects of ALK5 inhibition and mechanism of neuroprotection in neonatal hypoxic-ischemic..., Kim [/bib_ref] or in adult stroke. Thus, in vitro and in vivo evidence suggests that TGF-β can have a dual role in the developing brain by inducing oligodendrocyte maturation directly through TGF-β receptors on OPCs on the one hand, but on the other hand triggering astrocytes to produce Jagged-1 that preserves OPC immaturity by inducing proliferation (e.g., [bib_ref] Notch receptor activation inhibits oligodendrocyte differentiation, Wang [/bib_ref]. TGF-β-based therapies to combat EoP should therefore be aimed at oligodendrocyte-lineage cells specifically, for example, by using NG2+-targeted nanoparticles [bib_ref] Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with..., Rittchen [/bib_ref] , or TGF-β should be given in combination with a Jagged-1 antagonist.
Besides its effect on oligodendrocytes and astrocytic scar formation, TGF-β is also involved in microglia homeostasis and activation [bib_ref] Microglia in brain development, homeostasis, and neurodegeneration, Bohlen [/bib_ref]. TGF-β is often mentioned as an anti-inflammatory cytokine, either dampening the proinflammatory response, or skewing microglial activation to the neuroprotective M2-phenotype [bib_ref] The neuroprotective functions of transforming growth factor beta proteins, Dobolyi [/bib_ref]. Administration of TGF-β1 has been shown to specifically induce microglial apoptosis in vitro [bib_ref] Transforming growth factor-β1 induces apoptosis of rat microglia without relation to bcl-2..., Xiao [/bib_ref]. In addition, TGF-β2 administration reduced spontaneous myelin phagocytosis by microglia in vitro, while it had no effect on the amount of microglia that were recruited [bib_ref] Lesion-associated expression of transforming growth factor-beta-2 in the rat nervous system: Evidence..., Stoll [/bib_ref]. Substantive evidence demonstrating the protective potential of TGF-β isoforms against excitotoxicity is also available [bib_ref] The neuroprotective functions of transforming growth factor beta proteins, Dobolyi [/bib_ref].
Together, these results underline a potential protective effect for TGF-β in EoP. As of yet, no clinical trials using TGF-β or ALK-5 antagonists have been registered in the clinical trials database (clinicaltrials.gov).
## | bone morphogenetic proteins and noggin
BMPs form the second major protein class of the TGF-β superfamily, and BMP receptors are present on oligodendrocytes during all developmental stages [bib_ref] Oligodendrocyte maturation is inhibited by bone morphogenetic protein, See [/bib_ref].
In vitro studies illustrate the inhibitory role of BMPs on oligodendrocyte maturation. It has been shown that BMP-4 applied to primary oligodendrocytes or brain slices irreversibly inhibits OPC differentiation into myelinating cells [bib_ref] Inducible expression of noggin selectively expands neural progenitors in the adult SVZ, Morell [/bib_ref] [bib_ref] Delayed myelination in an intrauterine growth retardation model is mediated by oxidative..., Reid [/bib_ref] [bib_ref] Oligodendrocyte maturation is inhibited by bone morphogenetic protein, See [/bib_ref]. Similarly, BMP-2 cooperates with mitogen PDGF to restrict oligodendrocyte differentiation [bib_ref] Influence of LIF and BMP-2 on differentiation and development of glial cells..., Adachi [/bib_ref]. Oxidative stress has been reported to cause BMP-4 expression leading to oligodendrocyte maturational arrest [bib_ref] Delayed myelination in an intrauterine growth retardation model is mediated by oxidative..., Reid [/bib_ref]. Since the BMP-family negatively impacts oligodendrocyte maturation, it was hypothesized that the BMP-2/4-antagonist Noggin could protect against white matter damage.
Indeed, Noggin overexpression led to increased oligodendrocyte differentiation in an in vitro model using neurospheres [bib_ref] Inducible expression of noggin selectively expands neural progenitors in the adult SVZ, Morell [/bib_ref].
In a mouse model of late preterm hypoxic-ischemic injury, transgenic mice that overexpressed Noggin were protected against WMI, as shown by preservation of cells across the oligodendrocyte-lineage, and subsequent increased myelination [bib_ref] The bone morphogenetic protein antagonist noggin protects white matter after perinatal hypoxiaischemia, Dizon [/bib_ref]. In a rabbit model of IVH, treatment with Noggin similarly rescued oligodendrocyte maturational arrest, preserved myelination, and decreased astrogliosis (Dummula et al., 2011).
Besides their role halting oligodendrocyte maturation, BMPs have been implicated in neuroinflammation in MS and amyotrophic lateral sclerosis (ALS) pathophysiology [bib_ref] Bone morphogenetic proteins in multiple sclerosis: Role in neuroinflammation, Eixarch [/bib_ref] [bib_ref] Antagonizing bone morphogenetic protein 4 attenuates disease progression in a rat model..., Shijo [/bib_ref]. BMP-4 expression by astrocytes was found to be increased in a mutant rat model for ALS, and Noggin was effective at dampening the neuroimmune response, reducing astrogliosis and microglial activation [bib_ref] Antagonizing bone morphogenetic protein 4 attenuates disease progression in a rat model..., Shijo [/bib_ref]. In an in vitro model of oxygen/glucose deprivation and reperfusion, Noggin induced release of iron from microglia, aiding the process of remyelination after injury . BMP signaling affects differentiation of interneuron subpopulations differently, stimulating differentiation of parvalbumin interneuron differentiation, but halting somatostatin interneuron development [bib_ref] Differential effects of BMP signaling on parvalbumin and somatostatin interneuron differentiation, Mukhopadhyay [/bib_ref]. This suggests upregulation of BMPs in EoP may alter interneuron development as well as oligodendrocyte maturation.
In humans, elevation of BMP-4 in the brain has been associated with IVH in preterm infants [bib_ref] Bone morphogenetic protein inhibition promotes neurological recovery after intraventricular hemorrhage, Duchatel [/bib_ref]. Although results from preclinical studies show that BMP-antagonist Noggin is a promising option for the treatment of EoP, more research in preclinical models is warranted before clinical trials can be initiated (clinicaltrials.gov).
## | glial cell line-derived neurotrophic factor family
The glial cell line-derived neurotrophic factor (GDNF) family has also recently emerged as a potential candidate for treatment of white matter pathologies. GDNF-family members have ranging affinities to the GDNF family receptor α (GFRα) subtypes, and these receptors are differentially expressed on cells of the oligodendrocyte lineage, suggesting that each GDNF-family member may play a unique role during oligodendrocyte development [bib_ref] Neurotrophic factors and their effects in the treatment of multiple sclerosis, Razavi [/bib_ref] [bib_ref] Ischemia induced neural stem cell proliferation and differentiation in neonatal rat involved..., Strelau [/bib_ref]. When bound by a GDNF-family member, the desig- GDNF is produced by astrocytes and microglia in response to injury [bib_ref] GDNF, a neuron-derived factor upregulated in glial cells during disease, Duarte Azevedo [/bib_ref] and has been found to be protective after brain ischemia through promotion of neuronal survival (see review by [bib_ref] Neuroprotection by GDNF in the ischemic brain, Duarte [/bib_ref]. However, sustained GDNF activation may prolong neuroinflammation [bib_ref] GDNF, a neuron-derived factor upregulated in glial cells during disease, Duarte Azevedo [/bib_ref].
## | neurotrophins
The neurotrophin (NT) family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5, and NT-6.
Each NT acts with high affinity on a preferred receptor of the tropomyosin-related kinase (Trk) family and with low affinity to the p75 NT receptor present on oligodendrocytes [bib_ref] IGF-I and NT-3 signaling pathways in developing oligodendrocytes: Differential regulation and activation..., Ness [/bib_ref]. NTs and their receptors are upregulated starting from early embryonic neurodevelopment [bib_ref] The role of neurotrophins during early development, Bernd [/bib_ref] and likely provide trophic support to neurons and oligodendrocytes throughout life.
## After cns injury, expression of nts and their receptors has been
shown to be increased [bib_ref] Exploring the role of nerve growth factor in multiple sclerosis: Implications in..., Acosta [/bib_ref] [bib_ref] Neurotrophins: Roles in neuronal development and function, Huang [/bib_ref] [bib_ref] IGF-I and NT-3 signaling pathways in developing oligodendrocytes: Differential regulation and activation..., Ness [/bib_ref].
In vitro results indicate that NTs, through their preferred receptors, may have different effects on cells of the oligodendrocyte lineage. BDNF, well-known for its trophic effect on the brain, has been shown to enhance myelination in a model where DRG neurons are cocultured with oligodendrocytes [bib_ref] Brain-derived neurotrophic factor promotes central nervous system myelination via a direct effect..., Xiao [/bib_ref]. Furthermore, it was found that the effect of BDNF on myelination was mediated by TrkB expression on oligodendrocytes, as myelination of both DRG subtypes expressing either TrkA or TrkB occurred after BDNF supplementation, and this effect was only suppressed by a TrkB-inhibitor. In an in vitro model of chemically induced hypoxic injury, astrocyteconditioned medium containing BDNF promoted OPC differentiation [bib_ref] Astrocytes promote oligodendrogenesis after white matter damage via brain-derived neurotrophic factor, Miyamoto [/bib_ref]. This study also showed that the beneficial effect of BDNF was halted by use of the PI3K-inhibitor LY294002 or when astrocyte-conditioned medium was depleted of BDNF with a TrkB-Fc decoy receptor [bib_ref] Astrocytes promote oligodendrogenesis after white matter damage via brain-derived neurotrophic factor, Miyamoto [/bib_ref]. Not much is known about the effect of NT-4/5 on oligodendrocyte maturation, but this NT shares about 95% sequence homology with BDNF and also binds to TrkB, suggesting it may have a similar beneficial effect [bib_ref] Neurotrophins: Role in adverse pregnancy outcome, Dhobale [/bib_ref]. Similar to BDNF, NT-3 induced substantial axonal myelination when oligodendrocytes were cocultured with DRG neurons, as well as survival and proliferation of OPCs and mature oligodendrocytes [bib_ref] A crucial role for neurotrophin-3 in oligodendrocyte development, Barres [/bib_ref] [bib_ref] Novel role of sphingosine kinase 1 as a mediator of neurotrophin-3 action..., Saini [/bib_ref] [bib_ref] NT-3 weakly stimulates proliferation of adult rat O1-O4+ oligodendrocyte-lineage cells and increases..., Yan [/bib_ref]. In an isolated OPC culture, biofabricated sponges emitting NT-3 subtly increased differentiation of primary OPCs toward the pre-OL stage, but the population of MBP+ mature oligodendrocytes was similar to the untreated condition, indicating NT-3 only affected oligodendrocyte differentiation but not final maturation [bib_ref] Purine-crosslinked injectable chitosan sponges promote oligodendrocyte progenitor cells' attachment and differentiation, Mekhail [/bib_ref]. In another experiment where DRG neurons were cocultured with oligodendrocytes, administration of NGF indirectly reduced spontaneous myelination through TrkA present on DRG neurons, whereas NGF had no effect on myelin expression of purified OPCs, or when oligodendrocytes were cocultured with DRG neurons lacking TrkA [bib_ref] NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes, Chan [/bib_ref]. This is in contrast to BDNF, which directly affects myelination of oligodendrocytes [bib_ref] Brain-derived neurotrophic factor promotes central nervous system myelination via a direct effect..., Xiao [/bib_ref]. NGF has also been reported to cause apoptosis of mature oligodendrocytes cultured from neonatal rat brains through p75/JNK (see Box 2; [bib_ref] Death of oligodendrocytes mediated by the interaction of nerve growth factor with..., Casaccia-Bonnefil [/bib_ref] , but apoptosis was not seen after NGF administration to postmitotic oligodendrocytes isolated from human tissue [bib_ref] p75 neurotrophin receptor expression on adult human oligodendrocytes: Signaling without cell death..., Ladiwala [/bib_ref].
Trophic effects of NTs on myelination were largely confirmed in animal models. In BDNF+/− mice, oligodendrocyte numbers are reduced and myelination is impaired in the forebrain during the postnatal period [bib_ref] Brain-derived neurotrophic factor promotes central nervous system myelination via a direct effect..., Xiao [/bib_ref]. TrkB is likely a mediator of myelination through BDNF, as conditional knockout of TrkB in oligodendrocytes restricted formation of thick myelin sheaths during development [bib_ref] Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation, Wong [/bib_ref]. In models of neonatal WMI, knockdown of BDNF expression in astrocytes reduces oligodendrogenesis and exacerbates WMI [bib_ref] Astrocytes promote oligodendrogenesis after white matter damage via brain-derived neurotrophic factor, Miyamoto [/bib_ref] , whereas upregulation of BDNF and TrkB through induced histone acetylation increases OPC proliferation, oligodendrocyte maturation and myelination, and improved behavioral outcome . In a model of neonatal hypoxic-ischemic injury, genetically modified MSCs that overexpressed BDNF did not have a superior effect on reducing white matter damage compared to emptyvector MSCs, but did decrease lesion size and motor outcome compared to empty vector-MSCs [bib_ref] Therapeutic potential of genetically modified mesenchymal stem cells after neonatal hypoxic-ischemic brain..., Van Velthoven [/bib_ref]. NT-3 has been proven effective in rescuing myelination and enhancing remyelination in mouse models of MS [bib_ref] Repeated injuries dramatically affect cells of the oligodendrocyte lineage: Effects of PDGF..., Fressinaud [/bib_ref] [bib_ref] Neurotrophin-3 specifically increases mature oligodendrocyte population and enhances remyelination after chemical demyelination..., Jean [/bib_ref] [bib_ref] Neurotrophin-3 gene modified mesenchymal stem cells promote remyelination and functional recovery in..., Zhang [/bib_ref] and spinal cord injury [bib_ref] Sonic hedgehog and neurotrophin-3 increase oligodendrocyte numbers and myelination after spinal cord..., Thomas [/bib_ref]. Finally, TrkA-agonist BNN27, which mimics the effects of NGF but with improved pharmacokinetics, protects the brain from demyelination and stimulates oligodendrocyte maturation in a cuprizone-induced mouse model of MS [bib_ref] The novel synthetic microneurotrophin BNN27 protects mature oligodendrocytes against cuprizone-induced death, through..., Bonetto [/bib_ref].
A vast amount of literature is available about the role of NTs, particularly BDNF, in neuroinflammation. It is generally accepted that NT release functions as a protective mechanism following neuroinflammation, as NT insufficiency is linked to a wide array of neuropsychiatric conditions with a neuroinflammatory component [bib_ref] Brain-derived neurotrophic factor in brain disorders: Focus on neuroinflammation, Lima Giacobbo [/bib_ref]. While neuroinflammation can induce NT release in astrocytes, an excess of pro-inflammatory cytokines, particularly IL-1β, can also halt neurotrophic support [bib_ref] Neuroimmunologic and neurotrophic interactions in autism spectrum disorders: Relationship to neuroinflammation, Ohja [/bib_ref] [bib_ref] Brain-derived neurotrophic factordependent synaptic plasticity is suppressed by interleukin-1β via p38 mitogen-activated..., Tong [/bib_ref]. Nonetheless, NGF and BDNF have been shown to promote a neuroprotective phenotype in microglia [bib_ref] Regulatory effects of neuroinflammatory responses through brain-derived neurotrophic factor signaling in microglial..., Lai [/bib_ref] [bib_ref] Neurotrophins inhibit major histocompatibility class II inducibility of microglia: Involvement of the..., Neumann [/bib_ref] [bib_ref] NGF steers microglia toward a neuroprotective phenotype, Rizzi [/bib_ref] and reduce astrogliosis in in vitro studies [bib_ref] Nerve growth factor attenuates proliferation of astrocytes via the p75 neurotrophin receptor, Cragnolini [/bib_ref]. These results support the notion that NTs contribute to dampening the immune response in the injured brain.
In human studies, lowered maternal and cord blood BDNF, NGF, and NT-3 levels have been reported in preterm compared to term born infants [bib_ref] Neurotrophins: Role in adverse pregnancy outcome, Dhobale [/bib_ref]. However, postnatal inflammation in extremely preterm neonates has been associated with an upregulation of NTs in blood [bib_ref] Antecedents and correlates of blood concentrations of neurotrophic growth factors in very..., Leviton [/bib_ref] , which likely serves as an endogenous protective response, as increased systemic NT levels have been linked to improved cognitive outcomes at 10 years of age [bib_ref] Among children born extremely preterm a higher level of circulating neurotrophins is..., Kuban [/bib_ref]. In sum, endogenous production of NTs, particularly BDNF, has been associated with improvement of neurodevelopmental outcome following preterm birth, and exogenous NT supplementation could provide a valuable treatment option to enhance oligodendrocyte maturation and reduce neuroinflammation. Systemic administration of NTs is likely complicated by restricted passage of these large molecules over the (intact) BBB [bib_ref] Glial cell line-derived neurotrophic factor does not enter normal mouse brain, Kastin [/bib_ref] [bib_ref] Neurotrophins, neuroprotection and the blood-brain barrier, Pardridge [/bib_ref]. Alternative strategies to ensure optimal delivery of NTs are discussed in Section 4. Currently, no clinical trials have been initiated in preterm infants, but several have been planned and/or conducted for the treatment of traumatic and degenerative brain diseases (clinicaltrials.gov).
## | glycoprotein 130 receptor cytokine family
Glycoprotein 130 (gp130) is a cytokine receptor subunit that interacts with several cytokine receptors like the soluble receptors for IL-6, IL-11, LIF, and CNTF, and is localized throughout the brain on both neurons and glial cells [bib_ref] Characteristic localization of gp130 (the signal-transducing receptor component used in common for..., Watanabe [/bib_ref]. For IL-6, the IL-6R is minimally present on human oligodendrocytes [bib_ref] Multiple sclerosis: Cytokine receptors on oligodendrocytes predict innate regulation, Cannella [/bib_ref] , therefore it mainly communicates through gp130 using a process [bib_ref] Soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro differentiation of purified..., Valerio [/bib_ref] [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref] [bib_ref] Enhancement of oligodendrocyte differentiation from murine embryonic stem cells by an activator..., Zhang [/bib_ref]. IL-11, CNTF, and LIF have also been shown to increase survival of oligodendrocyte lineage cells [bib_ref] Activation of oligodendroglial Stat3 is required for efficient remyelination, Steelman [/bib_ref] [bib_ref] Interleukin-11 potentiates oligodendrocyte survival and maturation, and myelin formation, Zhang [/bib_ref] [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref]. Interestingly, it was shown for IL-11 that its antiapoptotic effect is predominantly mediated by STAT3 in cells of the neural lineage such as OPCs, but can be pro-apoptotic through STAT1 signaling in CD11c+ dendritic cells [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref] , indicating a differential effect of IL-11 on downstream STAT subtype involved based on cell lineage. Whether this pro-apoptotic effect of IL-11 extends to (Cd11c+) microglia has not been examined yet. In an in vitro model where OPCs were exposed to TNF-α, IL-11 reduced caspase cleavage, suggesting that IL-11 is protective against apoptosis even in the presence of an inflammatory stimulus [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref]. Additionally, in a BV-2 microglial cell line culture, IL-11 dose-dependently inhibited myelin phagocytosis [bib_ref] Local overexpression of interleukin-11 in the central nervous system limits demyelination and..., Maheshwari [/bib_ref]. In a glial coculture, activation of the TNF-α receptor TNFR2 on astrocytes led to enhanced LIF production, which consequently stimulated oligodendrocyte maturation, suggesting TNFR2 mediates a protective effect of LIF in response to TNF-α [bib_ref] Astrocyte-specific activation of TNFR2 promotes oligodendrocyte maturation by secretion of leukemia inhibitory..., Fischer [/bib_ref].
In vivo evidence implicating gp130 ligands in oligodendrocyte maturation and myelination comes largely from animal models of MS. Knockout of IL-11Rα, LIF, or CNTF in MS models exacerbated demyelination and oligodendrocyte loss compared to wild-type mice, while increasing glial reactivity [bib_ref] Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss, Butzkueven [/bib_ref] [bib_ref] CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic..., Linker [/bib_ref] [bib_ref] Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory..., Zhang [/bib_ref]. In line with the knockout studies, local overexpression of IL-11 increased mature oligodendrocytes and remyelination, and decreased microglial activation in a cuprizone-induced mouse model of MS [bib_ref] Local overexpression of interleukin-11 in the central nervous system limits demyelination and..., Maheshwari [/bib_ref]. LIF has also been shown to be a beneficial trophic factor for myelination during postnatal development, as LIF knockout mice showed reduced optic nerve myelination [bib_ref] Leukemia inhibitory factor regulates the timing of oligodendrocyte development and myelination in..., Ishibashi [/bib_ref]. Therapeutic targeting of NG2+ OPCs/pre-OLs with nanoparticles containing LIF has proven to be effective in promoting remyelination after focal demyelinating injury [bib_ref] Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with..., Rittchen [/bib_ref].
In contrast, LIF activation has also been shown to adversely influence oligodendrocyte maturation after perinatal hypoxic-ischemic injury through the induction of Notch signaling, which promotes NSC/OPC proliferation rather than maturation [bib_ref] Leukemia inhibitory factor participates in the expansion of neural stem/progenitors after perinatal..., Covey [/bib_ref].
Although the effect of gp130-ligands has been well established in relation to oligodendrocyte survival and maturation (e.g., D'Souza, [bib_ref] Ciliary neurotrophic factor selectively protects human oligodendrocytes from tumor necrosis factor-mediated injury, D'souza [/bib_ref] [bib_ref] Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and..., Mayer [/bib_ref] , they are upregulated during neuroinflammation and have contrasting effects [bib_ref] The role of the leukemia inhibitory factor receptor in neuroprotective signaling, Davis [/bib_ref] [bib_ref] Interleukin-11 induces preterm birth and modulates decidual inflammasome gene expression in mice, Winship [/bib_ref]. IL-11 treatment decreases TNF-α expression and skews microglia toward a protective M2 phenotype in the adult rodent brain [bib_ref] Spreading depression requires microglia and is decreased by their M2a polarization from..., Pusic [/bib_ref]. Through gp130, LIF has also been shown to increase expression of antioxidant genes in oligodendrocytes, which may decrease damage caused by oxidative stress from excessive ROS production by reactive microglia [bib_ref] Leukemia inhibitor factor promotes functional recovery and oligodendrocyte survival in rat models..., Rowe [/bib_ref]. In contrast, IL-6 and CNTF/soluble CNTFRα have been shown to induce pro-inflammatory microglial activation [bib_ref] Ciliary neurotrophic factor (CNTF) plus soluble CNTF receptor α increases cyclooxygenase-2 expression,..., Lin [/bib_ref] , which may exacerbate neuroinflammation during EoP.
In humans, elevated levels of IL-11 in decidua have been found after preterm compared to term birth [bib_ref] Progestin suppresses thrombinand interleukin-1β-induced interleukin-11 production in term decidual cells: Implications for..., Cakmak [/bib_ref]. These
## | interleukin-4 and interleukin-10
Due to their canonical status as anti-inflammatory cytokines, much research has been conducted on IL-10 and IL-4 in neuroinflammatory pathologies (e.g., [bib_ref] The therapeutic potential of interleukin-10 in neuroimmune diseases, Kwilasz [/bib_ref].
Production of IL-10 and IL-4 by immune cells shifts the microglial phenotype to enhance recovery [bib_ref] The therapeutic potential of interleukin-10 in neuroimmune diseases, Kwilasz [/bib_ref] , thereby counteracting the effects of pro-inflammatory cytokines such as IL-1β, TNF-α, and granulocyte-macrophage colony stimulating factor (GM-CSF; [bib_ref] Microglia activated by IL-4 or IFN-γ differentially induce neurogenesis and oligodendrogenesis from..., Butovsky [/bib_ref] [bib_ref] IL-10 inhibits granulocyte-macrophage colony-stimulating factor-dependent human monocyte survival at the early stage..., Hashimoto [/bib_ref]. All in all these anti-inflammatory cytokines contribute to a more favorable environment for neuronal and oligodendrocyte survival [bib_ref] Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in..., Yang [/bib_ref] [bib_ref] Interleukin-10 provides direct trophic support to neurons, Zhou [/bib_ref] , which might also hold true for oligodendrocytes. Aside from their antiapoptotic effects, IL-10 and IL-4 promote neurogenesis and oligodendrogenesis [bib_ref] Microglia activated by IL-4 or IFN-γ differentially induce neurogenesis and oligodendrogenesis from..., Butovsky [/bib_ref] [bib_ref] Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in..., Yang [/bib_ref]. Specifically, oligodendrogenesis is instigated via IGF-1 production by IL-4-conditioned microglia [bib_ref] Microglia activated by IL-4 or IFN-γ differentially induce neurogenesis and oligodendrogenesis from..., Butovsky [/bib_ref]. The studies on direct effects of IL-4 on OLs are conflicting as some studies find no direct effects of IL-4 on cells of the oligodendrocyte-lineage in vitro [bib_ref] Microglia activated by IL-4 or IFN-γ differentially induce neurogenesis and oligodendrogenesis from..., Butovsky [/bib_ref] [bib_ref] IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven..., Psachoulia [/bib_ref] , others report inhibition of differentiation of these cells [bib_ref] Reducing Th2 inflammation through neutralizing IL-4 antibody rescues myelination in IUGR rat..., Zanno [/bib_ref] or stimulation of OPC differentiation through transcription factor PPARγ .
Treatment with IL-10 in vivo has shown promise in several models of neonatal brain injury. For instance, IL-10 decreased white matter lesion size after neonatal excitotoxic brain injury in mice [bib_ref] Effects of interleukin-10 on neonatal excitotoxic brain lesions in mice, Mesples [/bib_ref]. A study by Pang, Rodts-Palenik, [bib_ref] Suppression of glial activation is involved in the protection of IL-10 on..., Pang [/bib_ref] showed that IL-10 decreased apoptosis and enhanced presence of pre-OLs, mature oligodendrocytes, and MBP density in a rat model of intrauterine infection. Microglial activation and astrogliosis were also reduced after IL-10 treatment [bib_ref] Suppression of glial activation is involved in the protection of IL-10 on..., Pang [/bib_ref]. NSCs modified to overexpress IL-10 rescued demyelination in a model for MS, by suppressing the infiltration and stimulating the apoptosis of peripheral immune cells and causing a more favorable intracerebral milieu [bib_ref] Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in..., Yang [/bib_ref]. IL-10 overexpression also favored differentiation of NSCs into neurons or oligodendrocytes instead of astrocytes in vitro and in vivo, thereby reducing astrogliosis and stimulating remyelination.
It has been shown that IL-4 may elicit a different response in neonates versus adults in in vivo experimental models. In a rat model of intrauterine growth restriction, IL-4 was significantly increased in the brain of growth-restricted pups, and treatment with a neutralizing IL-4 antibody directly after birth improved oligodendrocyte maturation and white matter development, by decreasing the Th2 neuroinflammatory response [bib_ref] Reducing Th2 inflammation through neutralizing IL-4 antibody rescues myelination in IUGR rat..., Zanno [/bib_ref]. In contrast, IL-4-based therapies show great promise in regenerating white matter in models of adult inflammatory brain injury .
Preterm infants that homozygously carry the IL-10 "high producer" allele were less vulnerable to development of periventricular WMI as assessed by ultrasound echodensities, and showed a better neurodevelopmental outcome at 2 years of age [bib_ref] Interleukin-10 high producer allele and ultrasound-defined periventricular white matter abnormalities in preterm..., Dördelmann [/bib_ref]. In conclusion, IL-10 and IL-4 protect oligodendrocytes against inflammatory conditions, either by increasing survival or by halting neuroinflammatory processes. Although both IL-10 and IL-4 seem to positively affect remyelination in adults, only IL-10 is deemed safe in neonatal models. To our knowledge, IL-4 and IL-10 have not been explored in clinical trials of preterm brain injury (clinicaltrials.gov).
## | cxc chemokine family
The CXC-family is a subclass of chemotactic cytokines (i.e., chemokines) that are expressed in the CNS, and their receptors have been found on cells of the oligodendrocyte lineage, as well as other CNS cell types [bib_ref] Characterization of chemokines and their receptors in the central nervous system: Physiopathological..., Bajetto [/bib_ref] [bib_ref] Chemokines and brain functions, Banisadr [/bib_ref]. CXCRs have been reported to activate the RAS/ERK and PI3K/Akt pathways in oligodendrocytes [bib_ref] CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and..., Tian [/bib_ref].
CXCL1 was found to promote OPC proliferation in rat isolated OPC cultures [bib_ref] The chemokine growth-regulated oncogene-alpha promotes spinal cord oligodendrocyte precursor proliferation, Robinson [/bib_ref] and
human preterm fetal brain slices [bib_ref] The effect of CXCL1 on human fetal oligodendrocyte progenitor cells, Filipovic [/bib_ref] , but only in conjunction with astrocytes and/or PDGF, suggesting that CXCL1 acts together with other factors to mediate the proliferative effect on oligodendrocytes [bib_ref] Oligodendrocytes: Biology and pathology, Bradl [/bib_ref]. The direct effect of CXCL1 on oligodendrocytes was studied in transgenic mice, in which knocking out its receptor CXCR2 led to a reduced number of mature oligodendrocytes and consequent hypomyelination, but an increased population of OPCs (Padovani-Claudio, [bib_ref] Alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice..., Padovani-Claudio [/bib_ref]. In a study by , it was shown that a CXCR2 antagonist was effective in stimulating OPC proliferation and differentiation in a cuprizone-induced mouse model of MS. Only a small subpopulation of oligodendrocytes expresses CXCR2 in human fetal and adult MS tissue [bib_ref] GRO-alpha and CXCR2 in the human fetal brain and multiple sclerosis lesions, Filipovic [/bib_ref] [bib_ref] The effect of CXCL1 on human fetal oligodendrocyte progenitor cells, Filipovic [/bib_ref] , therefore CXCL1 acts mainly on human oligodendrocytes through other cell types such as astrocytes [bib_ref] Oligodendrocytes: Biology and pathology, Bradl [/bib_ref].
CXCL12, another member of the CXC-family, exerts different effects on OPCs and oligodendrocytes through its two receptors CXCR4 and CXCR7 [bib_ref] CXCR7 is involved in human oligodendroglial precursor cell maturation, Kremer [/bib_ref] [bib_ref] Modulation of rat oligodendrocyte precursor cells by the chemokine CXCL12, Maysami [/bib_ref]. Initially, CXCL12 stimulates migration of OPCs through CXCR4 that activates downstream MEK/ERK and PI3K/Akt pathways [bib_ref] CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and..., Tian [/bib_ref]. During normal development, CXCR4 is downregulated and CXCR7 is upregulated [bib_ref] A role for CXCR4 signaling in survival and migration of neural and..., Dziembowska [/bib_ref] [bib_ref] Activation of CXCR7 receptor promotes oligodendroglial cell maturation, Göttle [/bib_ref] , thereby switching the role of CXCL12 to promote differentiation and maturation through CXCR7 in later stages of development [bib_ref] Activation of CXCR7 receptor promotes oligodendroglial cell maturation, Göttle [/bib_ref] [bib_ref] CXCR7 is involved in human oligodendroglial precursor cell maturation, Kremer [/bib_ref]. Although downregulated during development, CXCR4 was also found to be crucial for oligodendrocyte maturation in a mouse model of cuprizoneinduced demyelination, where blockade of the CXCR4 receptor using either a pharmacological or genetic (lentiviral) approach, decreased the number of mature oligodendrocytes, while increasing the preoligodendrocyte population [bib_ref] CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination, Patel [/bib_ref].
Next to direct effects of CXC-chemokines on cells of the oligodendrocyte-lineage, they could indirectly affect EoP through their role in inflammatory processes. For example, CXCL1 and CXCL5
can contribute to neuroinflammation through their mutual receptor CXCR2. In a rat model of hypoxic-ischemic injury, antagonizing CXCR2 attenuated microglial activation which rescued myelination and BBB integrity, whereas supplementing CXCL5 caused the opposite effect [bib_ref] CXCL5 signaling is a shared pathway of neuroinflammation and blood-brain barrier injury..., Wang [/bib_ref]. Antagonizing CXCR2 similarly reduced neuroinflammation and preserved white matter microstructure in a mouse model of chorioamnionitis [bib_ref] CXCR2 blockade mitigates neural cell injury following preclinical chorioamnionitis, Yellowhair [/bib_ref]. CXCL12 secretion by microglia is increased in response to hypoxia in vitro and in neonatal and adult rodents after hypoxicischemic injury [bib_ref] Expression of syndecan-2 in the amoeboid microglial cells and its involvement in..., Kaur [/bib_ref] , but CXCL12 expression is downregulated in brain endothelial cells in vitro after stimulation with LPS or TNF-α [bib_ref] Novel insights into neuroinflammation: Bacterial lipopolysaccharide, tumor necrosis factor α, and ureaplasma..., Silwedel [/bib_ref]. In contrast to CXCL1 and CXCL5, decreased CXCL12 levels caused by neuroinflammation may cause additional harm, as was shown in an EAE model where antagonism of CXCL12 exacerbated WMI [bib_ref] CXCL12 expression within the CNS contributes to the resistance against experimental autoimmune..., Miljkovic [/bib_ref].
In humans, upregulation of certain CXCs have been implicated in several CNS diseases with an inflammatory hallmark, such as MS,
Alzheimer's disease, and ischemic stroke [bib_ref] CC and CXC chemokines are pivotal mediators of cerebral injury in ischaemic..., Mirabelli-Badenier [/bib_ref] , and are also associated with preterm birth [bib_ref] Differential expression of CXC chemokines CXCL10 and CXCL12 in term and pre-term..., Aminzadeh [/bib_ref] [bib_ref] Epithelial cell-derived neutrophilactivating peptide-78 is present in fetal membranes and amniotic fluid..., Keelan [/bib_ref] [bib_ref] Possible early prediction of preterm birth by determination of novel proinflammatory factors..., Malamitsi-Puchner [/bib_ref]. CXCfamily members have not yet been used in clinical trials for the prevention of EoP (clinicaltrials.gov).
## | other factors
Several other (growth) factors have been implicated in oligodendrocyte lineage development and survival, but evidence underlining their potential efficacy and working mechanisms to combat EoP are still limited. Factors such as bFGF, PDGF, granulocyte-CSF (G-CSF), and hepatocyte growth factor (HGF) are primarily known as (OPC) mitogens [bib_ref] Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white..., Armstrong [/bib_ref] [bib_ref] PDGF and FGF2 regulate oligodendrocyte progenitor responses to demyelination, Frost [/bib_ref] [bib_ref] NG2 cells in white matter but not gray matter proliferate in response..., Hill [/bib_ref] [bib_ref] Platelet-derived growth factor-AA mediates oligodendrocyte lineage differentiation through activation of extracellular signalregulated..., Hu [/bib_ref] [bib_ref] CD82 blocks cMet activation and overcomes hepatocyte growth factor effects on oligodendrocyte..., Mela [/bib_ref] [bib_ref] Mechanisms regulating the development of oligodendrocytes and central nervous system myelin, Mitew [/bib_ref] [bib_ref] Hepatocyte growth factor (HGF) promotes oligodendrocyte progenitor cell proliferation and inhibits its..., Ohya [/bib_ref] [bib_ref] Human oligodendrocyte precursor cells in vitro: Phenotypic analysis and differential response to..., Wilson [/bib_ref] , which keep OPCs in the cell cycle and restrict their differentiation. Concomitant upregulation of several of these factors after brain injury may help sustain the immature OPC population [bib_ref] Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white..., Armstrong [/bib_ref] [bib_ref] Fibroblast growth factor signaling in oligodendrocyte-lineage cells facilitates recovery of chronically demyelinated..., Furusho [/bib_ref] and thus inhibit myelination. However, some of these factors could contribute to oligodendrocyte maturation and subsequent myelination, either through a direct effect on oligodendrocytes or through facilitating a better intracerebral milieu.
bFGF and PDGF are significantly reduced in preterm compared to term umbilical cord blood [bib_ref] Differential secretion of angiopoietic factors and expression of microRNA in umbilical cord..., Gródecka-Szwajkiewicz [/bib_ref]. Both bFGF and PDGF are known to stimulate OPC proliferation, but bFGF may exert this effect through inhibition of maturation, keeping OPCs in the cell cycle, while PDGF induces both proliferation and maturation [bib_ref] PDGF and FGF2 pathways regulate distinct oligodendrocyte lineage responses in experimental demyelination..., Murtie [/bib_ref]. However, more recent studies with selective knockout mice have implicated FGFR1/2 in proper myelin formation, axonal ensheathment, and in remyelinating capacity after injury [bib_ref] Fibroblast growth factor signaling in oligodendrocyte-lineage cells facilitates recovery of chronically demyelinated..., Furusho [/bib_ref] [bib_ref] Fibroblast growth factor receptor signaling in oligodendrocytes regulates myelin sheath thickness, Furusho [/bib_ref]. FGFR3 is present on OPCs and is downregulated after hypoxic-ischemic injury [bib_ref] bFGF protects pre-oligodendrocytes from oxygen/glucose deprivation injury to ameliorate demyelination, Qu [/bib_ref]. Prolonged i.p. treatment with bFGF ameliorated WMI in a HI rat model, through increasing the number of myelinated axons and thickness of the myelin sheath, while upregulating FGFR3 expression in pre-OLs [bib_ref] bFGF protects pre-oligodendrocytes from oxygen/glucose deprivation injury to ameliorate demyelination, Qu [/bib_ref]. bFGF therapy has also shown some efficacy in restoring myelination in mouse models of MS [bib_ref] Basic fibroblast growth factor potentiates myelin repair following induction of experimental demyelination..., Dehghan [/bib_ref] [bib_ref] Fibroblast growth factor receptor signaling in oligodendrocytes regulates myelin sheath thickness, Furusho [/bib_ref]. Consistent with in vitro evidence, PDGF-A was found to be important for oligodendrocyte maturation as endogenous production was reduced after hyperoxia in neonatal rats, corresponding to a reduction in mature oligodendrocytes and hypomyelination [bib_ref] Oligodendroglial maldevelopment in the cerebellum after postnatal hyperoxia and its prevention by..., Scheuer [/bib_ref]. Similarly, PDGF-α+/− mice had a reduced number of mature oligodendrocytes in the corpus callosum after 6-weeks recovery from cuprizone treatment to mimic MS [bib_ref] PDGF and FGF2 pathways regulate distinct oligodendrocyte lineage responses in experimental demyelination..., Murtie [/bib_ref]. Although primarily known as OPC mitogens, novel roles for bFGF and PDGF in oligodendrocyte maturation and myelination that have been discovered more recently may warrant further investigation into these factors for the treatment of EoP.
An upregulation in G-CSF levels in umbilical cord blood and amniotic fluid has been associated with preterm brain injury [bib_ref] Relationship between premature brain injury and multiple biomarkers in cord blood and..., Lu [/bib_ref] [bib_ref] Contribution of histologic chorioamnionitis and fetal inflammatory response syndrome to increased risk..., Lu [/bib_ref]. Increased levels of G-CSF may be a protective response, as some results have indicated efficacy of G-CSF against hallmarks of EoP, such as neuroinflammation [bib_ref] Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure..., Jellema [/bib_ref] [bib_ref] Granulocyte colony-stimulating factor (G-CSF) protects oligodendrocyte and promotes hindlimb functional recovery after..., Kadota [/bib_ref] [bib_ref] G-CSF treatment promotes apoptosis of autoreactive T cells to restrict the inflammatory..., Peng [/bib_ref] [bib_ref] The effects of granulocyte-colony stimulating factor on regeneration in nerve crush injuries..., Song [/bib_ref] , excitotoxicity [bib_ref] Delayed application of the haematopoietic growth factors G-CSF/SCF and FL reduces neonatal..., Neubauer [/bib_ref] and apoptosis through activation of the JAK/STAT, PI3K/Akt, and ERK pathways (see Box 2; [bib_ref] Granulocyte stimulating factor attenuates hypoxicischemic brain injury by inhibiting apoptosis in neonatal..., Kim [/bib_ref] [bib_ref] Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats, Yata [/bib_ref]. Consequently, improved oligodendrocyte maturation and myelination after G-CSF treatment was observed in an ovine model of preterm brain injury [bib_ref] Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure..., Jellema [/bib_ref] and in traumatic nerve injuries [bib_ref] Granulocyte colony-stimulating factor (G-CSF) protects oligodendrocyte and promotes hindlimb functional recovery after..., Kadota [/bib_ref] [bib_ref] The effects of granulocyte-colony stimulating factor on regeneration in nerve crush injuries..., Song [/bib_ref]. Conflicting results have been obtained in rodent models of preterm brain injury, ranging from adverse effects of G-CSF on brain injury and apoptosis, to positive or absent effects after delayed administration [bib_ref] Systemic application of granulocyte-colony stimulating factor and stem cell factor exacerbates excitotoxic..., Keller [/bib_ref] [bib_ref] Delayed application of the haematopoietic growth factors G-CSF/SCF and FL reduces neonatal..., Neubauer [/bib_ref] [bib_ref] Systemic G-CSF treatment does not improve long-term outcomes after neonatal hypoxicischaemic brain..., Schlager [/bib_ref]. Together, these results indicate some positive effects of G-CSF on apoptosis and inflammation exist, but timing seems crucial to its efficacy in EoP. Enteral administration of G-CSF has already been utilized in preterm infants to induce feeding tolerance and reduce the risk of NEC [bib_ref] Enteral granulocyte-colony stimulating factor and erythropoietin early in life improves feeding tolerance..., El-Ganzoury [/bib_ref] , but studies on the effect of G-CSF on the preterm brain have not
been conducted yet (clinicaltrials.gov). However, G-CSF has been tested for the treatment of several other neurological diseases, such as Parkinson's disease and cerebral palsy [bib_ref] Serial changes of cytokines in children with cerebral palsy who received intravenous..., Koh [/bib_ref] [bib_ref] Neuroprotection of granulocyte colony-stimulating factor for early stage Parkinson's disease, Tsai [/bib_ref].
c-Met receptor ligands HGF and CD82 are also involved in oligodendrocyte lineage progression. HGF mostly contributes to OPC proliferation and migration, keeping OPCs in an immature state and decreasing after birth [bib_ref] CD82 blocks cMet activation and overcomes hepatocyte growth factor effects on oligodendrocyte..., Mela [/bib_ref] [bib_ref] Hepatocyte growth factor (HGF) promotes oligodendrocyte progenitor cell proliferation and inhibits its..., Ohya [/bib_ref] [bib_ref] Hepatocyte growth factor stimulates the proliferation and migration of oligodendrocyte precursor cells, Yan [/bib_ref]. In contrast, expression of the c-Met antagonist CD82 emerges later in postnatal brain development and is involved in oligodendrocyte maturation and myelination [bib_ref] The tetraspanin KAI1/CD82 is expressed by late-lineage oligodendrocyte precursors and may function..., Mela [/bib_ref]. However, no in vivo evidence is available on efficacy of CD82 administration in models of white matter pathologies.
## | future perspectives
In this review, we have illustrated that a wide range of growth factors and cytokines can impact oligodendrocyte lineage maturation and microglia/astrocyte activation during healthy brain development and after injurious conditions like dWMI [bib_ref] Multiple sclerosis, Compston [/bib_ref] [bib_ref] Stem cells for brain repair and recovery after stroke, Gutierrez-Fernandez [/bib_ref] [bib_ref] Oligodendrocyte pathophysiology and treatment strategies in cerebral ischemia, Mifsud [/bib_ref]. Thus, to make the next step toward clinical application, additional research on the efficacy of promising factors in clinically relevant models of preterm dWMI, in combination with in vitro experiments using human glial cells is strongly advised.
Another challenge in clinical translationability is determination of the most optimal treatment strategy for preterm dWMI. One option would be to select the most promising candidate factor for monotherapy, preferably an all-round factor that boosts oligodendrocyte maturation while simultaneously dampening neuroinflammation. As summarized in this review, BDNF, NRG1, IL-11, LIF, IL-10, and CXCL12 seem to possess these all-round properties, and are therefore, in our opinion, the most promising candidates for monotherapy.
Even though IGF-1 possibly does not tick both these boxes, we believe it should be considered as a monotherapeutic option, due to the extensive amount of evidence on its role in healthy white matter development and potential to boost myelination after neonatal brain injury, gathered from multiple in vitro and in vivo models. Moreover, recent clinical research has deemed IGF-1 administration to extreme preterm infants to be safe [bib_ref] A pharmacokinetic and dosing study of intravenous insulin-like growth factor-I and IGFbinding..., Lofqvist [/bib_ref].
Another option is administration of a cocktail of multiple promising factors, aimed at oligodendrocyte survival, differentiation, and reduction of neuroinflammation. The contents and timing of such a trophic/ immunomodulatory factor-cocktail could be given in a tailor-made way, following the patient's disease course, for example, by administration of factors with anti-inflammatory properties during episodes of inflammation. Tailor-made treatment could also reduce safetyrelated issues, by preventing adverse effects that could result from an interaction with the environment, such as a high dose of IGF-1 during an episode of acute inflammation. Another strategy to prevent side effects due to undesirable interactions could be targeted delivery of factors to specific cell types in the brain, using nanoparticles [bib_ref] Cell delivery of therapeutic nanoparticles, Mcmillan [/bib_ref] [bib_ref] Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with..., Rittchen [/bib_ref]. [bib_ref] Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with..., Rittchen [/bib_ref]. Although promising, some hurdles for clinical application of nanoparticles remain. Ligand-specificity might prove to be challenging, especially considering potential changes in receptor expression on the surface of target cells, either in development or in response to environmental cues (i.e., inflammation; Luo, [bib_ref] Dual and multi-targeted nanoparticles for site-specific brain drug delivery, Luo [/bib_ref] [bib_ref] Ligand-installed nanocarriers toward precision therapy, Mi [/bib_ref]. Moreover, additional challenges include reliable reproducibility in nanoparticle syntheses, the avoidance of nanoparticle clearance by macrophages using nanoparticle coatings, and the optimization of biodegradability and subsequent clearance of nanoparticles after administration [bib_ref] Active cellular and subcellular targeting of nanoparticles for drug delivery, Nag [/bib_ref]. The proposed route of personalized medicine to ensure optimal treatment efficacy and safety for each individual patient could be supported by identification of biomarkers, for instance the low serum IGF-1 observed in (extreme) preterm infants.
Aside from serum concentrations in preterm patients, analysis of genetic profiles could contribute to development of personalized medicine, for example, by selecting children that lack the protective NRG1 SNP discussed earlier, or children with genetic variants that are associated with exacerbation of neuroinflammation [bib_ref] Impaired oligodendrocyte maturation in preterm infants: Potential therapeutic targets, Van Tilborg [/bib_ref]. Apart from factor-based therapy, other therapies that affect cerebral growth factor and cytokine concentrations during brain development have been identified, such as nutritional interventions [bib_ref] Postnatal nutrition to improve brain development in the preterm infant: A systematic..., Hortensius [/bib_ref] [bib_ref] Impact of nutrition on brain development and its neuroprotective implications following preterm..., Keunen [/bib_ref] and MSC therapy [bib_ref] The potential of stem cell therapy to repair white matter injury in..., Vaes [/bib_ref]. These treatments could be used in conjunction with additional trophic factor or cytokine supplementation or possibly serve as an overarching alternative. Even though stem cell therapy and/or nutritional interventions could be considered as a more desirable option, due to a more continuous secretion of trophic and/or immunomodulatory factors, these therapies limit manipulation of the exact other factors or cell types such as astrocytes). Abbreviations: bFGF, basic fibroblast growth factor; CD82, cluster of differentiation 82; CNTF, ciliary neurotrophic factor; CXCL, CXC ligand; EGF, epidermal growth factor; G-CSF, granulocyte colony-stimulating factor; GGF2, glial growth factor-2; GDNF, glial cell-line derived neurotrophic factor; Gp130, glycoprotein 130; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor 1; IL, interleukin (6, 4, 10, and 11); LIF, leukemia inhibitory factor; NRGs, neuregulins; OL, oligodendrocyte; PDGF, platelet-derived growth factor; TGF, transforming growth factor (α and β).
protein cocktail composition [bib_ref] Postnatal nutrition to improve brain development in the preterm infant: A systematic..., Hortensius [/bib_ref] [bib_ref] Impact of nutrition on brain development and its neuroprotective implications following preterm..., Keunen [/bib_ref] [bib_ref] The potential of stem cell therapy to repair white matter injury in..., Vaes [/bib_ref]. For this reason, some studies propose genetic modification of MSCs, inducing (transient) overexpression of specific beneficial factors in a controlled manner [bib_ref] The potential of stem cell therapy to repair white matter injury in..., Vaes [/bib_ref].
Additional preclinical studies with back-to-back comparison of the efficacy of monotherapy, cocktails of factors or overarching therapies like stem cell therapy in models of preterm dWMI are needed to substantiate these statements.
Treatments with a small number applied to transiently open the BBB in targeted areas, promoting entry of drugs from the circulation into the brain [bib_ref] Focused ultrasound-mediated drug delivery through the blood-brain barrier, Burgess [/bib_ref] [bib_ref] Ultrasound-induced blood-brain barrier opening, Konofagou [/bib_ref]. Similarly, nanoparticles were shown to induce local toxic effects, leading to BBB permeabilization, or support BBB passage through facilitation of endothelial trans-or endocytosis [bib_ref] Nanoparticle-mediated brain drug delivery: Overcoming blood-brain barrier to treat neurodegenerative diseases, Saraiva [/bib_ref] [bib_ref] Crossing the blood-brain barrier with nanoparticles, Zhou [/bib_ref]. Aside from these options, one could speculate that loss of trophic or immunomodulatory factors in the periphery following intravenous administration could still benefit the preterm patient with multiorgan failure, by dampening systemic inflammation and aiding in development of other organs. Optimal CNS delivery could be attained by local intracerebral injection, preferably using viral vectormediated gene transfer, inducing long-lasting expression of the transgene, avoiding repeated invasive injections [bib_ref] Functional reinnervation from remaining DA terminals induced by GDNF lentivirus in a..., Brizard [/bib_ref] [bib_ref] Viral vectors for neurotrophic factor delivery: A gene therapy approach for neurodegenerative..., Lim [/bib_ref]. Intranasal administration provides a noninvasive method of local delivery of therapeutic agents to the brain, bypassing the BBB, and therefore might prove to be the most favorable route to directly target the CNS [bib_ref] Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the..., Hanson [/bib_ref] [bib_ref] Intranasal delivery of biologics to the central nervous system, Lochhead [/bib_ref] [bib_ref] Delivery of insulin-like growth factor-I to the rat brain and spinal cord..., Thorne [/bib_ref] [bib_ref] The potential of stem cell therapy to repair white matter injury in..., Vaes [/bib_ref].
Although not our primary focus, preterm birth-related events, that is, hypoxia and inflammation, have been shown to disrupt the development of other immature cell types, such as cortical interneurons [bib_ref] Bone morphogenetic protein inhibition promotes neurological recovery after intraventricular hemorrhage, Duchatel [/bib_ref] [bib_ref] Interneuron development is disrupted in preterm brains with diffuse white matter injury:..., Stolp [/bib_ref]. Interestingly, interneurons have been shown to play a role in oligodendrocyte development [bib_ref] The cerebral cortex is a substrate of multiple interactions between GABAergic interneurons..., Benamer [/bib_ref] [bib_ref] GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white..., Zonouzi [/bib_ref] and could therefore be essential for healthy white matter development. Thus, interventions aimed to target the common pathophysiological pathways are likely to positively impact multiple aspects of EoP, potentially restoring both myelination failure and interneuron deficits.
Despite the challenges yet to overcome for clinical translation, this review proposes a broad range of potential therapeutic trophic/ immunomodulatory targets, known to aid myelination by directly boosting oligodendrocyte lineage development and/or by modulation of neuroinflammation. Future studies are urgently needed to refine these treatment strategies for preterm dWMI, in order to improve the neurodevelopmental prospects for these vulnerable patients.
## Conflict of interest
The authors declare no conflicts of interest.
# Data availability statement
Data sharing is not applicable to this article as no new data were created or analyzed in this study.
## Orcid
Cora H. Nijboer https://orcid.org/0000-0003-1223-1861
[table] Table 1: For some factors, such as IGF-1, a large body of evidence supporting its beneficial potential in dWMI is available from both in vitro and in vivo models of (neonatal) WMI. Other factors like GDNF and CNTF have been implicated in healthy oligodendrocyte development, or increased expression of factors like IL-11 and EGF, has been shown as an (inadequate) endogenous protective response following (diffuse) WMI but currently lack vigorous evidence in models of preterm dWMI. Moreover, for some of these factors, like LIF, NGF, TGF-α, and GDNF, evidence seems inconclusive as some studies describe beneficial properties where others describe detrimental effects of the factor on oligodendrocyte lineage development or neuroinflammation. With the current knowledge, many challenges still lie ahead when translating these experimental in vitro and in vivo studies on trophic and immunomodulatory factors to the field of EoP and clinical application further down the line.One of these challenges is the interpretation of data obtained in experimental rodent models of oligodendrocyte development to the human developing brain and to interpret data of other (adult) white matter pathologies to preterm dWMI. Even though rodent and human white matter development share similarities, such as the origin and migratory pattern of OPCs and the caudal-to-rostral pattern of myelin formation, there are also some apparent dissimilarities(van Tilborg et al., 2018). For example, while rodent oligodendrocytes widely express CXCR2, the receptor that mediates the pro-differentiation effect of CXCL1, CXCR2 expression in human oligodendrocytes is scarce(Filipovic & Zecevic, 2008). Similarly, the pro-differentiating effect of bFGF was shown to be limited in human OPCs(Wilson et al., 2003), making these factors less attractive for clinical application. Thus, potential differences between species should be considered carefully when translating a promising factor from experimental studies to the clinic. To facilitate translationability from rodent studies to humans,van Tilborg et al. (2018) proposed the use of human iPSCderived OPCs to complement the limited supply of fetal human tissue, as an alternative to study the efficacy of these factors on human oligodendrocyte lineage development. iPSCs could also be used to generate cerebral organoids, providing a more complex structure to study the therapeutic potential of trophic factors, including the interaction with other glial cell types(Kim et al., 2019;Madhavan et al., 2018;Ormel et al., 2018). Moreover, when interpreting data from other (adult) animal models, differences in the pathophysiology of preterm dWMI compared to other (adult) white matter diseases should be considered. In preterm dWMI, myelin formation is hampered due to a developmental oligodendrocyte maturation arrest, while in neonatal stroke or HIE, loss of white matter volume is accompanied by pronounced oligodendrocyte apoptosis. In addition, preterm dWMI is caused by impaired a priori myelination, whereas in adult models of stroke or other neurodegenerative diseases preexisting myelin sheaths are damaged and need to be remyelinated by the trophic therapies. Furthermore, other white matter disease models like MS have a considerable different pathophysiology in which immune system dysfunction causes demyelination [/table]
|
NUFEB: A massively parallel simulator for individual-based modelling of microbial communities
We present NUFEB (Newcastle University Frontiers in Engineering Biology), a flexible, efficient, and open source software for simulating the 3D dynamics of microbial communities. The tool is based on the Individual-based Modelling (IbM) approach, where microbes are represented as discrete units and their behaviour changes over time due to a variety of processes. This approach allows us to study population behaviours that emerge from the interaction between individuals and their environment. NUFEB is built on top of the classical molecular dynamics simulator LAMMPS (Large-scale Atomic/Molecular Massively Parallel Simulator), which we extended with IbM features. A wide range of biological, physical and chemical processes are implemented to explicitly model microbial systems, with particular emphasis on biofilms. NUFEB is fully parallelised and allows for the simulation of large numbers of microbes (10 7 individuals and beyond). The parallelisation is based on a domain decomposition scheme that divides the domain into multiple sub-domains which are distributed to different processors. NUFEB also offers a collection of post-processing routines for the visualisation and analysis of simulation output. In this article, we give an overview of NUFEB's functionalities and implementation details. We provide examples that illustrate the type of microbial systems NUFEB can be used to model and simulate.Author summaryIndividual-based Models (IbM) are one of the most promising frameworks to study microbial communities, as they can explicitly describe the behaviour of each cell. The development of a general-purpose IbM solver should focus on efficiency and flexibility due to the unique characteristics of microbial systems. However, available tools for these PLOS Computational Biology | https://doi./1 from UK Engineering and Physical Sciences Research Council (https://epsrc.ukri.org/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. purposes present significant limitations. Most of them only facilitate serial computing for single simulation, or only focus on biological processes, but do not model mechanical and chemical processes in detail. In this work, we introduce the IbM solver NUFEB that addresses some of these shortcomings. The tool facilitates the modelling of much needed biological, chemical, physical and individual microbes in detail, and offers the flexibility of model extension and customisation. NUFEB is also fully parallelised and allows for the simulation of large complex microbial system. In this paper, we first give an overview of NUFEB's functionalities and implementation details. Then, we use NUFEB to model and simulate a biofilm system with fluid dynamics, and a large and complex biofilm system with multiple microbial functional groups and multiple nutrients. This is a PLOS Computational Biology Software paper.A simulator for individual-based modelling of microbial communities PLOS Computational Biology | https://doi.org/10.
[formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula]
# Introduction
Microbial communities are groups of microbes that live together in a contiguous environment and interact with each other. The presence of microbial communities on the planet plays an important role in natural processes, as well as in environmental engineering applications such as wastewater treatment, waste recyclingand the production of alternative energy source. Therefore, studies on how these communities form and behave have become increasingly important over the past few decades.
Work on microbial communities has revealed that the community's emergent behaviour arises from a variety of interactions between microbes and their local environment. In vitro experiments offer a way to gain insights into these complex interactions, but at great expense in time and resources. On the other hand, in silico computational models and numerical simulations could help researchers to investigate and predict how complex processes affect the behaviour of biological systems in an explicit and efficient way. Different approaches have been developed for modelling microbial communities. One of the most promising strategies is to develop a mathematical model from the description of the characteristics and behaviour of the individual microbes, usually referred to as Individual-based Models (IbM). In conventional IbM, the microbes are represented as rigid particles, each of which is associated with a set of properties such as mass, position, and velocity. These properties are affected by internal or external processes (e.g., diffusion), resulting in microbial growth, decay, motility, etc. Therefore, IbM are particularly useful when one is interested in understanding how individual heterogeneity and local interactions influence an emergent behaviour.
The development of an IbM solver should focus on the following aspects. First, the solver needs to be flexible. Depending on the purpose of the model, IbM may involve multiple microbial functional groups, nutrients and sophisticated biological, chemical and physical processes, or sometimes it may be a simple model that describes mono-functional group or focuses on a few processes. Thus, it is important for the solver to be highly customisable (for building IbM) and extendible (with new IbM features). Second, the solver should be scalable. Simulation of large microbial communities is difficult since they contain a very high number of individuals. Different modelling strategies have been proposed to overcome this limitation, including using super-individuals and statistically representative volume elements. However, there is very little work on the development of a scalable IbM solver. Parallel computing can help scalability by using multiple computer resources to simulate many individuals simultaneously. This is accomplished by breaking the problem domain into discrete sub-domains which separate out the individuals as much as possible, allowing each processing element to simulate the local interactions between individuals whilst minimizing the interactions between subdomains. In this way each sub-domain can largely run concurrently with the others.
In this work, we present a three-dimensional, open-source, and massively parallel IbM solver called NUFEB that addresses these desired features above. The purpose of NUFEB is to offer a flexible and efficient framework for simulating microbial communities at the microscale, with an emphasis on biofilms. A comprehensive IbM is implemented in the solver which explicitly models biological, chemical and physical processes, as well as individual microbes. The present solver supports parallel computing and allows flexible extension and customisation of the model. NUFEB is based on the state-of-the-art software LAMMPS (Large-scale Atomic Molecular Massively Parallel Simulator). We selected LAMMPS because of its open-source, parallel, and extendible nature. There are several open-source IbM solvers that have been developed over the past decade and widely applied to microbiology research, such as iDynoMiCS, SimBiotics, BioDynaMo, and CellModeller. However, most of them only facilitate serial computing for single simulation, or focus only on biological processes, but do not model mechanical and chemical processes in detail. The NUFEB simulator instead includes all of these features. A prototype NUFEB implementation was used into study physical behaviour of microbial communities. In this manuscript we focus on a major improvement of the tool, in which new features and enhancements have been developed including three-way coupling with fluid dynamics (two-way coupled fluid-particle interactions plus particle-particle interactions), code parallelisation, chemical processes (pH dynamics, thermodynamics, and gas-liquid transfer), and post-processing routines.
## Model description
In this section, we describe and review the IbM implemented in NUFEB. The model is described through the ODD protocol (Overview, Design concepts and Details) which is a standard structure for clearly and efficiently describing IbM. We build on the ideas described previously inand extend them to cope with hydrodynamics and chemical processes. For the sake of accuracy, in the following we use the term "microbe" when describing biological and chemical processes, and use the term "particle" when describing physical process. However, the two terms effectively mean the same thing-"individual" of the IbM.
## Model overview
The purpose of the IbM implemented in NUFEB is to model a range of microbial systems (including biofilm and flocs) at the micro-scale, in order to study and predict their populationlevel properties and behaviours emerged from the interactions between individuals and their environment.
In the model, microbes are described as soft spheres, with each individual having a set of state variables including position, density, velocity, force, mass, diameter, outer-mass, outerdiameter, growth rate, yield, etc. These attributes vary among individuals and can change through time. Outer-diameter and outer-mass are used to represent an EPS (Extracellular Polymeric Substances) shell: in some microbes EPS is initially accumulated as an extra shell around the particle. Microbial functional groups (types) are groups of one or more individual microbes that share same characteristics or parameters (such as maximum growth rate), which are constant throughout a simulation. The separation of individuals into different functional groups is based on their specific metabolism.
The computational domain is the environment where microbes reside and the biological, physical and chemical processes take place. It is defined as a micro-scale 3D rectangular box with dimensions L X × L Y × L Z . The size of the domain normally ranges from hundreds to thousands micrometers for micro-scale simulation. Therefore, the computational domain is considered as a sub-space of a macro-scale bioreactor or any other large-scale microbiological system. We assume that the macro-scale ecosystem is made up of replicates of the micro-scale domain if the bioreactor is perfectly mixed. Within the domain, chemical properties such as nutrient concentration, pH, and Gibbs free energies for microbial catabolism and anabolism are represented as continuous fields. To resolve their dynamics over time and space, the domain is discretised into Cartesian grid elements so that the values can be calculated at each discrete voxel on the meshed geometry. The style of domain boundary can be defined as either periodic or fixed. The former allows particles to cross the boundary, and reappear on the opposite side of the domain, while a fixed wall prevents particles to interact across the boundary.
The IbM allows to model a biofilm system where different regions may be defined within the computational domain. The biofilm region is the volume occupied by microbial agents and their EPS, in which the distribution of soluble chemical species is affected by both diffusion, reaction and advection (due to biofilm porosity) processes. The boundary layer region lies over the biofilm, so that nutrient diffusion and advection is resolved in this space. The bulk liquid region is situated at the top of the boundary layer, and nutrients in this area are assumed to be perfectly mixed with the same concentration as in the macro-scale bioreactor. We also assume that the boundary layer region stretches from the maximum biofilm thickness to the bulk liquid, with the height of the region specified by the user. The boundary between the diffusion layer and bulk liquid regions is parallel with the bottom surface (substratum), but its location needs to be updated when the biofilm thickness changes.
## Design concepts
Here we address several important concepts in the NUFEB model design.
Emergence. The morphology of the microbial system, and the spatial distribution of its microbial functional groups, emerge from local interactions due to microbial growth, division, decay and mechanical interaction.
Sensing. The growth of each individual depends on a number of chemical properties within the voxel in which the individual resides, including nutrient concentration, pH, and Gibbs free energy. The motion of each individual results from local mechanical interactions and fluid flow.
Observation. At each given step, the physical and biological states of each individual (e.g., location, mass, size, type, growth rate) and the chemical state of each voxel (e.g., nutrient concentration, pH) are stored.
Interaction. The direct interactions among individuals are driven by mechanical forces, for example, contact force, EPS adhesive force, drag force, etc.
Stochasticity. The stochastic processes considered in the model include the size and location of daughter microbes after division, the location of EPS particle after EPS production, and the initial distribution of microbes.
## Details of the sub-models
The processes that influence microbe activities are considered under three main sub-models: biological, physical, and chemical.
Biological processes. The NUFEB biological sub-model handles microbial metabolism, growth, decay, and reproduction (cell division and EPS formation). Details of the biological sub-model are given below.
Microbe growth and decay. An individual microbe grows and its mass increases by consuming nearby nutrients. The process of growth and decay is described by the following ordinary differential equation:
[formula] dm i dt ¼ m i m i ;ð1Þ [/formula]
where m i is the biomass of the i th microbe, and μ i is the specified growth rate. To determine μ i , two growth models are implemented: (i) Monod-based and (ii) energy-based. The user can choose one of the growth models when configuring the simulation to run. For exemplification, the Monod-based growth model implements the work described inand. Three functional groups of microbes and two inert states are considered. They include: active heterotrophs (HET), ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), and inactive EPS and dead cells. Microbial growth is based on Monod kinetics driven by the local concentration of nutrients (S substrate , S NH 4 þ , S NO 2 À , S O 2 ) at the voxel in which each microbe resides. The decay rate is assumed to be first order.
The energy-based growth model implements the work proposed in . In this model, the growth rate of each microbe μ i is determined not only by nutrient availability but also by the amount of energy available for its metabolism:
[formula] m i ¼ Y i � ðq met i À m req i Þ ;ð2Þ [/formula]
where the maximum growth yield Y i is estimated by using the Energy Dissipation Method, q met i is the metabolic rate which depends on the availability of nutrients (in particular, their dissociation forms), and m req i is the average maintenance requirement. Thus, a microbe grows if it harvests more energy than the necessary for its maintenance requirement. On the other hand, the microbe decays when the energy requirement is not met.
Microbe division and death. Microbe division is the result of biomass growth, while death is the result of biomass decay. Both are considered as instantaneous processes. Division occurs if the diameter of a microbe reaches a user-specified threshold value; the cell then divides into two daughter cells. The total mass of the two daughter cells is always conserved from the parent cell. One daughter cell is (uniformly) randomly assigned 40%-60% of the parent cell's mass, and the other gets the rest. Also, one daughter cell takes the location of the parent cell while the centre of the other daughter cell is (uniformly) randomly chosen at a distance d (distance between the centres of the two agents) corresponding to the sum of the diameter of the daughters.
The size of a microbe decreases when nutrients are limited or the energy available is not sufficient to meet maintenance requirements. Microbes which shrink below a user-specified minimum diameter are considered as dead. Their type then changes to the dead type. Dead cells do not perform biological activities, but their biomass will linearly convert to substrate to be consumed by other microbes. For computational efficiency, decaying cells are removed from the system when their size is sufficiently small (defined as 10 times smaller than the death threshold diameter).
EPS production. The Monod-based growth model allows active heterotrophs to secrete EPS into their neighbouring environment. The EPS play an important role in microbial aggregation by offering a protective medium. The production process follows the approach presented inandwith the simplification that EPS are secreted by heterotrophs only. Initially, EPS is accumulated as an extra shell around a HET particle (note that EPS density is lower than microbe density). When the relative thickness of the EPS shell of the HET particle exceeds a certain threshold value, around half (uniformly random ratio between 0.4-0.6) of the EPS mass excretes as a separate EPS particle and is (uniformly) randomly placed next to the HET.
Physical processes. NUFEB's physical sub-model includes two key features and the dependencies between them: microbes (particles) and fluid. Microbes interact among themselves and with the ambient fluid. The physics of microbial motion is solved by using the discrete element method (DEM). Fluid momentum and continuity equations are solved based on computational fluid dynamics (CFD) and coupled with particle motion. The model developed in this study allows us to describe biofilm formation, detachment, and deformation based on mechanical interactions between particles. This is more realistic than many conventional IbMs where mechanistic approaches are not considered, for example, implementing detachment as probability or rate functions.
Mechanical relaxation. When microbes grow and divide, the system may deviate from mechanical equilibrium (i.e., non-zero net force on particles) due to particle overlap or collision. Hence, mechanical relaxation is required to update the location of the particles and minimise the stored mechanical energy of the system. Mechanical relaxation is carried out using the discrete element method, and the Newtonian equations of motion are solved for each particle in a Lagrangian framework. The equation for the translational and rotational movement of particle i is given by:
[formula] m i dṽ i dt ¼ F c;i þ F a;i þ F d;i þ . . . ;ð3Þ [/formula]
where m i is the mass, andṽ i is the velocity. The type of force acting on the particle varies according to different biological systems. For example, the above equation takes into account three commonly used forces in microbial system. The contact force F c,i is a pair-wise force exerted on the particles to resolve the overlap problem at the particle level. The force equation is solved based on Hooke's law, as described in:
[formula] F c;i ¼ X N i j¼1 ðK n dn i;j À m i;j g n v i;j Þ ;ð4Þ [/formula]
where N i is the total number of neighbouring particles of i, K n is the elastic constant for normal contact, δn i,j are overlap distance between the center of particles i and its neighbour particle j, m i,j is the effective mass of particles i and j, γ n is the viscoelastic damping constant for normal contact, and v i,j is the relative velocity of the two particles. The EPS adhesive force F a,i is a pair-wise interaction, which is modelled as a van der Waals force:
[formula] F a;i ¼ X N i j¼1 H a r i;j 12h 2 min;i;j n i;j ;ð5Þ [/formula]
where H a is the Hamaker coefficient, r i,j is the effective outer-radius of particles i and j, h min,i,j is the minimum separation distance of the two particles, and n i,j is the unit vector from particle i to j. The drag force F d,i is the fluid-particle interaction force due to fluid flow, with direction opposite the microbe motion in a fluid. It is formulated as:
[formula] F d;i ¼ V p;i � f ;i � s;i b i ðu p;i À U f ;i Þ ;ð6Þ [/formula]
where � s,i is the particle volume fraction, � f,i = 1 − � s,i is the fluid volume fraction, V p,i and u p,i are volume and velocity of particle i, respectively, U f,i is the fluid velocity imposed on particle i, and β i is the drag correction coefficient. Apart from the forces above, the LAMMPS framework offers mechanical interactions that one can apply directly to particles (see LAMMPS' user manual for more details. In the model, we assume that the mechanical equilibrium is obtained when the average pressure of the microbial community reaches the equilibrium state. The average pressure of the system due to mechanical interactions is calculated as:
[formula] P ¼ 1 3V ð X N i¼1 m iṽi �ṽ i þ X N i¼1 X N j>ir i;j �F i;j Þ ;ð7Þ [/formula]
here V is the sum of the volumes of particles. The first term in the bracket addresses the contribution from kinetic energy, where m i is the mass of particle i,ṽ i is the velocity. The second term addresses the interaction energy, wherer i;j andF i;j are the distance and force between two interacting particles i and j, respectively. Fluid dynamics. Hydrodynamics is an important factor in microbial community modelling as many microorganisms are found in water where their behaviour is influenced by fluid flow in two ways: transport of nutrients and detachment. Nevertheless, accurate hydrodynamics has rarely been considered in 3D microbial community modelling due to its computational complexity. In NUFEB, with the support of code parallelisation, hydrodynamics is introduced by using the two-way coupled CFD-DEM approach: the fluid flow affects the motion of particles and the particles in turn affect the motion of the fluid. DEM solves the motion of Lagrangian particles based on Newton's second law, while CFD (computational fluid dynamics) tracks the motion of fluid based on locally averaged Navier-Stokes equations.
The particle velocity at each voxel in space is replaced by its average, and the locally averaged incompressible continuity and momentum equations for the fluid phase are given by:
[formula] r � ð� s U s þ � f U f Þ ¼ 0 ;ð8Þ [/formula]
and
[formula] @ð� f U f Þ @t þ r � � f U f U f � � ¼ 1 r f À rP þ � f r � R þ � f r f g þ F f � � ; ð9Þ [/formula]
where � s , U s , and F f are the fields of the solid volume fraction, velocity and fluid-particle interaction forces (e.g., drag force) of microbes, respectively. They are obtained by averaging discrete particle data in DEM; � f is the fluid volume fraction and U f is the fluid velocity. Besides the fluid-particle interaction, the terms on the right-hand side of Eq 9 also include the fluid density ρ f , the pressure gradient rP, the divergence of the stress tensor R and the gravitational acceleration g. The fluid momentum equations are discretised and solved on an Eulerian grid by a finite volume method. The results, in particular the velocity field and the particle drag force, are used for solving nutrient transport and mechanical relaxation, respectively. Chemical processes. Nutrient transport is described using the diffusion-advection-reaction equation. To improve the representation of microbial growth, NUFEB also implemented the work proposed into allow pH dynamics and gas-liquid transfer to be considered. In this section, we briefly address the main ideas of this chemical sub-model. Nutrient consumption. The rate of nutrient consumption (or reaction rate) is calculated at each voxel. The reaction rates in the Monod-based growth model are defined according to Monod kinetics, where the stoichiometric matrix for particulate and soluble components is given in the S1 File. By contrast, in the energy-based growth model the consumption/formation rate of the i th microbe for each soluble species is driven by the microbial growth rate and the stoichiometric coefficients of the overall growth reaction, and is formalised as follows [22]:
[formula] R i ¼ m i 1 Y i Cat i þ Ana i � � X ;ð10Þ [/formula]
where Cat i is the free energy supplied by the microbial catabolic reactions, Ana i is the free energy required by the anabolic reactions, and X is the biomass density. Nutrient mass balance. Nutrient concentration at each point within the computational domain is affected by different processes. For example, nutrient advection due to fluid flow, microbial growth causing nutrient consumption, or solute component transferred into gas. On the other hand, the variation of the concentration also affects biological activities. A typical example would be that aerobes such as AOB can survive and grow in aerobic regions but not in anoxic regions.
To solve the nutrient distribution for each soluble component, the following diffusionadvection-reaction equation for the solute concentration S is employed in the model:
[formula] @S @t ¼ r � ðDrSÞ À r � ðṽSÞ þ R :ð11Þ [/formula]
On the right-hand side of the equation, the first part is the diffusion term which describes nutrient movement from an area of high concentration to an area of low concentration, where r is the gradient operator and D is the diffusion coefficient. The second part is the advection term which describes nutrient motion along the fluid flow, whereṽ is the fluid velocity field. Finally, R is the reaction term which is governed by both biological activities and chemical activities. Nutrient concentration in the bulk region is dynamically updated according to the following mass balance equation:
[formula] dS b dt ¼ Q V ðS in À S b Þ þ A f VL X L Y Z Z Z Rðx; y; zÞdxdydz :ð12Þ [/formula]
The bulk concentration S b of each soluble component is influenced by the nutrient inflow and outflow in the bioreactor (Q is the volumetric flow rate, V is the bioreactor volume and S in is the influent nutrient concentration), as well as the total consumption rate in the biofilm volume in the bioreactor (A f is the surface area of the biofilm, and R(x, y, z) is the reaction rate at each voxel). pH dynamics. We assume the influent enters the bulk liquid with a fixed pH value. However, the pH varies in space and time due to change in nutrient concentration as a result of microbial activity. The model considers acid-base reactions as equilibrium processes. The kinetic expressions are amended to consider only the non-charged form of the nutrients (e.g., HNO 2 but not NO 2 − ). The concentration of all dissociated and undissociated forms can be expressed as a function of the proton (H + ) concentration. Then, the proton concentration can be determined by finding the root between 1 and 10 −14 of the following charge balance equation, using an implicit Newton-Raphson approximation:
[formula] ½H þ � þ X p i¼1 m½S mþ i � ¼ ½OH À � þ X q j¼1 n½S nÀ j � ;ð13Þ [/formula]
where p and q are the total number of cations and anions contributing to the pH, respectively, m and n are the charges corresponding to the cations and anions considered in the dissociation equilibrium, and S is the concentrations of cations and anions calculated based on the Gibbs free energy adjusted for ambient temperature. Gas-liquid transfer. A gas field can be defined in NUFEB to describe the rate of nutrient mass transfer from gas to liquid or vice-versa. The phase is modelled as a volume fraction inside the biofilm region. The equilibrium between liquid and gas is disturbed by the acid-base reaction and the microbial activity taking place in the biofilm and bulk liquid. On the other hand, mass transfer may also affect microbial growth due to varying nutrient concentration in the liquid phase. The reaction rate from gas to liquid R G!L of a given nutrient can be expressed by the following equation:
[formula] R G!L ¼ K L a � ðS gas À S liq K H Þ :ð14Þ [/formula]
The rate is determined by the mass transfer coefficient of chemical component K L a, the gas concentration S gas in the head space, the saturation liquid concentration S liq , and Henry's constant K H . Mass transfer from liquid to gas is formalised as follows:
[formula] R L!G ¼ À R G!L V gas R g T ;ð15Þ [/formula]
where V gas is the volume of the reactor head space, which is considered of equal size to the computational domain, and R g and T are the ideal gas constant and temperature, respectively.
## Design and implementations
NUFEB is developed in C++ as a user package within the LAMMPS platform. In this section, we summarise the NUFEB functionalities and give some implementation details.
## Ibm in lammps
NUFEB is built on top of LAMMPS and extends it with IbM features. LAMMPS is a classical molecular dynamics simulator and primarily solves particle physics, including a wide range of inter-particle interactions and potentials. In NUFEB, a new sphere-like particle type is defined with additional attributes (e.g., outer-diameter, outer-mass) to represent a microbe. Microbes are grouped into different functional groups, and members of each group share the same biological parameters. The computational domain is restricted to a 3D rectangular box with user-specified dimensions, Cartesian grid size and boundary conditions. In LAMMPS, a "fix" is any operation that applies to the system during time integration. Examples include the updating of particle locations, velocities, forces. NUFEB defines a series of new fixes to perform the IbM related processes previously described. The user can also extend the tool with other processes by implementing new fix commands. During the simulation, fixes are invoked at a user-defined frequency to update field quantities and microbe attributes. For the purpose of computational efficiency and different modelling systems, NUFEB allows users to customise a simulation by enabling/disabling any of the fix commands from the input setting.
In order to execute a NUFEB simulation, an input script (a text file) is prepared with certain commands and parameters. NUFEB will read those commands and parameters, one line at a time. Each command causes NUFEB to take some action, such as setting initial conditions, performing IbM processes, exporting results, or running a simulation.we show an excerpt of an input script used in this work (Case Study 2). The details of the input format are described in S1 Manual. An example of (partial) input script for NUFEB simulation. A list of "fix" commands defines IbM processes that apply to the simulation, which includes Monod-based growth (k1), nutrient mass balance (k2), cell division (d1), and EPS production (e1). Each fix command may require one or more parameters for the model specification, such as EPS density (EPSdens), division diameter (divDia), and HET reduction factor in anoxic condition (etaHET). The last parameter is introduced due to the fact that the observed growth rate of HET in anoxic condition was always smaller than that in aerobic condition. The procedure of a classical IbM simulation in NUFEB is presented in Algorithm 1. Note that when solving nutrient mass balance (step 5), if energy-growth model is applied, the computation is accomplished with pH dynamics and gas-liquid transfer to update the concentration of dissociation forms, pH and reaction rate. The Monod-based model, however, does not couple with pH and gas-liquid transfer in the current implementation. The model's processes can be operated sequentially as they are on different timescales. The mechanical timestep is typically of the order of 10 −7 s; the diffusion timestep is of the order of 10 −4 s, while the biological timestep is much larger ranging from minutes to hours. The coupling between multiple timescales relies on the pseudo steady-state approximation and the frozen state. For example, when a steady state solute concentration is reached at each biological timestep, the concentration is assumed to remain unchanged (frozen state) until the next biological step. In this way, the computational load for solving fast dynamic processes can be significantly reduced.
Algorithm 1 (IbM simulation procedure) 1: input: initial states of computation domain, microbes and fields 2: output: states of all microbes and fields at each output time step 3: while biological time step t bio < t end do 4: solve fluid dynamics to update velocity field and particle (drag) force 5: solve nutrient mass balance to update solute concentration field 6: update nutrient concentration in bulk based on the total consumption rate in biofilm region 7: perform microbe growth to update biomass and size 8: perform microbe division, death and EPS production 9: perform mechanical relaxation to update microbe position and velocity 10:
update boundary layer location and neighbour list 11: t bio = t bio + Δt 12: end while Mechanical relaxation is resolved by using the Verlet algorithm provided by LAMMPS. The computation of interaction forces between particles, such as contact force, relies on LAMMPS' neighbour lists. During the Verlet integration, instead of iterating through every other particle, which would result in a quadratic time complexity algorithm, LAMMPS maintains a list of neighbours for each particle. The computation of the interaction force is only performed between a particle and its corresponding neighbours. The neighbour lists must be updated from time to time depending on the microbe's displacement and division.
The nutrient mass balance equation is discretised on a Marker-And-Cell (MAC) uniform grid and the concentration scalar is defined at the centre of the cubic voxel. The temporal and spatial derivatives of the transport equation are discretised by Forward Euler and Central Finite Differences, respectively. Further details about the calculations are given in the S1 File. Depending on the physical situation, different boundary conditions can be chosen when solving the equation. For instance, a biofilm system would normally uses non-flux Neumann conditions through the bottom surface to model impermeable support material, Dirichlet boundary conditions at the top surface as it is assumed to connect with bulk environment, and periodic boundary conditions for the rest of the four surfaces.
## Coupling with fluid dynamics
NUFEB employs and extends the existing CFD-DEM solver SediFoam for the simulation of hydrodynamics. SediFoam provides a flexible interface between the two open-source solvers LAMMPS and OpenFOAM. LAMMPS aims to simulate particle motions, while OpenFOAM (Open Field Operation and Manipulation) is a parallel CFD solver that can perform three-dimensional fluid flow simulations. Inbetween them, SediFoam offers efficient parallel algorithms that transfers and maps the properties of Lagrangian particles to an Eulerian mesh, and vice versa. In this work, we also extend SediFoam for compatibility with IbM features, in particular, to transfer and map the information of new divided particles and velocity field between the solvers.
The schema of the CFD-DEM coupling is shown inAt each fluid timestep, particle information (e.g., mass, force, velocity) is transferred from the DEM module to the CFD module. A particles list maintained by OpenFOAM is updated based on the obtained information. An averaging procedure is then performed to convert the properties of discrete particles to the Eulerian CFD mesh. The fluid momentum equations are discretised and solved on the Eulerian mesh by the finite volume method. The PISO (Pressure-Implicit with Splitting of Operators) algorithm is followed to solve the equations. In the DEM module, the solutions of drag force and velocity field obtained from OpenFOAM are assigned to each particle and corresponding mesh grid, respectively. The drag force will be taken into account in evolving the motion of the particles. The velocity field is used for solving the advection-diffusion-reaction equation (Eq 11).. Fluid dynamics is solved in the CFD module and particle motion is solved in the DEM module. Particle and field information are transferred between the two modules based on an averaging procedure.
https://doi.org/10.1371/journal.pcbi.1007125.g003
## Code parallelisation
The IbM implementation in NUFEB involves particle-based functions (e.g., contact force, division, and EPS excretion) and continuum-based functions (e.g., nutrient mass balance, and pH). The parallelisation of the former function group is based on LAMMPS' parallel mechanism. The computational domain is spatially decomposed into multiple MPI (Message Passing Interface) processes (sub-domains). Each sub-domain contains local particles as well as ghost particles. Local particles are those residing in the owned sub-domain, and each process is responsible for updating the status of their local particles. Ghost particles are copies of particles owned by neighbouring processes. During the simulation, the local particles obtain information from their ghost (and neighbour) counterparts for calculating and updating their physical properties (e.g., forces). The neighbour lists require updating when particles are moved due to mechanical interactions, and created/deleted due to microbe division, decay, etc.
Continuum-based functions have their variables computed using a uniform grid. Parallelism is achieved by spatially decomposing the computational domain into sub-domains. Computations such as diffusion and advection require solute information from adjacent cells. Therefore, like particle-based functions, grid cells on the boundary of each sub-domain need to be communicated to neighbouring sub-domains and are treated locally as ghost cells. The implementation of grid decomposition also considers the sub-domain box to be always conforming to the uniform grid boundary.
Automatic vectorisation was employed to further optimise computation intensive routines, such as pH calculation. The loops in the routines are vectorised and can be performed simultaneously. The vectorisation is achieved together with the use of control directives (i.e., #pragmas) to instruct the compiler on how to handle data dependencies within a loop.
## Other features
During simulation, NUFEB allows to output any state variable of microbes or voxels based on the keywords given in the input script. The output results can be stored into various formats for visualisation or analysis. The supported formats are VTK, POVray and HDF5. The VTK binary format is readable by the VTK visualisation toolkit or other visualisation tools, such as ParaView (all biofilm figures shown in the Results section were produced from ParaView). A post-processing routine is implemented to convert the LAMMPS default format to POVray image, which supports high quality rendering of particles. HDF5 is a hierarchical, filesystemlike data format supported by a number of popular software platforms, including Java, MATLAB and Python. This allows the user to directly import simulation data to any of the platforms for further analysis.
To understand the morphological dynamics of microbial systems, characteristics such as biofilm average height, biofilm surface roughness, floc equivalent diameter and floc fractal dimension can be measured and exported during simulation. These aggregated characteristics are essential factors to study and design microbial system. Parallelisation of the characteristics measurements is supported by NUFEB and based on domain decomposition.
NUFEB also supports most of the LAMMPS default commands, which can be useful in microbial simulations. For example, the restart and read restart functions write out the current state of a simulation as a binary file, and then start a new simulation with the previously saved system configuration; the lattice and create atoms functions allow to automatically create large numbers of initial microbes based on a user-defined lattice structure; load balance is performed with the objective of maintaining the same number of particles in each sub-domain in parallel runs. During the simulation, this function adjusts the size and shape of the subdomains to balance the computational cost in the processors.
# Results
We have successfully validated NUFEB against two biofilm benchmark problems BM2 and BM3 proposed by the International Water Association (IWA) task group on biofilm modelling. The validation results can be found in the S1 File. This section shows two further examples implemented using NUFEB. The case studies are based on biofilm systems, which are microbial communities of single or multiple microbial functional groups in which cells stick together and attach to a substratum by means of EPS.
## Case study 1: biofilm deformation and detachment
One of the outstanding questions in biofilm research is understanding how fluid-biofilm interactions affect the mechanical properties of biofilms. In this section, we describe how to use NUFEB to simulate a biofilm system with fluid dynamics by using three-way fluid and particle coupling. This is different from previous studies, where the coupling was from flow field to biofilm structure but not the other way, or assumes biofilm as a collection of 1D springs.
To simulate a hydrodynamic biofilm, we apply fluid flow to a pre-grown biofilm that consists of heterotrophs and their EPS production. The biofilm is grown from 40 microbes inoculated on the substratum to a pre-determined height (80 μm) without flow and in an oxygenlimited condition (1 × 10 −4 kg m −3 ). In this way, a mushroom-shaped biofilm structure can be developed to model liquid filled voids and channels (seeand S1 Video). Then, we impose a fluid flow to the biofilm. During the fluid stage, any biological process is considered to be in the frozen state due to the small time scale of hydrodynamic calculations, and nutrient mass balance is omitted for the sake of simplicity. The motion of microbes is driven by both particle-particle and particle-fluid interactions, including EPS adhesion, contact force and drag force, as described in Eqs (4)-. Note that the EPS adhesive force also exists between HETs due to their EPS shells. The physical model parameters are kept constant throughout the simulation and can be found in the S1 File. For boundary conditions, we impose a fixed. The biofilm deforms and microbes detach along with the flow direction. In the early stage of the detachment process, the top of the biofilm is highly elongated and forms filamentous streamers. However, most of the microbes are still connected together with cohesion, and there is only a small number of clusters detached from the head of the streamers due to cohesive failures. As the fluid continues to flow, large chunks of microbes detach from the biofilm surface. These detached microbe chunks may also break-up again, re-agglomerate with other clusters or re-attach to the biofilm surface. Such deformation and detachment events observed from our NUFEB simulation show qualitative agreement with both experimental resultsand other numerical simulations using different methods. The deformation reaches a pseudo-steady-state when the mushroom-shaped biofilm protrusions are removed from the system. As a result, the biofilm morphology changes dramatically from a rough to a flat surface. During the deformation, the fluid, represented as red arrows, travels around the biofilm. Due to the irregular shape of the biofilm and the high fluid velocity, small vortexes can be observed at the biofilm surface on both the upstream and downstream sides. This phenomenon has been observed in previous studies.
For a more quantitative measurement of the deforming biofilm, we evaluated the area density and surface roughness of the biofilm at different fluid velocities. The biofilm surface roughness is calculated by:
[formula] roughness ¼ ð 1 L X L Y Z Z ðhðx; yÞ À � hÞ 2 dxdyÞ 1=2 ;ð16Þ [/formula]
where h(x, y) is the biofilm height in the z direction at location (x, y) on the substratum, and � h is the average biofilm height:
[formula] average height ¼ 1 L X L Y Z Z hðx; yÞdxdy ;ð17Þ [/formula]
As expected, when the fluid velocity increases the removed biomass also increases. For example, when U f = 0.4 m s −1 is applied, the area density reaches steady-state after 0.004s, and the value decreases by 13% . Note that the decrease does not take into account the change of basis. By contrast, the area density decreases less than 1% if U f = 0.1 m s −1 is applied. The biofilm surface roughness shows a similar trend: the roughness decreases with increasing velocities , indicating that biofilm morphology tends to be more flat in high-velocity fluid conditions as most of the mushroom protrusions can be removed.
## Case study 2: biofilm growth with 10 7 particles
In this case study, we first show the development of a large and complex biofilm system and then focus on the parallel efficiency of the simulations. The aim of this case study is to demonstrate the capability and performance of NUFEB in the simulation of larger biological systems. Biofilm development. The system is defined as a multi-functional group and multi-nutrient biofilm. In order to represent a more realistic biofilm, we explicitly consider nitrification as a two-step oxidation process that is performed by different groups of microbes: ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). In addition, the biofilm includes heterotrophs (HET) and their EPS production. The reaction model contains five soluble species, nutrients and products during microbial metabolism. The catabolic reactions include oxidation of ammonium NH 4 + to nitrite NO 2 − by AOB, oxidation of nitrite to nitrate NO 3 − by NOB, and HET aerobic and anaerobic growth by consuming organic substrate in oxygenated conditions or nitrate in anoxic denitrifying conditions. The kinetics and reaction stoichiometry of the modelled processes and their corresponding parameters are detailed in the S1 File. The computational domain is divided into three regions. In the bulk region, nutrients are assumed to be completely mixed and their concentration is updated dynamically at each biological timestep, except for oxygen. We also assume that there is sufficient O 2 and NH 4 + but no NO 2 − and NO 3 − in the reactor influent. So the concentration of the two N compounds can only come from the transformation of NH 4 + . In the boundary layer region, a 20 μm distance from the maximum biofilm thickness to the bulk liquid is defined for solving the nutrient gradient. In the biofilm region, instead of using the super particle method, a small division diameter (1.3 μm) is chosen to represent real microbe sizes (on average 1 μm.
The simulation is run on an in-house HPC system at Newcastle University. In and S5 Video. we present the biofilm development over time. The system reaches 2.3 × 10 7 particles after 160 hours (CPU time = 30 hours). The initial particles are randomly placed on the substratum. In the early stages of biofilm formation, due to high growth rate and sufficient supply of substrate from bulk liquid, heterotrophs grow faster than nitrifiers (0, 60 and 120 hours). As the biomass grows, the system turns to substrate-limited condition for the HET group, while there is still sufficient NH 4 + due to its high initial concentration that favours nitrifier growth.
As a result, biofilm surface coverage of heterotrophs becomes smaller than nitrifiers (160 hours). This phenomenon matches previous experimental results where the nitrifying population can be significantly higher than heterotrophs in a substrate-limited reactor. The biofilm geometry forms a wavy structure after 160 hours. This is because of a self-enhancing process from the non-uniform initial microbial distribution. The spatial distribution of NO 2 − concentration is shown in It is clear that the NO 2 − concentration field differs according to the AOB distribution, where areas with high NO 2 − concentration are the locations where AOB clusters are present.shows a quantitative evaluation of the total biomass accumulation over time. The trend shows linear biomass increase which indicates that the total microbial growth rate is not yet balanced by the decay rate. Therefore, a biomass steady state is not achieved after 160 hours. This is due to the high-oxygen environment (1 × 10 −2 kg m −3 ) and the thinin the bulk liquid relax to steady state before the total biomass concentration relaxes, as bulk concentrations are mainly determined by biomass in the top biofilm layers, which ensures a high growth rate. The NO 2 − profile is influenced by both AOB synthesis and NOB consumption. Thus, the bulk concentration decreases with the increase of NOB populations. Parallel performance. Parallel performance is crucial to IbM solvers for simulating large and complex problems. To investigate the performance of NUFEB, a scalability test was performed based on Case Study 2. The test aims at evaluating how simulation time varies with the number of processors for a fixed problem size. The speed-up of the scalability test is defined as t p0 /t pn , where t p0 and t pn are the actual CPU times spent on the baseline case and the test case respectively. Then the efficiency is the ratio between the speed-up of the baseline and the test cases obtained when using a given number of processors, i.e., N p0 t p0 /N pn t pn , where N p0 and N pn are the number of processors employed in these cases.
In order to reach a considerable number of microbes (4 millions), the baseline case is performed using 4 processors, and the subsequent tests were performed with increasing numbers of processors, ranging from 8 to 256. As mentioned previously, NUFEB implements two distinct solutions for the parallelisation of continuum-based and particle-based processes. The performance of the two solutions are studied separately, and are presented inIt can be observed that when employing a small number of processors, both solutions are close to the ideal speed-up (linear increase) and the parallel efficiency is over 90%. As more processor nodes are added, each processing node spends more time doing inter-processor communication than useful processing, and the parallelisation becomes less efficient. In the tests using 128 and 256 processors, the parallel efficiency decreases to around 42%, but it still shows a good scalability. Note that we assume the oxygen concentration in bulk liquid is kept constant by aeration. The slow growth of NOBs is due to the nutrient limitation, their low growth rate, and spatial distribution. At the early stage of the biofilm formation, the system is in NO 2 − limited condition. As the biofilm growth, the bottom area where NOBs reside turns to oxygen-limited condition. Both conditions inhibit NOBs growth.
https://doi.org/10.1371/journal.pcbi.1007125.g008
## Availability and future directions
In this paper, we have presented the NUFEB tool for modelling and simulating Individualbased Models. The tool, documentation, and examples are publicly available on the GitHub repository: https://github.com/nufeb/NUFEB. To date, NUFEB has been adopted to model microbial communities in a variety of studies. In, we studied the influence of nutrient gradients on biofilm structure formation, and the influence of shear flow on growing biofilm. Bacteria twitching motility under fluid flow was investigated in. We also successfully applied the tool to study the influence of thermodynamics and pH on microbial growth. Inand, we used micro-scale NUFEB simulations to emulate the behaviour of biofilm/floc in the macro-scale. An emulation strategy for parameter calibration of NUFEB against iDy-noMiCS has been applied in.
The main advantage of the 3D microbial system model is to study effects of spatial behaviours of microbial systems. Our ongoing work includes modelling biofilm streamer oscillation, modelling nitritation anammox system, and coupling microbial growth with fluid condition. The goal for NUFEB development in the future would be to deliver an even more general, efficient and user-friendly platform. This will include, for example, the development of an intuitive Graphical User Interface which will significantly improve the user experience. In order to make NUFEB available for power-efficient HPC architecture, we will also focus on a Kokkos port for the NUFEB code. This would allow the code to run on different kinds of hardware, such as GPUs (Graphics Processing Units), Intel Xeon Phis, or many-core CPUs. The initial conditions and the model parameters are kept the same in all cases. The average achieved particle number of all cases is 4.77 × 10 6 with 0.5% standard deviation. The difference is due to the randomness in subdomains. The simulation with 4 processors is regarded as baseline case for computing speed-up. Doubling the numbers of processors results in almost double speed-up when small processor numbers are employed (e.g., less than 32). However, the speed-up and parallel efficiency decrease with increasing numbers of processors due to inter-processor communication.
https://doi.org/10.1371/journal.pcbi.1007125.g009
The computational demands of IbM will always place a limit on the scale at which they can be applied. However, it is now evident that this limit can overcome by the use of statistical emulators. A statistical emulator is a computationally efficient mimic of an IbM that can run thousands of times faster. In principle this new approach will allow the output of an IbM to be used at the metre scale and beyond and thus to make predictions about systems level performance in, for example, wastewater treatment or biofilm-fouling and drag on ships. This is a strategically important advance that creates a new impetus for the development of IbM that can credibly combine chemistry, mechanics, biology and hydrodynamics in a computationally efficient framework. NUFEB is, we believe, the first generation of IbM to meet that need and will help IbM transition from a research to application.
## Supporting information
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Development and validation of a pain monitoring app for patients with musculoskeletal conditions (The Keele pain recorder feasibility study)
Background: Assessing daily change in pain and related symptoms help in diagnosis, prognosis, and monitoring response to treatment. However, such changes are infrequently assessed, and usually reviewed weeks or months after the start of treatment. We therefore developed a smartphone application (Keele Pain Recorder) to record information on the severity and impact of pain on daily life. Specifically, the study goal was to assess face, content and construct validity of data collection using the Pain Recorder in primary care patients receiving new analgesic prescriptions for musculoskeletal pain, as well as to assess its acceptability and clinical utility. Methods: The app was developed with Keele's Research User Group (RUG), a clinical advisory group (CAG) and software developer for use on Android devices. The app recorded pain levels, interference, sleep disturbance, analgesic use, mood and side effects. In a feasibility study, patients aged > 18 attending their general practitioner (GP) with a painful musculoskeletal condition were recruited to use the app twice per day for 28 days. Face and construct validity were assessed through baseline and post-study questionnaires (Spearman's rank correlation coefficient). Usability and acceptability were determined through post-study questionnaires, and patient, GP, RUG and CAG interviews. Results: An app was developed which was liked by both patients and GPs. It was felt that it offered the opportunity for GPs to discuss pain control with their patients in a new way. All participants found the app easy to use (it did not interfere with their activities) and results easy to interpret. Strong associations existed between the first 3 days (Spearman r = 0.79) and last 3 days (r = 0.60) of pain levels and intensity scores on the app with the validated questionnaires. Conclusions: Collaborating with patient representatives and clinical stakeholders, we developed an app which can be used to help clinicians and patients monitor painful musculoskeletal conditions in response to analgesic prescribing. Recordings were accurate and valid, especially, for pain intensity ratings, and it was easy to use. Future work needs to examine how pain trajectories can help manage changes in a patient's condition, ultimately assisting in self-management.
# Background
Annually, 15-20% of all adults attending general practice present with musculoskeletal conditions [bib_ref] International comparisons of the consultation prevalence of musculoskeletal conditions using populationbased healthcare..., Jordan [/bib_ref]. Around 40% of these patients are prescribed analgesia during their first consultation for musculoskeletal pain, half of whom will receive a non-steroidal anti-inflammatory drug (NSAID), and 29% a moderate to strong opioid containing analgesic [bib_ref] Pain medication management of musculoskeletal conditions at first presentation in primary care:..., Ndlovu [/bib_ref] [bib_ref] The association between pain intensity and the prescription of analgesics and non-steroidal..., Muller [/bib_ref]. The general practitioner's (GP's) intention is to relieve their patient's pain and the decision to use analgesia, for example in low back pain, is often based on the patient's verbal report of their pain and their personal analgesic preferences [bib_ref] Deciding on analgesic prescription dosing for acute back pain: once daily or..., Perrot [/bib_ref]. Any subsequent review of analgesic efficacy is similarly limited by the GP relying on the patient's reports of their pain progress, often quantified by asking patients to rate their pain on a scale from 0 (no pain) to 10 (worst pain) [bib_ref] Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36..., Hawker [/bib_ref]. However, this existing approach to pain monitoring fails to account for the multiple dimensions of pain experience in relation to its onset (acute, or gradually developed over time), course over time (stable or highly variable), severity, and impact on everyday life [bib_ref] Long-term trajectories of back pain: cohort study with 7-year follow-up, Dunn [/bib_ref].
Obtaining more detailed pain experience information could be an important and useful tool for clinical practice. For example, symptom trajectories have been shown to help obtain an accurate diagnosis in headache; monitor the severity/impact of symptoms in asthma or abnormal vaginal bleeding [bib_ref] Characteristics of prospectively measured vaginal bleeding among women trying to conceive, Mikolajczyk [/bib_ref] ; or assess short-term responses to treatment [bib_ref] Transcutaneous electrical nerve stimulation as adjunct to primary care management for tennis..., Chesterton [/bib_ref]. Furthermore, information on short-term symptom trajectories has the potential to provide important prognostic information, and is likely to result in better long-term prediction of health outcomes [bib_ref] Repeat assessment improves the prediction of prognosis in patients with low back..., Dunn [/bib_ref] [bib_ref] Brief pain re-assessment provided more accurate prognosis than baseline information for low-back..., Mansell [/bib_ref].
Despite a wide variation in musculoskeletal pain trajectories over time, early changes in painful symptoms after prescribing an analgesic (such as an opioid) are not routinely collected as part of musculoskeletal follow-up assessments, which often take place several weeks or even months later [bib_ref] Predictors of outcome in neck and shoulder symptoms: a cohort study in..., Bot [/bib_ref] [bib_ref] Characterizing the course of low Back pain: a latent class analysis, Dunn [/bib_ref] [bib_ref] Defining discriminative pain trajectories in hip osteoarthritis over a 2-year time period, Verkleij [/bib_ref]. Monitoring of these changes in response to opioids is very important, and now underpins current guidelines, especially since evidence for the long-term effectiveness of opioids is lacking [bib_ref] The effectiveness and risks of long-term opioid therapy for chronic pain: a..., Chou [/bib_ref] , and there is an increasing recognition of the harms related to opioid use [bib_ref] Relationship of opioid use and dosage levels to fractures in older chronic..., Saunders [/bib_ref] [bib_ref] Opioid prescriptions for chronic pain and overdose, Dunn [/bib_ref]. Although many studies have reported on the long-term (6-12 months) outcomes of musculoskeletal pain [bib_ref] Repeat assessment improves the prediction of prognosis in patients with low back..., Dunn [/bib_ref] [bib_ref] Characterizing the course of low Back pain: a latent class analysis, Dunn [/bib_ref] , and investigated long-term trajectories using repeated pain assessment [bib_ref] Characterizing the course of low Back pain: a latent class analysis, Dunn [/bib_ref] [bib_ref] Prediction of outcome in patients with low back pain -a prospective cohort..., Kongsted [/bib_ref] [bib_ref] The course of chronic and recurrent low back pain in the general..., Tamcan [/bib_ref] [bib_ref] Trajectories of low back pain, Axén [/bib_ref] , little is known about short-term pain trajectories following primary care consultations, how they relate to long-term outcomes, and in what way they might be used to support the primary care management of musculoskeletal pain.
When pain trajectories are measured, data have often been collected using paper diaries, which are cumbersome, have low completion rates, and may be completed retrospectively resulting in inaccurate and potentially biased data [bib_ref] Daily process methodology for measuring earlier antidepressant response, Lenderking [/bib_ref] [bib_ref] Daily diaries vs retrospective questionnaires to assess asthma control and therapeutic responses..., Okupa [/bib_ref] [bib_ref] for the ProAct65+ Study Team. Completion and return of fall diaries varies..., Perry [/bib_ref]. In recent years alternative methods for daily data collection have been proposed, including the use of text messaging [bib_ref] Comparison between data obtained through real-time data capture by SMS and a..., Johansen [/bib_ref] [bib_ref] Identifying clinical course patterns in SMS data using cluster analysis, Kent [/bib_ref] , palm top computers [bib_ref] An electronic diary on a palm device for headache monitoring: a preliminary..., Allena [/bib_ref] , or Smartphone technology [bib_ref] Smartphone applications for chronic pain management: a critical appraisal, Alexander [/bib_ref]. These approaches are gaining popularity given the increasing use of Smartphones and particularly now with two thirds of British adults owning one. One systematic review identified 55 articles reporting the design, evaluation, or use of smartphone-based software for healthcare professionals, students, or patients. The authors highlight the increasing use of Smartphone technology in healthcare and their potential role in patient education, disease self-management, and remote monitoring of patients [bib_ref] A systematic review of healthcare applications for smartphones, Mosa [/bib_ref]. Limitations of Smartphone technology have also been reported, most importantly the lack of personalised feedback, usability issues (e.g. ease of data entry), and poor integration of Smartphone data with electronic health records [bib_ref] Identifying preferences for mobile health applications for self-monitoring and self-management: focus group..., Ramanathan [/bib_ref] [bib_ref] Mobile applications for diabetes selfmanagement: status and potential, El-Gayar [/bib_ref]. A systematic review of currently downloadable pain monitoring apps has highlighted the lack of scientific rigour used to ensure validity and reliability with respect to pain measurement [bib_ref] mHealth: a strategic field without a solid scientific soul. A systematic review..., De La Vega [/bib_ref]. However, where attention to validation has been robust, moderate to high reliability and validity has been reported [bib_ref] Construct validity and reliability of a real-time multidimensional smartphone app to assess..., Stinson [/bib_ref] [bib_ref] Development and testing of Painometer: a smartphone app to assess pain intensity, De La Vega [/bib_ref]. Therefore, in this feasibility study, our aim was to:
## Develop a smartphone application ("keele pain
Recorder") for use by patients with painful musculoskeletal conditions to record daily information on their pain severity and the impact of pain on daily life. 2. assess the acceptability and clinical utility of the Pain Recorder in terms of completion rates, feasibility of its use, and its influence on GP decision-making. Even if an app is found to have a high level of validity in collecting data, its clinical usefulness is only as good as its level of acceptability to the user in day to day use [bib_ref] Assessing the quality and usability of smartphone apps for pain self-management, Reynoldson [/bib_ref]. It is therefore, as part of any app development, essential to examine how acceptable and useful to the user it is. 3. assess face and content validity and explore construct validity of data collection using the Keele Pain Recorder in musculoskeletal patients presenting to primary care receiving new analgesic prescriptions. We hypothesised that single Pain Recorder items were highly correlated with validated questionnaires measuring the same domain of interest i.e. whether there was a strong correlation between the Keele Pain Recorder scores and questionnaire scores at baseline (day 1-3 for the Pain Recorder) and follow-up (last 3 days for the pain app), and between changes over time in scores from the app and questionnaires (longitudinal validity). Recorder. This is an area of data collection that is important since information on short-term symptom trajectories might help in establishing the possible cause of symptoms (diagnosis); estimate the future course of a condition (prognosis) [bib_ref] Transcutaneous electrical nerve stimulation as adjunct to primary care management for tennis..., Chesterton [/bib_ref] [bib_ref] The Nordic Back pain subpopulation program: can patient reactions to the first..., Axén [/bib_ref] ; monitor the severity of symptoms in patients with chronic conditions; and assess early response to treatment (intervention) [bib_ref] Transcutaneous electrical nerve stimulation as adjunct to primary care management for tennis..., Chesterton [/bib_ref]. However, early changes in pain and other symptoms are often not assessed until several weeks or even months after the first consultation or start of treatment [bib_ref] Predictors of outcome in neck and shoulder symptoms: a cohort study in..., Bot [/bib_ref] [bib_ref] Characterizing the course of low Back pain: a latent class analysis, Dunn [/bib_ref] [bib_ref] Defining discriminative pain trajectories in hip osteoarthritis over a 2-year time period, Verkleij [/bib_ref] , and development of the Keele Pain Recorder (KPR) offers a clear opportunity to examine these short-term trajectories in relation to these areas of clinical assessment.
## Assess the daily
# Methods
## Development of the "pain recorder" design of the pain recorder
A workshop with 9 members of Keele University's Institute for Primary Care and Health Sciences (IPCHS) Research User Group (RUG) was organised to obtain the views and opinions of people with experience of living with chronic musculoskeletal pain to underpin the design of the app. During the workshop, drafts (mock-ups) of screens which might be potentially used for the Pain Recorder, and mock-up examples of possible pain trajectories were presented to the RUG and a range of aspects of the design and content of the proposed app were discussed. These included: content and phrasing of questions regarding pain and the impact of pain on everyday life to include in the app; response options; appearance and functionality of the app; content of help functions; how completion rates could be optimised. The app developer as well as members of the research team attended the workshop. Consensus on the final content was achieved through the use of electronic voting (using Turning Point) which allowed the RUG to vote independently, ranking options in order of preference. The software calculates a weighted response giving greater importance to higher ranked options and thereby a clear indication of the group's overall choice on any aspect of the app discussed. Discussions were audio-recorded, and written reports drafted summarising feedback and advice from the RUG. The app developer used the outcomes from this workshop to develop a first alpha-version of the Pain Recorder.
## Symptom measures
Though no formal reference to IMMPACT pain measurements were made as this was a pain monitoring app that was developed entirely by patients, the content of the app reflects accurately those pain measurements suggested as outcome measures in its recommendations [bib_ref] Core outcome measures for chronic pain clinical trials: IMMPACT recommendations, Dworkin [/bib_ref]. Consequently, the KPR records data on all domains including pain intensity, analgesic use, the temporal nature of pain, its effect on physical function, emotional functioning (mood), pain trajectories assessing change, and a record of adverse events. All measures were derived by the RUG and clinical advisory group (CAG). Pain recording was measured using a standard numerical scale (0 = no pain, 10-worse pain imaginable) [bib_ref] Assessment of pain, Breivik [/bib_ref]. Pain impact was recorded in terms of interference of pain with activities at home, leisure, or work. A scale of 1-5 where 0 = none and 5 = extremely was felt most appropriate. This scale was developed by the RUG amended from the SF36. If the patient had been asleep in the previous 12 h this could be recorded and whether their pain had interfered with sleep or not. This was based on the Jenkins sleep scale [bib_ref] A scale for the estimation of sleep problems in clinical research, Jenkins [/bib_ref]. For psychometric assessment the WHO-5 Well-Being Index assessing cheerfulness was used, scored from 0 = none of the time, to 5 = all of the time. The WHO-5 psychometric properties have been assessed and found to have adequate validity in screening for depression and in measuring outcomes in clinical trials. It has good construct validity as a unidimensional scale measuring well-being. The RUG voted against using the 'pain bothersomeness' question commonly used in research.
A novel suggestion from the RUG related to assessing medication adherence. Rather than recording the number of tablets taken, the group felt this was too complex and time consuming and it was suggested the patient record if they had taken their medication 'as prescribed' , 'less than' , or 'more than' recommended by their GP. Finally, perceived side effects could be recorded at each data entry point, or at any time the user wished to.
## Face and content validity, beta-testing
The alpha version of the Pain Recorder was demonstrated, including examples of downloads of pain recordings (trajectories), during further workshops with our patient advisory group (6 RUG members), and an additional clinical advisory group (13 participants) of (academic) GPs, physiotherapists, research nurses, primary care researchers, research facilitators, and an IT manager. The following issues were discussed: (a) content and wording of items included in the Pain Recorder (face validity); (b) whether the app incorporated all relevant aspects of pain interference (content validity); (c) its utility for discussing symptoms and medication use with patients (including adherence and possible adverse effects); (d) its utility for supporting decisions regarding treatment; and (e) opportunities for using the app for research purposes. These meetings were recorded, and written reports produced to summarise comments and suggestions made by the group.
Following these meetings, a beta version of the Pain Recorder app suitable for Android smartphones/tablets was developed by the study team based solely on the recommendations from these groups.
The clinical advisory group (CAG) reviewed the app and overall it was liked. It was felt that it offered the opportunity for GPs to discuss pain control with their patients in a new way that related medication use and activity. An interesting point was made that in fact the app might encourage adherence to decisions made on pain medication use through its repeat recording reminder. It was agreed that all elements were clinically useful and informative. They did not suggest further changes to the context of the app.
Beta testing was undertaken over a four-week period using 6 'Pain Recorder' loaded tablets by study team members, RUG members, and additional members of the public of varying age, educational level, and familiarity with smartphone technology. Following identification of 'bugs' , typographic errors and any elements of the Pain Recorder that could cause confusion, the developer produced the 'Gold' version of the app which was to be used in the feasibility study.
Feasibility study "Pain recorder" deployment and data collection Study population Patients aged 18 years and over who consulted at their general practice with a new episode of musculoskeletal pain (defined as no consultation for musculoskeletal conditions in the previous 3 months) were invited to take part in the study, if they were prescribed a stronger class of analgesic (Non-Steroidal Anti-Inflammatory Drug (NSAID) or strong opioid/opioid combination medication containing more than 8 mg of codeine per tablet). Each GP system was programmed to notify the GP when a suitable patient for the study was identified through an appropriate prescription being issued for one of the stronger analgesics or NSAIDs during a consultation. This would also tag the patients record identifying them as a suitable candidate for the study with a searchable code. An automated message would appear on the GP's computer screen (a 'pop-up') during the consultation and accordingly the GP could invite the patient to the study. If the GP did not invite the patient, weekly searches of the GPs computer system identified these individuals through the code and they were then invited to the study via a letter from the GP's surgery. Patients were excluded by the GP if they had symptoms or signs indicative of pathology requiring urgent medical attention; pain because of cancer or other non-musculoskeletal condition; pain due to an acute injury; inability to read and speak English; vulnerability (e.g. dementia, terminal illness, severe mental health problems); or travelling outside Europe/for longer than 30 days following the consultation. Eligible patients were identified either during the consultation by the GP or nurse practitioner, or after the consultation through regular searches of consultation records. If the patient was considered suitable, the patient was informed about the study and provided with a participant information sheet and referral form. Patients signing this form provided written consent to be contacted by the research nurse. The GP/nurse practitioner prescribed pain medication as planned and continued to provide care as usual during the study. Four general practices in North Staffordshire participated in the study. Ethical approval for this study was obtained from the NRES Committee West Midlands (REC Reference: 14/WM/1214). Patients involved in the RUG group and workshops after data collection received travelling expenses whilst the GPs involved and users of the app in the study were not remunerated.
## Data collection
The research nurse contacted patients interested in the research project, provided further information about the study and made an appointment for a baseline visit at the general practice. When signed informed consent was obtained, participants completed a baseline questionnaire, which included questions on sociodemographic variables, lifestyle factors (alcohol use, physical activity level); height and weight (for Body Mass Index, BMI); history of musculoskeletal pain; use of analgesics; and baseline levels of pain intensity, pain interference, sleep, and mood using validated questionnaire items. The baseline pain assessment recorded how long the patient had been experiencing pain (less than 2 weeks to more than 12 month), and how long since they had had no pain for more than a month. The main measure relating to the app was pain severity in the last 24 h.
The research gave the participant's a tablet pre-loaded with the app for the duration of the study (4 weeks). The Pain Recorder was demonstrated and set up for the participant, who was then invited to enter recordings twice daily for a period of 4 weeks.
An appointment was made for the participant to attend a follow-up meeting with the research nurse and a repeat consultation with the GP or nurse prescriber 1 month after receiving the Pain Recorder. During the follow-up meeting the research nurse exported anonymised data from the Pain Recorder as a password protected file and sent this to a secure NHS account, accessible only by the GP, the research nurse and members of the study team responsible for data management and analysis. Graphical presentations of the data were produced allowing the GP or nurse prescriber to discuss the pain trajectories and response to prescribed pain medication with the participant. The GPs and Nurse practitioners received no formal training in how to interpret the graphs, or use them in their management. This allowed us to examine how this novel data collection method would be used, or not used, in their day to day practice. The participant was given a follow-up questionnaire to be completed at home, including follow-up questions on pain intensity, pain impact, sleep and mood; course of pain over the past month, acceptability of the Pain Recorder, and the participants' views regarding the use of the Pain Recorder in the management of their pain problem.
## Acceptability, usability and clinical utility
Acceptability was assessed by descriptively summarising Pain Recorder completion rates and responses by study participants to questions regarding acceptability and usefulness in the follow-up questionnaire. Furthermore, after completion of data collection a workshop was organised with study participants, which was facilitated by members of our RUG, and semi-structured telephone interviews were conducted with GPs from recruiting practices to discuss: (a) acceptability and feasibility of using the Pain Recorder in clinical practice; (b) its utility for discussing symptoms and medication use; (c) its utility for supporting treatment decisions; (d) opportunities for using the Pain Recorder for research purposes and (e) its usability in terms of ease of use and interference with patients' daily activities. Usability is a key strength of any smartphone application as it will ultimately determine whether the app will be used in daily practice by patients. The full potential of any app is unlikely to be realized unless the development and design take into account usability [bib_ref] Assessing the quality and usability of smartphone apps for pain self-management, Reynoldson [/bib_ref]. The workshop group with users of the app and RUG members followed an open forum. The meetings were led by the RUG members who had developed the content of the app using headline topics that were then detailed using the thinking aloud technique. The agenda here was driven by patients, and reflects their experiences with no input from clinicians such that the perspectives that evolved were personal to users of the app. GPs interviews followed a specific set of questions relating to their interaction with the user and were purely their own opinion.
Workshop discussions and interviews were audio-recorded, and reports written to summarise responses to questions and feedback provided during discussions.
## Initial testing of construct validity
The pain trajectories generated by the app were downloaded and assessed by a second clinical advisory group (CAG) to discuss the validity of recordings and determine if subgroups of participants with distinct patterns of short term pain could be identified.
Construct validity was explored by comparing scores for pain, sleep interruption, and mood collected at baseline and one-month follow-up using validated questionnaires with entries on the Pain Recorder. The questionnaires used the same validated questions as those used in the app, for example the WHO 5 well-being index, a RUG adapted SF36 pain interference scale and numerical pain rating scale [bib_ref] Assessment of pain, Breivik [/bib_ref]. Other validated questions included in the questionnaires included pain duration [bib_ref] The importance of symptom duration in determining prognosis, Dunn [/bib_ref] , pain trajectories, the Jenkin's sleep questionnaires [bib_ref] A scale for the estimation of sleep problems in clinical research, Jenkins [/bib_ref] , and physical activity (GPAQ) [bib_ref] Validation of the Global Physical Activity Questionnaire for self-administration in a European..., Wanner [/bib_ref].
Statistical analysis Descriptive statistics were used to characterise the study population, in terms of age, gender, BMI, work status, pain characteristics, global health and lifestyle factors. Potential subgroups of participants with distinct patterns of short-term pain were identified using blind voting. Nine members, including GPs, a pain consultant, and physiotherapists were shown trajectories of pain intensity, pain interference and mood for each participant, with blind voting (Turning Point software) being used to identify distinct trajectories and classify participants according to these proposed subsets. Spearman's correlation coefficient was used to assess the strength of correlations between Pain Recorder scores and questionnaire scores.
## The 'gold' version of the keele pain recorder app
The final version of the app had an alarm built in to remind the user to record their pain experience at 8 am and 8 pm. This could not be switched off, but the tablet/ phone could be if the patient did not wish to be disturbed. There was an initial set up which was completed with the research nurse to demonstrate the app. The patients personal ID was entered and in the set-up gender and date of birth were recorded. Then each screen subsequently appeared to ask in order about (1) Average pain level in the last 24 h (2) Level of pain interference or whether their pain disturbed sleep (3) Well-being questions. At the set alarm times the patient would then complete a similar set of questions (1) Average pain in the last 12 h (2) Level of pain interference or had the pain disturbed sleep (3) Well-being questions (4) Medication use (more than, less than or as prescribed) (5) Did the patient feel they has side effects and if yes a diary record of these could be entered (date and time recorded) (6) A screen asking if they wanted to record anything else and if 'yes' they could enter a written note (date and time recorded). The patient had the option to enter a pain recording at any other time they wished other than the pre-set times.
From the front page of the app patients could access a help section for each page, giving advice on completing the page, and this could be accessed from each page when being competed as well. There was also in this section a Frequently Asked Questions (FAQ) page which answered questions such as who to contact if there is a problem, and who had access to the information recorded (Screenshot 8, Additional file 1). The last page of the app advised patients if they felt severely unwell during the study for any reason to contact urgent medical help, ensure the tablet/phone was charged at least once per day, if the patients missed a recording not to worry and just complete the next.
After 1 month the patient returned to the Research Nurse on the day of their appointment with their GP, the graphical output from the app was downloaded and passed to the GP for use in the consultation. The follow up questionnaire was completed.
# Results
Development and testing of the "pain recorder"
The 'gold' version of the Pain Recorder developed following the RUG and CAG workshops consisted of baseline information; 6 questions to assess pain severity, impact of pain on sleep or activities during the day, mood, use of analgesics, and experience of adverse effects from analgesics; two help functions and a diary. Screenshots are available in Additional file 1 (Screenshot 1-7). The RUG felt that it would not be onerous for users to record data twice per day, every day (between 8 and 10, am and pm), and that an alarm would be helpful to remind them.
The RUG felt that baseline information should be kept to a minimum to improve completion rates and consisted of age, gender and number of days per week where the user was physically active for more than 30 min per day.
The RUG indicated that it was imperative instructions for all sections were easily accessible and written in plain English. It was also felt that a frequently asked questions (FAQ) section would be helpful, and in this section information about contacting the study team should be easily accessible.
One suggestion from the RUG was to link physical activity, events, and other unusual activities to the recordings as this might explain strong fluctuations in pain trajectories. The conclusion was to allow patients access to a diary which would record the date of entry and in which such information, and any other that the patient felt necessary, could be recorded and made available to the GP.
Beta testing of the app revealed 38 'bugs' and issues that required remedy by the developer. The majority of these were phraseology within the instructions and help sections in the app, two major 'bugs' concerned functionality (e.g. pressing 'help' on the side effects page led to the diary and not the appropriate help page). Correction of these errors produced the final 'Gold' version which was used in the feasibility study.
Finally, the study team and developer produced instructions for installing the Pain Recorder onto Smartphones, how to complete the app, and how to download pain recordings for both the research nurses and patients to use. Downloads from the app could be copied and pasted into GP medical records, printed for the GP to use with the patient in a follow up consultation, as well as emailed to the GP through a secure NHS.net account used by the research nurse.
Acceptability and clinical utility of the "pain recorder" Completion rates Five general practices (17 general practitioners, 2 nurse practitioners) agreed to take part in the Keele Pain Recorder study, four of which recruited participants to the study. The computer-generated message identifying potential candidates for the study (pop-up) fired on 167 occasions from which 27 suitable patients were invited to the study and 25 consented to take part. Three withdrew consent and one was lost to follow up when they did not attend the baseline clinic. Of the 21 participants, 13 were females and 8 males, with a median age of 62 (IQR 50 to 70) years old [fig_ref] Table 1: Patients self-reported characteristics at baseline [/fig_ref]. Their median baseline pain intensity was 6 (IQR: 4-7). Eighteen participants attended the follow up clinic and returned the 1-month follow-up questionnaire with three being lost to follow up at this point. The participants entered 862 records, 53.1% in the morning, and 46.9% in the evening. Of these records, 255 (30%) were entered during the first week, 198 (23%) the second week, 198 (23%) the third week, and 211 (24%) the fourth week. Median number of records per participant were 23 in the morning and 22 in the evening over the recording period of 4 weeks, indicating that recordings were made on 73.3% of days. There was no association between completion rates and gender or baseline pain intensity levels, but older participants tended to record more often than younger participants (Spearman correlation coefficient 0.47, p = 0.03). [fig_ref] Table 2: Summary of results from the follow-up questionnaire [/fig_ref] gives a summary of results from the 1-month follow-up questionnaire (response n = 18; 86%). All participants found the Pain Recorder easy to read, with the majority using the app daily or often. Six participants reported that the use of the Pain Recorder had interfered with their daily routine. The majority discussed the graphs with their GP, and 11 reported that the GP showed interest in the results. Most found the graphs easy to understand, but opinions varied regarding their impact on helping to discuss their pain or changes on medication.
No technical errors within the app occurred during testing other than with two users where we were unable to download the graphical output at the follow up nurse research clinic, and these were later downloaded and given directly to the GP who consulted the patient in the following week.
## Workshop with participants
Two users attended the workshop with 2 RUG members who developed the app. All study participants who attended the participants' meeting indicated that the Pain Recorder had been very easy to use ('child's play'), self-reporting that it took only 2 min on average to complete (max 5 min), and not interfering with daily life or sleep. They had not needed the 'Help Function' , nor had required help from the study team or other people (these two users were not those who reported it did interfere or who had used the help function in the questionnaires). There was a discussion regarding the usefulness of the diary function: although this was considered helpful to provide context to pain impacting on specific activities, it was not needed on a daily basis. In terms of utility the Pain Recorder was perceived as a tool to inform the GP how they had been managing their pain, and it contributed to decisions by the GP regarding medication changes. They felt the graphs were useful in aiding the discussion and understanding of their pain. They also indicated that the app did not directly influence their thoughts, feelings or actions related to mood, pain interference, or medication usage. Important suggestions for future use of the Pain Recorder included [bib_ref] International comparisons of the consultation prevalence of musculoskeletal conditions using populationbased healthcare..., Jordan [/bib_ref] to make data recorded by users available to them in graphical output at any time, and (2) to make the app a 'real time' monitoring tool which might offer advice on treatment when required, either from protocols in the app or from a medical professional.
## Results from gp interviews
Results from semi-structured telephone interviews (20-30 min each) with one GP from each of the four recruiting practices showed that they felt the graphs generated by the Pain Recorder were easy to interpret, and most felt that the graphs were useful in helping patients make choices about their use of medication. GPs were confident that patients were happy to bring these graphs to the consultation in the expectation of discussing the results with their GP. Two GPs however felt they could get the same information from taking a history from the patient, and only one felt it influenced their management strategy of the patients' conditions. One GP who did not use the graphs in the consultation did not do so as they felt there was not enough time to do this, so chose to ignore them. All four GPs said they would recommend the Pain Recorder to patients for self-monitoring of their condition. In terms of usefulness for research, two felt it would be more useful in investigating and managing chronic rather than acute pain, whilst one GP thought it had a place in trials of analgesics to monitor patients' response. They suggested that in future iterations patients should be able to see trends in their pain levels at any time they wished. Face and construct validity of the "pain recorder"
At both baseline and follow-up (4 weeks), the correlations of pain intensity scoring between questionnaire and Pain Recorder were very strong (Spearman's correlation coefficient ≥ 0.79, P < 0.0001). For pain interference and mood, although weaker, significant or potential correlations were still seen based on this relatively small group of participants . The correlation was even weaker for sleep where the question examined related to 'trouble staying asleep'. The number of entries in this category were fewer with missing data and probably accounts from some of the issues. Overall there were 862 responses in total. 840/862 (97.5%) responded with prescribing information. Among the 862, in total 93(10.7%) reported a side effect. Those using analgesia "as prescribed" (n = 585) and "less than prescribed" (n = 186) had similar side effect rates (10.0 and 11.9%, respectively). Side effect rate appeared to be higher in "more than prescribed", but there were only 6 such records. No significant difference of side effect was observed between the three (P < 0.33).
## Assessment of pain trajectories from the "pain recorder"
A wide variety of short-term pain trajectories were evident from recordings obtained from participants of the feasibility study. The clinical advisory group proposed to classify these into four main groups : (a) recovering (five participants); (b) fluctuating low to moderate level of pain (eight participants); (c) deteriorating; (d) unable to classify (two participants), when there was more than 7 days of consecutive data missing. This CAG visually inspected the temporal associations between changes in daily pain score, mood, and pain interference (for example . The subjective impression in this example was that the level of pain was reflected in scores for mood score and interference of pain on everyday activities, suggesting face validity of the pain trajectories.
# Discussion
In close collaboration with patient representatives and clinical stakeholders, we developed a smartphone/tablet app, which can be used to help clinicians and patients monitor painful musculoskeletal conditions in response to analgesic prescribing. Early testing in a small sample of people consulting with musculoskeletal pain in general practice showed promising results in terms of face and content validity, acceptability, and clinical usefulness. Correlation between Pain Recorder and questionnaire scores for assessing construct validity Despite there being more than 270 pain apps available to download [bib_ref] There's a pain app for that": review of patient-targeted smartphone applications for..., Lalloo [/bib_ref] , there have been few studies that have validated pain monitoring apps. Often existing apps are designed by software engineers and there is little input from patients and clinicians in the design and evaluation of these apps [bib_ref] There's a pain app for that": review of patient-targeted smartphone applications for..., Lalloo [/bib_ref] [bib_ref] 181) utilization of a smartphone app for chronic pain care: does this..., Jurcik [/bib_ref]. A recent review and Editorial examining pain app design and use highlighted this fact, particularly the finding that apps sometimes appear to offer solutions to pain management with little thought given to the content of the app being validly related to clinical factors [bib_ref] Smartphone applications for chronic pain management: a critical appraisal, Alexander [/bib_ref] [bib_ref] A quality review of smartphone applications for the management of pain, Portelli [/bib_ref]. This same review concluded that the development of apps needs to be evidence-based with rigorous evaluation of outcomes being important in enhancing the understanding of the potential of these apps [bib_ref] A quality review of smartphone applications for the management of pain, Portelli [/bib_ref]. Our study directly addresses this issue in that the design of the Keele Pain Recorder was driven by both patient and clinician experience and advice, whilst its clinical usefulness, acceptability and validity was assessed by several primary care clinicians in conjunction with the app users. One recent study has offered a more vigorous examination of a pain monitoring App. Suso-Ribera found that weekly monitoring of pain over a 30-day period found similarly high levels of compliance, acceptability, ease of use and construct validity to the KPR [bib_ref] Reliability, Feasibility, and usefulness of pain monitor: a multidimensional smartphone app for..., Suso-Ribera [/bib_ref]. Jamison also found that a pain monitoring app was acceptable to patients and easily utilised [bib_ref] A pilot comparison of a smartphone app with or without 2-way messaging..., Jamison [/bib_ref]. This is encouraging as it suggests that pain monitoring apps in varying forms are devices that patients will be willing to engage with when managing their pain.
A series of iterative workshops and interviews with patients, clinicians and musculoskeletal researchers were used to (i) discuss content and functionality of the app, (ii) further improve the design of the app, and (iii) explore opinions regarding acceptability, validity, and usefulness. Patients confirmed that they felt discussing recordings from the app helped with their GP's understanding of their pain condition, whilst GPs considered it useful in helping their patients make choices about medication. Patients found the Keele Pain Recorder easy to use, and the GP found the graphical output easy to interpret. As in a study from the USA, both groups found using a pain recorder app acceptable in clinical practice [bib_ref] 181) utilization of a smartphone app for chronic pain care: does this..., Jurcik [/bib_ref].
The individual pain trajectories of patients varied widely, reflecting wide differences in the impact and experience of pain, even over the course of only 1 month after consulting in primary care. However, the trajectories were determined through a visual analysis and a consensus exercise amongst experienced clinicians rather than using statistical methods. The number of trajectories available to examine were too few and precluded this. Therefore, it is possible that the trajectories determined were subject to individual bias, however, agreement in each case was by majority, and each participant had extensive experience in the management of musculoskeletal pain. There may be value in assessing early symptom trajectories in people with musculoskeletal conditions, though further research is needed before any potential clinical usefulness can be established. Many prognostic models developed in studies of low back pain have been shown to have limited predictive performance [bib_ref] What is the prognosis of back pain?, Hayden [/bib_ref] , although some tools, such as the Start Back Screening Tool, have been extensively tested and are now also available as a smartphone app. Although its predictive performance has been confirmed in several populations, one study showed that it was no better than clinical acumen in predicting low back pain outcomes [bib_ref] Prediction of outcome in patients with low back pain -a prospective cohort..., Kongsted [/bib_ref]. One reason for limited predictive performance of prognostic tools may be that most are based on only a single assessment of pain. In our study, the Keele Pain Recorder app demonstrated three main short-term pain patterns: improving, fluctuating or worsening, which reflect those previously reported in patients with low back pain [bib_ref] Long-term trajectories of back pain: cohort study with 7-year follow-up, Dunn [/bib_ref] [bib_ref] The course of chronic and recurrent low back pain in the general..., Tamcan [/bib_ref] [bib_ref] Trajectories of low back pain, Axén [/bib_ref]. Potentially the use of a smartphone/ tablet pain app might allow for more frequent and detailed characterisation of pain trajectories shortly after healthcare consultation. This, therefore, might be used in the future to help develop more accurate predictive models and early identification of patients likely to do well (preventing unnecessary treatment) versus those who may benefit from early, more intensive treatment [bib_ref] Characterizing the course of low Back pain: a latent class analysis, Dunn [/bib_ref].
An important limitation is the small sample size of the feasibility study, which limits generalisability and precision of our findings regarding construct validity. Though the sample was small, we found a good correlation between established and validated pain measures in the baseline and follow-up questionnaires. However, there was no significant correlation with interference, mood and sleep. It is possible that in all these 3 domains (mood, interface and sleep), this might have occurred because we averaged results from the app over the first and last 3 days of the study. Therefore, due to the potential variability in these factors recorded in the app over this time, they may not reflect those recordings in the baseline and follow-up questionnaire. Small numbers in the study will also have limited our power to detect any correlation. Further testing of the Pain Recorder in larger groups of patients is needed to more formally and quantitatively investigate construct validity of the app, and to establish its clinical utility for monitoring pain by investigating impact of its use on clinical decision making and patient outcomes. Additionally, we only tested the app amongst patients with musculoskeletal pain, so the generalisability of its use in other conditions such as headache or pelvic pain cannot be assured. Similarly, our study focused on monitoring pain following prescription of a stronger class of analgesics, but the app could also be used to study pain trajectories following other types of treatments for pain.
Assessing acceptability and usability of the KPR presents problems in common with other smartphone apps. Both factors are interdependent, and it is likely that testing these elements 1 week after starting using the app would provide different results to our assessment which was at 1 month of use. There may be issues for users when they first use the software due them being unfamiliar with it, and therefore limit ease of use. Consequently, acceptance of the app might be diminished. However, after a month's use, familiarity with the mechanics of the software might improve usability for the patients. Equally though, with loss of novelty, the user might lose interest in it and so its acceptance as a daily activity might be lost. Future research might overcome these issues through testing at both points in time to give a more comprehensive view of an apps acceptability and usability.
Low completion rates were a limiting factor in this study. This might have been compounded by the intrusion of the technology into daily life with it being perceived as an interference in the user's normal routine. There are ways in which this might be overcome, for example gamification has been shown to improve engagement and retention in app use [bib_ref] Using a gamified mobile app to increase student engagement, retention and academic..., Pechenkina [/bib_ref]. Equally if the app had been used on the patient's own mobile smartphone, the more immediate access to this device (rather than a tablet kept elsewhere) might have improved completion rates. An additional limitation relates to the comparison of the baseline and follow-up questionnaire pain scores with those recorded in the app to determine how valid these were. The most valid figure would have been to equate the single baseline/follow-up figure with the first and last day score in the app giving a direct contemporaneous comparison. However, we chose to use the initial and final 3-day average of the study. This might have led to errors in the comparison due to the potential variability of the patient's pain during that period when compared to the single recording at baseline and follow-up. However, due the possibility that we might recruit low total numbers to this novel research, we chose to use the 3-day average which would potentially give at least 1 record during the 3 days. If we had only used the 1st or last day alone, there might have been more missing values.
Two recordings could not be classified due to a lack of consecutive data (> 7 days). This limitation was not overcome by using reminder on the tablet and is likely to be due to external circumstances, or interference of the technology in the participants' everyday life, which was indicated to occur sometimes according to 6 of 18 participants in the feasibility study. This may have been compounded by the fact that the users did not have direct access to their pain graphs, which might have acted in a positive way to reinforce use of the app. Concerns have been expressed regarding the potential negative impact of frequent pain reporting on physical health and work productivity [bib_ref] Prevalence of pain reporting and associated Health outcomes across emerging markets and..., Goren [/bib_ref]. When asked specifically, participants reported that they felt using the app had not directly influenced their thoughts, feelings or actions related to mood, pain interference, or medication usage. However, further research should investigate to what extent the use of the Keele Pain Recorder is associated with consultation rates, healthcare resource use, and changes in physical or mental health.
We developed secure methods for archiving, downloading and emailing pain trajectories from the Pain Recorder to the GP and patient to be used in their consultation. These methods will now be extended to allow open-access to the Pain Recorder and support use of the app on both Android and Apple phones or tablets. Future research, however, needs to examine how such data may be accessed in a 'live' format such that GPs or other health care professionals may use information regarding pain trajectories to manage a patient's condition when it deteriorates, for example during an acute attack of gout or a flare of knee osteoarthritis. Research may also focus on the potential of using the Keele Pain Recorder in self-management, such that software might independently recognise when a patient is at risk of developing disabling pain, offering feedback and advice to the patient without the input of a third party such as the GP. However, these devices will require rigorous assessment to ensure the advice is safe, relevant, and does not miss the possibility of 'red-flag' conditions such as cancer pain or other conditions (e.g. inflammation) that need medical attention.
# Conclusions
In conclusion, within this limited sample of users, we have successfully developed the Keele Pain Recorder tablet app which both patients and clinicians considered easy to use. Early testing shows promise in terms of validity, acceptability and clinical usefulness, with clear priorities identified for further testing and investigation of its potential role and impact in the clinical and self-management of musculoskeletal conditions and other pain problems.
[fig] Figure 1, Figure 2: Baseline questionnaire "staying asleep" & Pain Recorder "staying asleep" first 1-Follow-up questionnaire pain intensity & Pain Recorder pain intensity last 1-3 a days (n = 18) 0.92 < 0.0001 6. Follow-up questionnaire pain interference & Pain Recorder pain interference last 1-3 a days (n = 18) 0.40 0.11 7. Follow-up questionnaire mood & Pain Recorder mood last 1-3 a days (n = 18) 0.15 0.56 8. Follow-up questionnaire "staying asleep" & Pain Recorder "staying asleep" last 1-3 a days (n = 15) Real life examples of different pain 4-week trajectories pain recorded by the app: (a): recovering; (b) fluctuating low to moderate pain; (c) deteriorating; (d) unable to classify (a) (b) (c) Real life changes in pain score recorded by the app (a) (1 = no pain, 10 = worst pain), mood (b) (1 = cheerful all the time 5 = none of the time) and interference (c) (1 = not at all, 5 = all the time) over a 28 day period following initiation of a new prescription analgesic Bedson et al. [/fig]
[table] Table 1: Patients self-reported characteristics at baseline [/table]
[table] Table 2: Summary of results from the follow-up questionnaire [/table]
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Who does not participate in telehealth trials and why? A cross-sectional survey
Background: Telehealth interventions use information and communication technology to provide clinical support. Some randomised controlled trials of telehealth report high patient decline rates. A large study was undertaken to determine which patients decline to participate in telehealth trials and their reasons for doing so. Methods: Two linked randomised controlled trials were undertaken, one for patients with depression and one for patients with raised cardiovascular disease risk (the Healthlines Study). The trials compared usual care with additional support delivered by the telephone and internet. Patients were recruited via their general practice and could return a form about why they were not participating.Results: Of the patients invited, 82.9 % (20,021/24,152) did not accept the study invite, either by returning a decline form (n = 7134) or by not responding (n = 12,887). In both trials patients registered at deprived general practices were less likely to accept the study invite. Decline forms were received from 29.5 % (7134/24,152) of patients invited. There were four frequently reported types of reasons for declining. The most common was telehealth-related: 54.7 % (3889/,7115) of decliners said they did not have access or the skills to use the internet and/or computers. This was more prevalent amongst older patients and patients registered at deprived general practices. The second was health need-related: 40.1 % (n = 2852) of decliners reported that they did not need additional support for their health condition. The third was related to life circumstances: 27.2 % (n = 1932) of decliners reported being too busy. The fourth was research-related: 15.3 % (n = 1092) of decliners were not interested in the research.Conclusions: A large proportion of patients declining participation in these telehealth trials did so because they were unable to engage with telehealth or did not perceive a need for it. This has implications for engagement with telehealth in routine practice, as well as for trials, with a need to offer technological support to increase patients' engagement with telehealth. More generally, triallists should assess why people decline to participate in their studies.Trial registration: The Healthlines Study has the following trial registrations: depression trial: ISRCTN14172341 (registered 26 June 2012) and CVD risk trial: ISRCTN27508731 (registered 05 July 2012).
Background "Telehealth is the remote exchange of data between a patient at home and their clinician(s) to assist in diagnosis and monitoring typically used to support patients with Long Term Conditions". Examples of telehealth include online cognitive behavioural therapy, home monitoring of health parameters such as blood pressure and internet-based health forums. Globally, telehealth is becoming a prominent part of healthcare delivery.
Telehealth interventions are increasingly being tested in randomised controlled trials (RCTs). Some of these telehealth trials have reported high decline rates of over 75 % amongst potential participants [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref]. A recent review of 37 telehealth studies found an average decline rate of 32 %, but this varied between 4 % and 71 % for the individual studies; and only two studies were RCTs [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref]. Decline rates may vary because of the health condition under study. For example, some patient groups are known to be especially difficult to recruit, such as those with depression [bib_ref] Factors affecting recruitment into depression trials: systematic review and meta-synthesis of qualitative..., Hughes-Morley [/bib_ref]. An alternative explanation is that selection criteria may have differed between trials. It is also possible that the nature of the telehealth interventions being tested affected the decline rates due to varying levels of acceptability amongst potential participants.
High decline rates in RCTs generally are problematic because they may result in underpowered trials or extended recruitment periods which have resource implications [bib_ref] A reinvestigation of recruitment to randomised, controlled, multicenter trials: a review of..., Sully [/bib_ref]. Additionally, if those declining are not representative of the clinical population, external validity may be compromised [bib_ref] The external validity of published randomized controlled trials in primary care, Jones [/bib_ref]. High decline rates may also indicate problems with the acceptability of the intervention, with implications for its uptake when delivered in routine practice. This latter issue may be particularly relevant to telehealth trials, since there may be physical barriers in terms of accessing technology [bib_ref] Exploring barriers to participation and adoption of telehealth and telecare within the..., Sanders [/bib_ref] , as well as psychological barriers such as low confidence in using the technology [bib_ref] Are people with chronic diseases interested in using telehealth? A cross-sectional postal..., Edwards [/bib_ref]. Understanding which types of patients do not participate in telehealth trials, and why, may help to improve recruitment rates, external validity and interpretation of trial results.
There has been some research examining the types of patients who do not participate in telehealth trials. No gender differences have been found in telehealth trials [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] or telehealth studies which use other methodologies [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref]. Older adults are more likely not to participate in telehealth trials [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] due to technological demands and because they have less access to technology [bib_ref] Exploring barriers to participation and adoption of telehealth and telecare within the..., Sanders [/bib_ref] [bib_ref] Are people with chronic diseases interested in using telehealth? A cross-sectional postal..., Edwards [/bib_ref] [bib_ref] Patients' characteristics and rate of internet use to obtain cancer information, Mancini [/bib_ref]. This higher non-participation rate amongst older people also mirrors RCTs more generally [bib_ref] Age and response rates to interview sample surveys, Herzog [/bib_ref] [bib_ref] When "no" might not quite mean "no"; the importance of informed and..., Williams [/bib_ref]. In terms of differences in participation amongst socio-economic groups, one telehealth trial did not find any differences [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref]. However, studies of routine telehealth services report lower uptake amongst patients from lower socioeconomic groups [bib_ref] Factors that influence public engagement with eHealth: A literature review, Hardiker [/bib_ref]. This mirrors the evidence based more generally on healthcare research, where there are lower response rates from patients experiencing greater socio-economic deprivation [bib_ref] The effect of health, socio-economic position, and mode of data collection on..., Ekholm [/bib_ref] [bib_ref] Non-participation and mortality in different socioeconomic groups: the FINRISK population surveys in..., Harald [/bib_ref]. There is no evidence on participation in telehealth trials by ethnic minority groups. However, these groups are usually under-represented in RCTs in the United Kingdom and North America, partly because a common exclusion criterion is the inability to speak English fluently.
To date, only a handful of studies have explored why patients do not participate in telehealth studies [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref] [bib_ref] Exploring barriers to participation and adoption of telehealth and telecare within the..., Sanders [/bib_ref]. These have been fairly small quantitative studies of 625, 331 and 79 patients [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref] , with only one qualitative study with 19 patients [bib_ref] Exploring barriers to participation and adoption of telehealth and telecare within the..., Sanders [/bib_ref]. There was a review of why patients may not participate in telehealth research, but this was in relation to telehealth studies in general, rather than RCTs specifically [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref]. Eight main reasons for declining participation in telehealth trials have been identified. First, patients have concerns, and in some cases anxiety, regarding the technological aspects of the intervention [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Exploring barriers to participation and adoption of telehealth and telecare within the..., Sanders [/bib_ref]. Second, some patients do not perceive the intervention to be beneficial [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref]. Third, some patients prefer face-to-face care, rather than healthcare delivered remotely, which is an integral component of telehealth interventions [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref]. Fourth, some patients believe that there is no need for the intervention, for example, because routine healthcare is sufficient [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref]. Fifth, many patients decline due to poor health [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] A pilot randomised controlled trial of a telehealth intervention in patients with..., Bentley [/bib_ref] or perceive their older age to be a barrier [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref]. Sixth, patients are simply not interested [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref] , but it is not clear whether patients are not interested in the telehealth intervention or the research itself. Seventh, patients simply do not want to participate in the study. Finally, some patients are too busy to participate. These mirror some of the reasons why patients decline to participate in RCTs more generally: patients are not interested or are too busy, are too ill, perceive themselves as ineligible, have transport barriers, have concerns about the intervention or do not want to be randomised [bib_ref] Motives for (not) participating in a lifestyle intervention trial, Lakerveld [/bib_ref] [bib_ref] Drivers and barriers to patient participation in RCTs, Jenkins [/bib_ref] [bib_ref] Exploring patients' reasons for declining contact in a cognitive behavioural therapy randomised..., Barnes [/bib_ref].
Given the high decline rate in some telehealth trials and the limited literature investigating this topic, it is important to understand more about which patients do not choose to participate in telehealth trials and their reasons for this choice. We utilised recruitment data for two large linked RCTs of telehealth (the Healthlines Study) to undertake a quantitative study of who does not participate in telehealth trials and why.
# Methods
## The healthlines study
The Healthlines Study consists of two linked RCTs of a telehealth intervention, one for patients with depression and one for patients with a raised risk of cardiovascular disease (CVD risk) [bib_ref] Effectiveness and cost-effectiveness of a telehealth intervention to support the management of..., Thomas [/bib_ref]. The telehealth intervention consisted of up to 12 months of telephone support from a health information advisor and access to a range of online resources, including computerised cognitive behavioural therapy software packages, support for home monitoring (such as blood pressure monitoring) and links to online educational and support tools. To participate in the trials, patients had to have at least weekly access to an email account and the internet. The trials were approved by the National Research Ethics Service Committee South West-Frenchay (Reference 12/SW/0009) and have the following trial registrations: depression trial: ISRCTN14172341 (registered 26 June 2012) and CVD risk trial: ISRCTN27508731(registered 05 July 2012).
## Recruitment of patients
Patients were recruited via 43 general practices between June 2012 and July 2013. Searches of practice records were undertaken to identify potential participants. For the depression trial, patients were identified who were aged 18 to 100 years old, and in the preceding three months to the search visit were coded as having depression, anxiety or low mood, or who were issued an antidepressant prescription. Exclusion criteria were applied such as having psychosis, substance abuse issues or dementia. For the CVD risk trial, patients were selected who were aged 40 to 74 years and, based on data in routine general practice records, were estimated to have at least a 19 % risk of having a cardiovascular event in the next ten years and had one or more modifiable risk factors (being a smoker, overweight or having high blood pressure). Exclusion criteria were applied including being identified as part of the depression trial or having had a cardiovascular event, such as a heart attack or stroke. General practitioners at each practice were asked to exclude any patients they felt were unsuitable, for example, the recently bereaved [bib_ref] Effectiveness and cost-effectiveness of a telehealth intervention to support the management of..., Thomas [/bib_ref]. After exclusions, 16,570 patients were invited to participate in the depression trial, and 7582 patients in the CVD risk trial. A larger number of patients were invited in the depression trial because it was anticipated that there would be a lower participation rate.
Practice staff sent the selected patients an information pack, including a personalised invitation letter, a patient information booklet, an acceptance form, a decline form and a freepost envelope. Approximately three weeks later, patients who had not responded were re-sent the information packs.
## Acceptors, active decliners and non-responders
Invited patients could return an acceptance form (acceptors). On receipt of this form, they were contacted by a researcher to assess their eligibility. Alternatively, patients could return a decline form (active decliners), or choose not to respond (non-responders). The decline form was anonymous, but linked to demographic information from the general practices through a study ID number. Patients were asked to indicate why they were declining to participate by ticking one or more of the pre-specified reasons provided on the form, including: I do not have regular access to the internet or an email address I do not feel confident enough with computers I am too busy at the moment I do not feel I need any more support with my health at this time I am not interested in this research
The pre-specified reasons incorporated the common reasons for declining reported in previous literature on uptake in telehealth studies and RCTs more generally. In addition, there was an 'other reason' that responders could select, and they were asked to specify this reason in a free text box. A small proportion of patients (4.2 %, n = 1020/24,152) were sent a decline form with the additional pre-specified reason of 'I do not understand the study'. This was part of a sub-study where patients were sent a re-designed written patient information sheet to explore whether this increased recruitment [bib_ref] Effectiveness and cost-effectiveness of a telehealth intervention to support the management of..., Thomas [/bib_ref].
Data on the age, sex and ethnicity of invited patients was collected from the general practice records. However, ethnicity was poorly recorded in practice records, and the study had to reply on self-report from patients who returned forms. For this reason, ethnicity is missing for non-responders. Levels of socio-economic deprivation were derived from the general practice at which patients were registered.
# Analysis
A two-stage analysis was conducted. The first stage was a comparison of the socio-demographic characteristics of the different response types. This included comparing the acceptors with a combined category of active decliners and non-responders. A comparison of active decliners and non-responders was then conducted to understand the effect of non-response bias on our study of why patients declined. Data were entered into SPSS. Ethnicity was re-categorised from multiple ethnicity categories into a dichotomous variable of White or Black and Minority Ethnicity (BME). A variable was created to capture socio-economic deprivation based on the general practice at which a patient was registered. Deprivation was defined by the decile attributed to a general practice in the National Practice Profiles. Patients registered at practices in deciles 1-5 were coded as 'deprived' and patients registered at practices in deciles 6-10 were coded as 'affluent'. Chi-square and independent t-tests were undertaken to test for differences by age, gender, ethnicity and deprivation.
The second stage involved exploring the reasons why patients declined to participate in the trials. Frequencies and percentages were calculated to understand the prevalence of each reason. The other reasons patients provided for declining were coded by one researcher (AF) and checked by another researcher (KAH). Of the 979 patient-reported 'other' comments in the depression trial, the researchers initially disagreed on the coding for 204 . Of the 551 CVD risk 'other' comments, there was disagreement on 63 (11.4 %). These comments were discussed and consensus reached on coding.
As a considerable proportion of responders declined because of technology-related reasons, and this issue is intrinsic to telehealth, further analysis was conducted. Responders were categorised as declining for a technologyrelated reason if they had selected the pre-specified reasons of no internet access or no computer confidence, or if they provided their own reason related to technology issues, such as not having a computer. Technology-related decliners were compared with other types of decliners by age, gender, ethnicity and deprivation. Chi-square and independent t-tests were undertaken.
# Results
## Responses to the healthlines study invitation
Overall, 82.9 % (20,021/24,152) of the patients invited did not accept the study invite [fig_ref] Table 1: Frequency of response type by health condition [/fig_ref]. Most of these patients (n = 12,887) did not respond at all, but decline forms were received from 29.5 % (n = 7134) of all patients invited [fig_ref] Table 1: Frequency of response type by health condition [/fig_ref]. Response type differed by health condition, with patients with depression more likely not to accept than those with raised CVD risk (84 % depression versus 80 % CVD risk, P ≤ 0.001, X 2 = 452.8, df = 1).
## Socio-demographic characteristics decliners (active decliners and non-responders) compared to acceptors
All those that declined the invite (active decliners and non-responders) were compared with those accepting the invite [fig_ref] Table 2: The characteristics of the different response types for the depression trial [/fig_ref]. In the depression trial, males (86 %, n = 4570) were more likely to decline than females (84 %, n = 9419), P ≤ 0.001, X 2 = 11.43, df = 1. In the CVD risk trial, females (83 %, n = 1675) were more likely to decline than males (78 %, n = 4357 males), P ≤ 0.001, X 2 = 18.26, df = 1. Although the differences were statistically significant, they were not large for the depression trial. Of those whose ethnicity was known, in the depression trial there was no difference in decline rates between white patients (87 %, n = 4152) and BME patients 92 %, n = 82), P = 0.168, X 2 = 1.90, df = 1), although numbers were small. In contrast, there was some evidence in the CVD risk trial that patients from BME groups (82 %, n = 78) were more likely to decline than white patients (70 %, n = 2553), P = 0.08, X 2 = 6.94, df = 1). Patients from deprived general practices were more likely to decline compared with patients from affluent general practices in both the depression and CVD risk trials (Depression: 87 %, n = 3947 deprived versus 84 %, n = 10,042 affluent declined, P ≤ 0.001, X 2 = 21.4, df = 1; CVD risk: 85 %, n = 1725 deprived versus 78 %, n = 4307 affluent declined, P ≤ 0.001, X 2 = 42.93, df = 1). In the depression trial, there was no difference in the mean age of decliners compared to acceptors (Mean age: Decliners: 50.66 years old versus Acceptors: 50.79, P = 0.710, t = −0.371). In the CVD risk trial, however, there was a statistically significant difference, with decliners being younger than acceptors (Mean age: Decliners: 65.75 years versus Acceptors: 66.33 years, P = 0.001, t = −3.321), although the mean difference in age was less than a year.
## Active decliners compared to non-responders
Of the patients who did not accept the study invite, decline forms were received from 35.6 % (7134/20,021). The active decliners were compared to non-responders to understand how representative active decliners were of all patients who did not accept the study invite. In both the depression and CVD risk trials, females were more likely to return decline forms compared to males (Depression: 41 %, n = 3049 females versus 29 %, n = 1333 males, P ≤ 0.001, X 2 = 14.67, df = 1; CVD risk: 54 %, n = 907 females versus 42 %, n = 1845 males, P ≤ 0.001, X 2 = 67.95, df = 1). Ethnicity could not be compared because the data were not available for non-responders. In both trials, patients registered at affluent practices were more likely to return decline forms compared to patients registered at deprived practices (Depression: 32 %, n = 3239 affluent versus 29 %, n = 1143 deprived, P ≤ 0.001, X 2 = 14.31, df = 1; CVD risk: 46 %, n = 2001 affluent versus 43 %, n = 751 deprived, P = 0.039, X 2 = 4.24, df = 1), although differences were not large. In both trials, patients who returned decline forms were more likely to be older than non-responders (Depression: Mean age: Active decliners: 59.7 years versus Non-responders: 46.5 years, P ≤ 0.001, t = −45.679; CVD risk: Mean age: Active decliners: 67.4 years versus Non-responders: 64.4 years, P ≤ 0.001, t = −18.740).
## Reasons for declining number of reasons for declining
Of the 7134 decline forms received, 19 were blank. These have been included in the response type calculations above, but they have been excluded from the reasons for decline analysis below. Ninety-nine percent (n = 7045) of patients who returned a decline form provided a reason for declining to participate in the trial (Depression: 98. CVD risk: 29). These were added to the frequencies of the pre-specified reasons. A small number of responders ticked the other reason option, but instead of specifying a reason, they wrote a comment, such as "thank you, but no thank you" (Depression: 17; CVD risk: 8); these are not included below. People mainly ticked the pre-specified categories [fig_ref] Table 4: Pre-specified reasons for declining [/fig_ref] , with one in five giving other reasons for declining [fig_ref] Table 5: Other reasons for declining [/fig_ref]. Both the pre-specified reasons and the other reasons were categorised into four domains. These domains were developed thematically based on conceptual similarities.
# Telehealth-related reasons
Telehealth-related reasons, in terms of no computer confidence and/or no internet access, were prominent prespecified reasons [fig_ref] Table 4: Pre-specified reasons for declining [/fig_ref] , affecting 54.7 %, n = 3889 of active decliners. A small number of active decliners also specified that they were not participating because they did not like the telehealth intervention (1.4 %, n = 102), primarily because it was being delivered remotely rather than face-to-face; because of communication difficulties, such as a visual impairment, which meant that engagement in a telehealth intervention was problematic (1.3 %, n = 92); and because of practical barriers, such as not having a computer (1.3 %, n = 94).
## Health need
A lack of perceived health need was the second most frequent option in the pre-specified reasons (40.1 %, n = 2852). A small number of people also declined because they were satisfied with their current level of support, such as having regular health checks (3.1 %, n = 222), or they were currently receiving treatment for other health conditions (3.4 %, n = 245).
## Patients' lives
The fourth most common reason for declining was being too busy (27.2 %, n = 1932). Some decliners provided other related reasons such as not having space in their life to participate in a trial, which sometimes related to not having the emotional capacity to participate (3.4 %, n = 243); or indicating that it was an inconvenient time to join the study, for example, because of planned time away from home (2.7 %, n = 189).
# Research-related reasons
A lack of interest in the research was given as the fifth most common reason for declining (15.3 %, n = 1092). Related reasons were that patients perceived themselves as unsuitable for the trial, for example, because they did not have depression (1.9 %, n = 134); or the trial could be distressing, such as having to discuss health issues
# Technology-related reasons
Further analysis was conducted to compare responders who actively declined for technology-related reasons to those who actively declined for non-technology issues [fig_ref] Table 6: The characteristics of patients declining due to technology issues [/fig_ref]. Decliners in the CVD risk trial were more likely to offer a technology-related reason than those in the depression trial (P ≤ 0.001, X 2 = 126.93, df = 1). In the CVD risk trial, 63 % (n = 1729) of patients gave at least one reason for declining that was related to technology issues compared with 49 % (n = 2160) in the depression trial. There were some differences in the demographics of patients who declined for technology reasons compared with non-technology reasons. In the CVD risk trial, females were more likely to decline for a technology reason than males (P = 0.011, X 2 = 6.52, df = 1), although there was no such difference in the depression trial (P = 0.508, X 2 = 0.44, df = 1). Across both trials, there was no statistically significant difference in the ethnicity of patients who declined because of technology issues and those that did not (Depression: P = 0.590, X 2 = 0.29, df = 1; CVD risk: P = 0.281, X 2 = 1.16, df = 1), although the direction was that technology issues affected more people from BME communities. In both trials, patients were more likely to decline because of technology reasons if they were registered at deprived practices (Depression: P ≤ 0.001, X 2 = 30.98, df = 1; CVD risk: P ≤ 0.001, X 2 = 21.22, df = 1), or older (Depression: P ≤ 0.001, t = −35.683; CVD risk: P = 0.001, t = −3.352). This age difference was more pronounced in the depression trial, where there was a mean age difference of 15 years between patients who declined for technology reasons and those who declined for non-technology reasons compared with a one-year difference for CVD risk. This may be because there was a wider age range of patients included in the depression trial compared to the CVD risk trial.
# Discussion
Although non-participation in telehealth trials is a widespread problem and may introduce bias, this issue has only been explored within small-scale studies. Our study appears to be the largest to date to examine two key issues related to this problem in patients with long-term conditions: the characteristics of patients who do not participate in telehealth trials (comparing over 24,000 invited patients) and the main reasons for not participating (responses from over 7000 patients). In total, 82.9 % of patients did not accept the study invite, which is comparable to other telehealth trials [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref]. Patients from deprived general practices were less likely to accept the study invite than those from affluent general practices. This contrasts with one telehealth trial which found no difference [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] , but it is similar to findings in other health research generally [bib_ref] The effect of health, socio-economic position, and mode of data collection on..., Ekholm [/bib_ref] [bib_ref] Non-participation and mortality in different socioeconomic groups: the FINRISK population surveys in..., Harald [/bib_ref]. In the CVD risk trial, younger patients and BME patients were less likely to accept the study invite. However, the differences were small. Overall, reasons for declining could be grouped into those that were specific to the telehealth intervention, those that were health need-related, issues related to patients' lives and research-related reasons. Firstly, a large proportion of patients cited issues specific to telehealth interventions. They did not have access to a computer or the internet, or sufficient skill to use them; this has been found elsewhere [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref]. The proportion of patients in this study declining because of technology reasons was high compared with a recent survey on telehealth, which reported that 68.5 % had computer technology available [bib_ref] Are people with chronic diseases interested in using telehealth? A cross-sectional postal..., Edwards [/bib_ref]. Our study and this latter survey were both undertaken as part of the Healthlines Study, and both included patients with depression and raised CVD risk. It may be that people in our study gave this answer as an easy option to decline or that they felt they needed more access to, or confidence with, technology in order to engage in these telehealth trials. It is also surprisingly high because in the patient information booklets for the Healthlines trials, it was explained to potential participants that methods were in place to facilitate access. For example, researchers could help patients to set up email accounts, and patients were allowed to use a family member or friend's email address or computer.
Older patients were more likely to decline because of technology reasons, and this is consistent with previous research [bib_ref] Patients' characteristics and rate of internet use to obtain cancer information, Mancini [/bib_ref]. For example, internet availability rates have been reported as only 26.5 % amongst patients over 74 years old [bib_ref] Are people with chronic diseases interested in using telehealth? A cross-sectional postal..., Edwards [/bib_ref]. Consequently, telehealth may be more accessible for health conditions where patients are generally younger, for example, cystic fibrosis. Patients registered at general practices categorised as deprived were more likely to decline because of technology reasons, as found in research regarding the uptake of telehealth in general [bib_ref] Factors that influence public engagement with eHealth: A literature review, Hardiker [/bib_ref].
Secondly, a common reason for declining was patients not feeling a need for additional support with their health, reflecting previous literature [bib_ref] Research in home-care telemedicine: Challenges in patient recruitment, Subramanian [/bib_ref]. Health problems could also create a barrier, reflecting an obstacle to participation in trials in general [bib_ref] Recruitment difficulties in a home telecare trial, Mair [/bib_ref] [bib_ref] A pilot randomised controlled trial of a telehealth intervention in patients with..., Bentley [/bib_ref]. However, this result does contrast with other studies about the acceptability of telehealth, which found that poor health was not related to less interest in telehealth [bib_ref] Are people with chronic diseases interested in using telehealth? A cross-sectional postal..., Edwards [/bib_ref]. Consequently, it may be that declining the trial due to health issues is related to participation in the trial rather than receiving the intervention.
Finally, there were a number of reasons offered that were generic to trials, not just telehealth trials [bib_ref] Home telehealth uptake and continued use among heart failure and chronic obstructive..., Gorst [/bib_ref] [bib_ref] Drivers and barriers to patient participation in RCTs, Jenkins [/bib_ref] [bib_ref] Why patients don't take part in cancer clinical trials: an overview of..., Cox [/bib_ref] [bib_ref] Recruitment challenges and strategies in a home-based telehealth study, Oliver [/bib_ref]. Some were related to patients' lives, such as being too busy. We identified an issue from the free text answers around emotional capacity to participate in terms of not having 'space in their life' , for example, having caring responsibilities. A proportion of patients declined for geographical reasons, for example, because they were moving house or they were frequently away from home (for example, having a second home abroad). These are interesting both in the context of trials and routine practice of telehealth because they challenge a key principle of telehealth, which is that it is suitable for patients who have difficulty attending a general practice in person [bib_ref] Therapistdelivered internet psychotherapy for depression in primary care: a randomised controlled trial, Kessler [/bib_ref]. There were also some reasons given that were research-related, such as not being interested in participating. It was unclear whether this affected patients' desire to participate in the trial or to receive the telehealth intervention. A small number of patients declined because they had issues with the research procedures, such as not wanting to be randomised, confidentiality concerns or regarding the study as a waste of money. These reasons are consistent with previous literature [bib_ref] Why patients don't take part in cancer clinical trials: an overview of..., Cox [/bib_ref] and need to be better addressed in patient information booklets. However, such reasons were only a small proportion of the reasons that patients declined.
# Strengths and limitations
The key strength of the study is that it explored who does not participate in telehealth trials and the reasons why. It appears to be by far the biggest study exploring non-participation in telehealth trials, as the largest study we have identified included only 625 decliners. It also explores acceptability of telehealth trials in relation to two diverse long-term conditions, which is important as telehealth is often aimed at supporting patients with their long-term conditions [bib_ref] Submit your next manuscript to BioMed Central and take full advantage of:..., Mclean [/bib_ref]. There were some limitations. First, decline forms were only returned from 35.6 % of patients who did not accept the study invite. We do not know why patients did not respond. We have shown that active decliners were different from those who simply did not respond. These demographic differences may have an impact on the prevalence of the various reasons given for declining. For example, older people were more likely to return decline forms, but also to cite technology issues as the reason for declining. Therefore, technology reasons may be less of an issue in the wider patient population than in our sample. Second, we know little about the impact of ethnicity. The ethnicity of non-responders was not known in our study and there were only a small number of patients from BME groups in the decline population. Third, it was sometimes unclear whether patients were declining the trial or the telehealth intervention. Fourth, over 90 % of responders selected at least one of the prespecified reasons for declining. This may be because they identified the key reasons why patients choose not to participate in a telehealth trial. However, there is the risk that patients may have selected those reasons because they were easy to tick. It is recommended that some of the other key reasons cited, such as health issues, be included in future decline forms. Fifth, the sample size was large and sometimes statistically significant differences were very small differences and therefore unlikely to be important. Sixth, this analysis is based on participation in two trials and the findings may be specific to the health conditions and the selection criteria for inviting people.
## Implications for telehealth trials and routine practice
The main implication of this research for telehealth trials is that a large proportion of patients with long-term conditions, especially those in more deprived geographical areas, either do not have access to the internet or do not perceive themselves to have the necessary technological skills required for computer-based telehealth interventions. Telehealth interventions may be acceptable to more people if access is facilitated as part of any intervention, particularly for patients in areas of socioeconomic deprivation. The implication for routine health care provision is that at present telehealth should not be the only care option because there is a significant proportion of the population without sufficient technology skills or access to use it. For example, patients with depression would need to be offered cognitive behavioural therapy in forms other than computer-based ones (such as books) or offered considerable technological support.
Another important implication relates to the contextual issues of patients' lives, with patients choosing not to participate because of health or domestic issues. Given this, triallists could aim to minimise the commitment and burden to participants of both the intervention and the trial. Additionally, there were some differences in the reasons for declining between the depression and CVD risk trials, which indicates that whilst there are similarities in telehealth trials, the specific reasons may differ depending on the population from which the trial is recruiting. It is recommended that future telehealth trials include a similar process of exploring why patients decline to participate to help build a picture of issues affecting participation for different health conditions. Finally, a key reason for concern about low participation rates in trials is that this may lead to recruitment bias, with those patients included in the trial not being representative of those for whom the intervention would be provided in routine clinical practice. This was not the most pressing issue here. Most patients declining did so because they felt that telehealth was not suitable for them rather than for reasons related to the research.
# Conclusions
In both trials, patients from deprived general practices were more likely to decline the study invite. Patients provided a range of reasons for declining to participate in a telehealth trial, and these reasons were generally consistent with the literature from the few other telehealth trials, as well as from trials more generally. Some reasons were specific to telehealth, such as not having internet access; others were generic to all studies, such as being too busy. However, some reasons were specific to individuals' health needs, and so were different across the two groups of long-term conditions recruited to this study. The primary reason for declining was due to technology issues; this was the case for patients who were registered at general practices in deprived areas. This has implications for the feasibility of both telehealth trials and telehealth in routine practice. For some patients, it was not clear if they were declining to participate in the trial or the intervention per se, for example, patients who said that they were not interested. If the latter, it raises questions about the acceptability of the telehealth intervention. It is recommended that other trials also explore why patients are not participating to facilitate a greater understanding of non-participation.
Abbreviations BME: Black and minority ethnic; CVD: Cardiovascular disease; RCT: Randomised controlled trial.
[table] Table 1: Frequency of response type by health condition [/table]
[table] Table 2: The characteristics of the different response types for the depression trial [/table]
[table] Table 3: The characteristics of the different response types for the CVD risk trial [/table]
[table] Table 4: Pre-specified reasons for declining [/table]
[table] Table 5: Other reasons for declining [/table]
[table] Table 6: The characteristics of patients declining due to technology issues [/table]
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Understanding COVID-19: From Origin to Potential Therapeutics
Currently, a global pandemic era of public health concerns is going on with the Coronavirus Disease 2019 , which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The first case of COVID-19 was reported from Wuhan's Huanan seafood market in China late December 2019. Bats, pangolins, and snakes have been nominated as salient carriers of the virus. Thanks to its high pathogenicity, it can cause severe respiratory infections. Fever, dry cough, sore throat, pneumonia, septic shock, and ground-glass opacities are the foremost clinical manifestations of COVID-19. Immunocompromised patients are at high risk for COVID-19 infection and may lead to death. Scientist and government agencies around the globe are putting forward their best efforts and resources for the effective treatment of human coronavirus infections; however, neither vaccines nor antiviral drugs are available for the treatment of human coronaviruses (HCoV) infections such as SARS (severe acute respiratory syndrome), MERS (Middle Eastern respiratory syndrome), and COVID-19. Since the outbreak, a plethora of research and review articles have been published. Moreover, the mass media has bombarded the public with conflicting opinions about the pandemic. There is a dire need for accurate and reliable information concerning this pandemic. In this review, we have compiled the up to date information about the origins, evolution, epidemiology, and pathogenesis of this disease. Moreover, very few reports have addressed the clinical features and current status of treatment for COVID-19; we have adequately addressed these topics in detail in this review. Finally, a detailed account of clinical trials of vaccines and other therapeutics currently in progress has been delineated.
# Introduction
The recent pandemic has affected more than seventeen million people across 215 countries and territories and caused deaths of more than 751.399 people as per reported data from John Hopkins University Coronavirus Resource Center on August 13, 2020. World Health Organization declared this pandemic a Public Health Emergency of International Concern (PHEIC) on 31 January 2020. possesses a large RNA genome and exhibits a unique replication strategy. Specifically, the virus has a nonsegmented positive-sense RNA genome with a 5 cap structure along with a 3 poly (A) tail. This unique structure allows the replicase polyproteins to read the genome for translation. Two-thirds of the genome encodes nonstructural proteins (NSPs), while the remaining one-third encodes for structural and accessory proteins. Vital structural proteins are spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. In addition, two viral replicase polyproteins termed PP1a and PP1ab are produced by the virus, which are modified into 16 mature NSPs [bib_ref] Recent progress in studies of arterivirus-and coronavirus-host interactions, Zhong [/bib_ref]. [fig_ref] Figure 1: The life cycle of a coronavirus, modified, and used from Zhang et... [/fig_ref] illustrates the life cycle of the CoVs. the genome encodes nonstructural proteins (NSPs), while the remaining one-third encodes for structural and accessory proteins. Vital structural proteins are spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. In addition, two viral replicase polyproteins termed PP1a and PP1ab are produced by the virus, which are modified into 16 mature NSPs [bib_ref] Recent progress in studies of arterivirus-and coronavirus-host interactions, Zhong [/bib_ref]. [fig_ref] Figure 1: The life cycle of a coronavirus, modified, and used from Zhang et... [/fig_ref] illustrates the life cycle of the CoVs. [bib_ref] Recent progress in studies of arterivirus-and coronavirus-host interactions, Zhong [/bib_ref] with an openaccess journal license. Upon entry to the host lungs, the CoV attaches to angiotensin-converting enzyme (ACE-2) receptors and releases its nuclear content into the cytoplasm. Translation of the RNA makes replicase complex, which results in the formation of transcripts of several proteins (named in pink box). Translation of these transcripts makes spike (S), envelope (E), membrane (M), nucleocapsid (N), and other accessory proteins. Assembly of these proteins produces a new virion that is matured and is released out of the cell to infect other cells.
## Evolution of covid-19
It has been suggested that most of the coronavirus species infecting humans are found in the bat reservoir [bib_ref] Bats Are Natural Reservoirs of SARS-Like Coronaviruses, Li [/bib_ref]. Unsurprisingly, several scientist groups have certified the genetic resemblance between SARS-CoV-2 and coronaviruses found in bats. A recent study showed that the gene sequence of novel coronavirus has 96.2% similarity to the coronaviruses found in bat (RaTG13), [bib_ref] A review of coronavirus disease-2019 (COVID-19), Singhal [/bib_ref].1% similarity to SARS-CoV, and about 50% similarity to MERS-CoV [bib_ref] Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins..., Lu [/bib_ref]. It has been documented [bib_ref] Recent progress in studies of arterivirus-and coronavirus-host interactions, Zhong [/bib_ref] with an open-access journal license. Upon entry to the host lungs, the CoV attaches to angiotensin-converting enzyme (ACE-2) receptors and releases its nuclear content into the cytoplasm. Translation of the RNA makes replicase complex, which results in the formation of transcripts of several proteins (named in pink box). Translation of these transcripts makes spike (S), envelope (E), membrane (M), nucleocapsid (N), and other accessory proteins. Assembly of these proteins produces a new virion that is matured and is released out of the cell to infect other cells.
## Evolution of covid-19
It has been suggested that most of the coronavirus species infecting humans are found in the bat reservoir [bib_ref] Bats Are Natural Reservoirs of SARS-Like Coronaviruses, Li [/bib_ref]. Unsurprisingly, several scientist groups have certified the genetic resemblance between SARS-CoV-2 and coronaviruses found in bats. A recent study showed that the gene sequence of novel coronavirus has 96.2% similarity to the coronaviruses found in bat (RaTG13), [bib_ref] A review of coronavirus disease-2019 (COVID-19), Singhal [/bib_ref].1% similarity to SARS-CoV, and about 50% similarity to MERS-CoV [bib_ref] Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins..., Lu [/bib_ref]. It has been documented that both SARS-CoV-2 and SARS-CoV attach to the same receptor at the host named angiotensin-converting enzyme (ACE2). After analyzing and evaluating 103 genomes of SARS-CoV-2 by single nucleotide polymorphisms (SNPs), a recent study showed that the virus evolved into two L and S types. SNPs showed absolute linkage across the strain of SARS-CoV-2. Examination of these types revealed that the L-type (70%) is predominant as compared with the S-type (30%). The evolutionary inspection suggested that the S-type is the most ancient form of SARS-CoV-2. Several tests and analyses also showed that the L-type is more vigorous and cruel as compared with the S-type [bib_ref] On the origin and continuing evolution of SARS-CoV-2, Tang [/bib_ref].
An unimaginably high degree of transmission raised a question of whether evolution was stimulated by mutations. Genetic mutation analysis is essential for understanding evolution [bib_ref] Mutated COVID-19, May Foretells Mankind in a Great Risk in the Future, Dawood [/bib_ref]. A study was conducted to understand and evaluate the genetic mutations pattern, in which eighty-six genomes of SARS-CoV-2 were selected. All strains were identified in patients from Australia, China, France, England, Germany, Taiwan, Japan, Belgium, USA, and Vietnam. China was used as a reference to evaluate the sequence of the genome. SARS-CoV-2 presents a specific type of protein, a long ORF1ab polyprotein at the 5 end with additional structural proteins, including envelope protein, spike glycoprotein, matrix, and nucleocapsid protein. The genetic examination identified three deletions in the genomes collected from Australia, the USA, and Japan. Out of three deletions, two deletions were noted in the ORF1ab polyprotein, and one deletion was noted in the 3 end of the genome. Furthermore, changing antigenicity may be driven by the mutations that occurred in the spike glycoproteins, which may be the reason for the vigorous pathogenicity of SARS-CoV-2 [bib_ref] Genetic diversity and evolution of SARS-CoV-2, Phan [/bib_ref].
Two precarious mutations were observed in the bat coronaviruses considered highly transmissible so far and are attributed to the current COVID-19 pandemic. Out of the two, the first improved the structure of spike proteins that appeared as projections on the outer surface of the virus. These modified spike proteins allow the virus to attach to the ACE2 receptor, which spans the respiratory tract lining. The second critical mutation permitted the virus to develop a protein dagger named furin, which can slice other proteins to cause the virus to bind firmly to the lung and throat cells. It is presumed that furin protein is the reason that COVID-19 is so deadly and contagious to humans [bib_ref] A unique protease cleavage site predicted in the spike protein of the..., Wang [/bib_ref]. Those mutations could have appeared while the virus was moving in bats. Another possibility of mutation is that it could have occurred in a person who was infected by the former type of virus but showed no sign and symptoms [bib_ref] Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly..., Matyášek [/bib_ref].
## Epidemiology
As described earlier, SARS-CoV-2 originated from Wuhan, China, and has affected more than 20 million people across 212 countries and territories. The transmission rate in some countries is exceptionally high compared with others, and the number of infected patients is increasing gradually [bib_ref] Defining the epidemiology of Covid-19-Studies needed, Lipsitch [/bib_ref]. Chronologically, the first virus was obtained from a patient on 7 January 2020. In response, the sequence of the viral genome was published by Chinese scientists on 10 January 2020, on Global Initiative on Sharing All Influenza Data (GISAID). In China, five patients were admitted to the hospital between 18 and 29 December 2019, out of which one patient expired [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref].
Additionally, 41 more patients were admitted to the hospitals by 2 January 2020. The diagnosis of these patients confirmed COVID-19. Thailand was the first country that was infected after China, and the first case was reported in Thailand on 13 January 2020, in a patient who traveled to Thailand. Five hundred and seventy-one new cases of COVID-19 were reported by 22 January 2020, in 25 provinces of China [bib_ref] Drug treatment options for the 2019-new coronavirus (2019-nCoV), Lu [/bib_ref]. National Health Commission of China (NHC) confirmed the first 17 deaths due to COVID-19 by 22 January 2020. On 25 January 2020, there were a total of 1975 infected patients in China, out of which 56 expired [bib_ref] The extent of transmission of novel coronavirus in Wuhan, China, 2020, Nishiura [/bib_ref]. According to another report from 30 January 2020, a total of 7734 cases were reported in China, and 90 cases were confirmed from other countries including Vietnam, Nepal, Sri Lanka, Japan, Thailand, Republic of Korea, United States, United Arab Emirates, Malaysia, The Philippines, India, Australia, Canada, Cambodia, Finland, Singapore, France, Germany, and Taiwan [bib_ref] The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak, Rothan [/bib_ref].
The Chinese Center for Disease Control and Prevention reported 44,672 confirmed cases by 11 February 2020, in which 81% of deaths were seen in patients age over 60 years. Mortality rates in patients aged 70-79 and over 80 years were 8.0% and 14.8%, respectively [bib_ref] Open access epidemiological data from the COVID-19 outbreak, Xu [/bib_ref]. On 28 February 2020, WHO reported 82,000 confirmed cases worldwide, and the outbreak reached 45 countries other than China. Following the rapid transmission course, 90,000 infected cases and 60 countries were dealing with the COVID-19 outbreak by 2 March 2020. Previously, it was thought that children were less susceptible to infection owing to a lack of evidence, but on 5 March 2020, Chinese studies concluded that children are as susceptible as adults. By 13 March 2020, Europe was declared as the epicenter of the outbreak by WHO. There were a greater number of reported cases in Europe as compared with the rest of the world. In Europe, Italy was the largest outbreak territory. The United States President declared the national emergency in the United States on 13 March 2020. By 27 March 2020, the total number of infected patients reached half a million, and pandemic spanned about 175 countries. The one million mark globally was reached on 2 April 2020. The next million cases were reported in less than two weeks, and there were 2 million cases worldwide on 15 April 2020, with the United States being on the top of the list, followed by Italy and Spain. Currently, there are almost 21 million cases with 752,225 deaths around the globe, accessed on August 13, 2020 (https://coronavirus.jhu.edu/map.html).
Rapid transmission of COVID-19 reflects the high transmissibility and reproductive number (R o ). Chinese reports show a R o value of 2.2-2.7, meaning the infected cases double every 6-7 days. The rate of transmission was inconceivably high, so several medical facilities were established, and guidelines were reported by the CDC and the WHO to manage the escalating disease [bib_ref] The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus..., Yang [/bib_ref].
Another parameter for understanding the severity and prognosis of infection is the case fatality ratio (CFR), which is calculated by dividing the number of expired patients by the total number of diagnosed patients, multiplied by 100. According to the WHO situation report-185 of COVID-19, the overall CFR was 5.12 as of 23 July 2020. A remarkable difference in CFR was noted in some countries presented in [fig_ref] Table 1: Case fatality ratio of Coronavirus Disease 2019 [/fig_ref]. The exact cause for this variability is still a topic of investigation in the scientific community. CFR is useful in estimating the risk of death within a population owing to a specific disease. Data suggest increased mortality with advanced age has a well-known impact on the prognosis of the disease. The median age of any population reflects the variation in fatality rates [bib_ref] Demographic science aids in understanding the spread and fatality rates of COVID-19, Dowd [/bib_ref]. One of the Chinese reports revealed that the mortality rate could be 3% high in geriatrics, especially over 80 [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref].
## Pathogenesis
SARS-CoV-2 targets the lower respiratory tract and causes mild to severe symptoms. COVID-19 patients show significant clinical symptoms, including non-productive cough, myalgia, reduced leukocyte, dyspnea, fever, fatigue, and pneumonia. These symptoms are also shown by the patients infected by SARS-CoV and MERS-CoV [bib_ref] Severe acute respiratory syndrome, Peiris [/bib_ref]. Although the pathogenesis of novel coronavirus disease is not fully understood, the similarity of novel coronavirus with SARS-CoV can provide significant information about the pathogenesis of COVID-19 infection. The body shows aggressive inflammatory responses after viral entry, and it may cause severe damage to airways. The pathogenicity is increased by a large number of viral copies replicated in the cell. The life cycle of SARS-CoV-2 is divided into four stages: (i) entry of the virus, (ii) protein expression, (iii) transcription, and (iv) release of the virus from the cell [bib_ref] COVID-19): History, Current Knowledge and Pipeline Medications, Jahangir [/bib_ref].
Coronavirus enters the cell by its sole determinant viral S-protein and undergoes several steps to accomplish the replication. While S-protein is divided into two domains, S1 and S2 domain, the S1 domain is responsible for binding with the receptor, and the S2 domain accounts for the fusion of the viral membrane with the cell membrane [bib_ref] Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: Implication..., He [/bib_ref]. The viral spike glycoproteins attach themselves to the ACE2 receptor of the cell, for both viruses SARS-CoV [bib_ref] Angiotensin-converting enzyme 2: A functional receptor for SARS coronavirus, Kuhn [/bib_ref] and SARS-CoV-2 [bib_ref] A new coronavirus associated with human respiratory disease in China, Wu [/bib_ref]. Initially, the virus binds to the cell receptor and enters the cell through the process of membrane fusion of the virus and plasma membrane [bib_ref] Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry, Simmons [/bib_ref]. Belouzard et al. [bib_ref] Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at..., Belouzard [/bib_ref] showed that the proteolytic processes occur in the S2 domain, which accounts for fusion and infection. After the viral entry, the RNA genome of the virus is released in the cytoplasm and then undergoes translation, followed by transcription, through which the virus continues to replicate [bib_ref] Coronaviruses post-SARS: Update on replication and pathogenesis, Perlman [/bib_ref]. After the formation of viral proteins by translation, these new proteins are inserted into the endoplasmic reticulum or Golgi apparatus. When viral RNA is combined with the proteins, the nucleocapsid is formed. Finally, the newly formed viruses enclosed in the vesicles are released by exocytosis [bib_ref] Coronaviruses post-SARS: Update on replication and pathogenesis, Perlman [/bib_ref]. The viral release marks the time when infected patients start displaying significant symptoms and laboratory values. Higher leukocyte count, respiratory problems, and exaggerated pro-inflammatory cytokines are exhibited by COVID-19 patients. In a case report, a patient presented with fever for the past two days displayed abnormal breathing sounds and a temperature of 39.0 - C. The real time-polymerase chain reaction (RT-PCR) conducted on the sputum culture showed a positive result for COVID-19 and confirmed the disease [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Guan [/bib_ref].
The laboratory analysis showed a leukocyte count of 2.91 × 10 9 cells/L, of which neutrophils were 70%. Further, the C-reactive protein was 16.16 mg/L (normal range = 0-10 mg/L). The erythrocyte sedimentation rate (ESR) was also high with D-dimer. The primary pathogenesis of the nCoV is associated with respiratory infections, pneumonia, ground-glass opacities (GGOs), acute cardiac injury, ARDS, and RNAaemia [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref]. Additionally, high blood values of chemokines and cytokines were also observed in patients, including IL1-β, IL7, IL8, IL9, IL10, IL1RA basic FGF2, GMCSF, IFNγ, IP10, GCSF MCP1, MIP1α, MIP1β, PDGFB, and TNFα, as shown in [fig_ref] Figure 2: Int [/fig_ref]. Critical patients showed a high value of pro-inflammatory cytokines as a cytokine storm, including IL2, IL7, IL10, GCSF, IP10, MCP1, MIP1α, GCSF, and TNFα, which exaggerated the severity of the infection [bib_ref] COVID-19: Consider cytokine storm syndromes and immunosuppression, Mehta [/bib_ref]. This exaggerated immune response destroys the lung's infrastructure [bib_ref] COVID-19 illness in native and immunosuppressed states: A clinical-therapeutic staging proposal, Siddiqi [/bib_ref].
Current models give information about the three stages of the immune response. The first stage involves the early activation and persuasive interferon response for clearing the virus. The second stage exhibits the delayed response of interferon that may lead to tissue damage. The third stage leads to hyper-inflammation, followed by exaggerated macrophage activation, resulting in fibrosis dysregulation of tissue repair processes [bib_ref] COVID-19 illness in native and immunosuppressed states: A clinical-therapeutic staging proposal, Siddiqi [/bib_ref]. For each step, potential therapeutic options can be developed. Some are under clinical trials to evaluate their efficacy along with safety. Initially, the virus attaches to the ACE2 receptor. TMPRSS2 is responsible for the viral protein cleavage. Protease inhibitors can be designed and developed to prevent spike protein cleavage. Blocking the viral fusion through either TMPRSS2 or receptor ACE2 can prevent the infection. For removing pro-inflammatory cytokines, several novel models have been proposed in which the blood of a COVID-19 patient is passed through customized columns that recognize and trap the pro-inflammatory cytokines, and then the purified blood is administered back to the patient [bib_ref] The trinity of COVID-19: Immunity, inflammation and intervention, Tay [/bib_ref]. ARDS seen in several COVID-19 patients may lead to secondary infections, and respiratory failure leads to death in 79% of critical cases. Furthermore, the cytokine storm may lead to sepsis and cause death in 28% of critical cases. Scientists are trying to design therapeutics to prevent this exaggerated immune response responsible for the enhanced severity of the infection [bib_ref] The trinity of COVID-19: Immunity, inflammation and intervention, Tay [/bib_ref] [bib_ref] Race to find COVID-19 treatments accelerates, Kupferschmidt [/bib_ref]. Current models give information about the three stages of the immune response. The first stage involves the early activation and persuasive interferon response for clearing the virus. The second stage exhibits the delayed response of interferon that may lead to tissue damage. The third stage leads to hyper-inflammation, followed by exaggerated macrophage activation, resulting in fibrosis dysregulation of tissue repair processes [bib_ref] COVID-19 illness in native and immunosuppressed states: A clinical-therapeutic staging proposal, Siddiqi [/bib_ref]. For each step, potential therapeutic options can be developed. Some are under clinical trials to evaluate their efficacy along with safety. Initially, the virus attaches to the ACE2 receptor. TMPRSS2 is responsible for the viral protein cleavage. Protease inhibitors can be designed and developed to prevent spike protein cleavage. Blocking the viral fusion infection. SARS-CoV-2 attaches to the ACE2 receptor, and the spike protein is cleaved by the type II transmembrane serine protease (TMPRSS2), resulting in viral replication. Mature viruses are released from the cell by exocytosis. In the dysfunctional immune response, cells may undergo pyroptosis and release adenosine triphosphate (ATP), nucleic acids, and ASC oligomer. These impaired-associated molecular forms can be detected by the neighboring epithelial cells and other alveolar macrophages. In response, the pro-inflammatory cytokines are released along with chemokines, including IL-6, IP-10, macrophage inflammatory protein 1α (MIP1β), MCP1, and MIP1α. These released proteins draw the monocytes, T cells, and lymphocytes to the inflammatory site, causing exaggerated inflammation at the site of action. This exaggerated response damages the lung's infrastructure. This cytokine storm can move to other organs and may damages the other organs as well.
As discussed earlier, ACE2 is the receptor for SARS-CoV-2. It has been reported that the involvement of ACE2 receptors is also related to the renin-angiotensin system (RAS) in 2019-nCoV pathogenesis. Very recently, a meta-analysis was conducted to get insight into the relationship between the prevalence of COVID-19 disease and the genetic differences in the genes involved in the RAS system. The results of the study suggest that the increase of the I/D allele frequency ratio significantly increases the recovery rate (point estimate: 0.48, confidence interval (CI) 95%: 0.05-0.91, p = 0.027), but the I/D allele frequency ratio has no significant difference in the case of death rate (point estimate: 1.74, CI 95%: 4.5-1.04, p = 0.22). The I/D allele ratio of ACE receptors varies significantly across world regions, accounting for the different recovery rates across regions, but it also raises concerns that ethnic and genetic differences can impact the effectiveness of currently investigated RAS-associated medications in different regions.
## Clinical features
SARS-CoV-2 represents an incubation period of 6.4 days, fluctuating between 2.1 and 11.1 days with possible asymptomatic transmission. Infection is manifested after the incubation period. The period from the commencement of disease leading to death fluctuates between 6 and 41 days with an average of 14 days. The age of patient and condition of the immune system are the sole determinants of this period. Persons with a weaker immune system are more susceptible to infection. This period is shorter in geriatrics >60 years, as older patients have a weaker immune system [bib_ref] Imaging profile of the COVID-19 infection: Radiologic findings and literature review, Ng [/bib_ref]. The most common symptoms of COVID-19 are dry cough, myalgia, diarrhea, headache, and high-grade fever [bib_ref] Unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia..., Wang [/bib_ref] ; fever is reported in more than 90% of cases, cough in 75%, and dyspnea in 50% of the patients [bib_ref] Identification of a novel coronavirus causing severe pneumonia in human: A descriptive..., Ren [/bib_ref]. Fewer patients may also develop kidney damage, ARDS, septic shock, and acute cardiac injury requiring hospitalization [bib_ref] Identification of a potential mechanism of acute kidney injury during the COVID-19..., Pan [/bib_ref]. The clinical symptoms are presented in . . Clinical symptoms integrated with the severity of COVID-19 [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref] [bib_ref] Unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia..., Wang [/bib_ref].
## Clinical type symptoms
Mild disease No signs of pneumonia; it happened in 81% of patients.
## Severe disease
Oxygen saturation of blood ≤93%, infiltration of lungs ≥50% within one or two days. The ratio of PaO2/FiO2 (oxygen partial pressure/fraction of inspired oxygen) <300. GGO (ground glass opacities): 14% of cases acquired severe symptoms.
Critical disease Failure of the respiratory system, septic shock, concerted organ dysfunction, or multiple organ failure (MOF); it happened in 5% of patients.
Mutual laboratory findings include lymphopenia and leucopenia. These findings were common and observed in a large number of cases [bib_ref] The use of anti-inflammatory drugs in the treatment of people with severe..., Zhang [/bib_ref]. Chest computed tomography (CT) scan showed clinical features including pneumonia and other abnormal features, for instance, RNAaemia, acute cardiac injury, and occurrence of GGO, that set the path towards death. In several cases, multiple marginal GGOs were observed in both lungs, which expectedly triggers the systemic and localized immune reaction, causing severe inflammation [bib_ref] Imaging profile of the COVID-19 infection: Radiologic findings and literature review, Ng [/bib_ref]. Atypical CT scan showed single, multiple solid, and consolidated nodules in the middle of the lobe of right or left or both lungs, fenced by GGOs in the number of COVID-19 patients [fig_ref] Figure 3: Atypical CT imaging of a 56-year-old female presented with a history of... [/fig_ref] [bib_ref] Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province, Kui [/bib_ref]. Although there are some similarities, the symptoms manifested by earlier coronaviruses are not precisely similar to those of COVID-19. For instance, the distinctive features of COVID-19 are fever, fatigue, and dry cough. In contrast, upper respiratory tract symptoms like rhinorrhea, sore throat, and sneezing are uncommon, which were commonly observed in previous coronavirus infections. Moreover, COVID-19 patients present with intestinal symptoms like diarrhea, which were less Although there are some similarities, the symptoms manifested by earlier coronaviruses are not precisely similar to those of COVID-19. For instance, the distinctive features of COVID-19 are fever, fatigue, and dry cough. In contrast, upper respiratory tract symptoms like rhinorrhea, sore throat, and sneezing are uncommon, which were commonly observed in previous coronavirus infections. Moreover, COVID-19 patients present with intestinal symptoms like diarrhea, which were less prevalent in MERS-CoV and SARS-CoV infections [bib_ref] Systematic comparison of two animal-to-human transmitted human coronaviruses: SARS-CoV-2 and SARS-CoV, Xu [/bib_ref].
In addition, patients may also report anosmia and dysgeusia as early symptoms of COVID-19 [bib_ref] Anosmia and ageusia: Common findings in COVID-19 patients, Vaira [/bib_ref]. Anosmia (loss of sense of smell) and dysgeusia (alteration of the sense of taste) are also associated with COVID-19 patients. The American Academy of Otolaryngology-Head and Neck Surgery developed the COVID-19 Anosmia Reporting Tool for clinicians to conduct a pilot study. This tool allows clinicians to submit the anosmia and dysgeusia associated COVID-19 patients. They analyzed the first 237 entries and revealed that anosmia was observed in 73% of patients prior to the diagnosis of COVID-19 and was the early symptom in 26.6% of COVID-19 patients. Conclusively, this study suggested that anosmia can be the representative symptom of COVID-19 [bib_ref] Features of anosmia in COVID-19, Klopfenstein [/bib_ref].
## Diagnosis
The diagnosis and treatment program for COVID-19 was formulated by NHC of China [bib_ref] Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province, Kui [/bib_ref] [bib_ref] Review of Potential High-Leverage and Inexpensive Mitigations for Reducing Risk in Epidemics..., Manheim [/bib_ref] and was previously suggested by WHO for SARS and MERS [bib_ref] Coronavirus disease 2019 (COVID-19): A perspective from China, Zu [/bib_ref] [bib_ref] Evidence for camel-to-human transmission of MERS coronavirus, Azhar [/bib_ref]. The specific diagnostic measures have been implemented for suspected as well as confirmed cases: a patient with a minimum of two clinical symptoms and at least one exposure history is accounted as a suspected case. The suspected case should show at least three clinical symptoms if there is not any clear exposure history.
The serology testing for COVID-19 is helpful and provides the basis for diagnosis. In the case of the unavailability of molecular testing, the serological test provides a means to triage the suspected cases of COVID-19. A fast-serological test with specific performance features is highly important to avoid missing factual cases of COVID-19. A positive test for IgM or IgG antibody strongly indicates SARS-CoV-2 infection. This approach is extremely effective in individuals 5-10 days after the onset of symptoms. IgG or IgM positive patients are quarantined, and those who need critical care are referred to the hospital. Those with a negative serological antibody test have a swab collected for molecular testing. The experience in China has shown that the use of a serological antibody test can improve the sensitivity of COVID-19 case detection [bib_ref] Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease, Zhao [/bib_ref]. This approach allowed to test a huge number of symptomatic individuals rapidly in the community, minimizing the waiting time for molecular testing and preventing the health-management system from being overwhelmed.
Subsequently, when nucleic acids of SARS-CoV-2 by RT-PCR results come back positive, it is accounted for as a confirmed case [bib_ref] Coronavirus disease 2019 (COVID-19): A perspective from China, Zu [/bib_ref]. Meanwhile, a CT scan of the chest provides confirmed diagnostic evidence of COVID-19 disease as it has greater sensitivity and high specificity. Thus, the chest CT has been recognized as a significant indicator for COVID-19 diagnosis in different epidemic areas [bib_ref] Chest CT findings in coronavirus disease-19 (COVID-19): Relationship to duration of infection, Bernheim [/bib_ref] [bib_ref] On the origin and continuing evolution of SARS-CoV-2, Tang [/bib_ref] [bib_ref] Characteristics of and public health responses to the coronavirus disease 2019 outbreak..., Deng [/bib_ref]. It can be said that the chest CT is an essential procedure for the timely detection and management of this infection. However, it should be noted that a patient with an RT-PCR may have a negative chest CT at the time of admission [bib_ref] Patients with RT-PCR confirmed COVID-19 and normal chest CT, Yang [/bib_ref]. In this context, a study compared three RT-PCR analyses, targeting the RNA-dependent RNA polymerase RdRp/Helicase, spike, and nucleocapsid genes of the virus with testified RdRp-P2 assay, which is extensively used in more than 30 European countries. According to this study, RdRp/Hel is more specific and gives accurate results for SARS-CoV-2. Moreover, the RdRp/helicase assay of COVID-19 does not cross-react with other types of coronaviruses and respiratory microorganisms in the cellular medium. In contrast, the RdRp-P2 assay, reported for previous coronaviruses, reacts with the pathogens of the respiratory tract in the cell culture. Thus, the COVID-19-RdRp/Hel can be considered sensitive and specific, and this information helps fortify the diagnosis of COVID-19 [bib_ref] Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific..., Chan [/bib_ref].
Laboratory findings can be helpful in predicting COVID-19 cases with positive RT-PCR. ALT, C-reactive protein, neutrophils, AST, and lymphocytes counts have very good accuracy in predicting cases with positive RT-PCR for COVID-19 patients [bib_ref] Laboratory parameters in detection of COVID-19 patients with positive RT-PCR; a diagnostic..., Mardani [/bib_ref]. A study suggested that the number and percentage of white blood cells (WBCs), lymphocytes, and neutrophils were significantly different between positive and negative RT-PCR cases for COVID-19 patients [bib_ref] Platelet-to-lymphocyte ratio is associated with prognosis in patients with coronavirus disease-19, Qu [/bib_ref]. Another cohort study suggested that lymphopenia (low levels of lymphocytes) occurred in 80% of COVID-19 patients [bib_ref] Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in..., Yang [/bib_ref]. An increased level of AST and ALT was also noted in severely affected patients [bib_ref] Clinical features and short-term outcomes of 221 patients with COVID-19 in Wuhan, Zhang [/bib_ref]. High levels of LDH, C-reactive protein, neutrophils, and erythrocyte sedimentation rate (ESR) were also notable in COVID-19 patients [bib_ref] Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis, Rodriguez-Morales [/bib_ref]. Thus, these laboratory findings have great importance in terms of predicting COVID-19 cases with positive RT-PCR [bib_ref] Laboratory parameters in detection of COVID-19 patients with positive RT-PCR; a diagnostic..., Mardani [/bib_ref].
## Treatment
Currently, no specific and sensitive therapeutic regimen is available for the treatment of COVID-19. However, the primary step is isolation to prevent any type of contact that later may cause transmission of the disease. If the patient experiences any mild symptoms, it should be managed at their residence with proper counseling to patients about the critical signs and symptoms. The primary management protocols include the management of cough and fever [bib_ref] A review of coronavirus disease-2019 (COVID-19), Singhal [/bib_ref]. First-line therapy to manage the fever is acetaminophen, while expectorants such as guaifenesin are used for non-productive cough [bib_ref] Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in..., Wang [/bib_ref]. Patients with SARS, hypoxemia, respiratory distress or shock should be treated by the administration of instant oxygen therapy. The frequency of oxygen therapy should be 5 L/min to obtain SpO 2 of ≥92-95% in pregnant women, and ≥90% in adults and children [bib_ref] COVID-19: Lessons from SARS and MERS, Park [/bib_ref]. Antiviral therapy is also being tried to manage the infection [bib_ref] The First 75 Days of Novel Coronavirus (SARS-CoV-2) Outbreak: Recent Advances, Prevention,..., Yan [/bib_ref].
Critical patients show an unhealthy immune response that eventually causes hyper-inflammation. This exaggerated immune response should be controlled and checked by different approaches and procedures. As discussed earlier, inflammatory cytokines are released at the site of infection and cause infiltration of lungs. Clinical trials are underway to identify the benefits of blocking cytokine storm targeting IL-1β and IL-6, as shown in [fig_ref] Table 3: Potential therapeutic targets associated with macrophage activation under clinical trials[93][94][95] [/fig_ref]. Interfering cytokine storm signaling can prevent hyper-inflammation, thereby reducing infection. Current evidence shows that the overproduction of macrophages linked to monocytes accumulates in tissue that can cause lung infiltration. For instance, it is found that CCR2 activation contributes to the accumulation of monocytes in the tissue, causing severe inflammation. So monocytes' accumulation can be reduced by blocking the CCR2, resulting in minimizing hyper-inflammation [bib_ref] Monocyte emigration from bone marrow during bacterial infection requires signals mediated by..., Serbina [/bib_ref]. Different clinical trials are underway to reduce the hyper-inflammation by JAK inhibitors [bib_ref] COVID-19: Combining antiviral and anti-inflammatory treatments, Stebbing [/bib_ref]. Other relevant clinical trials are ongoing to evaluate the blocking of myeloid-derived cytokines, for example, TNF [bib_ref] Trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed, Feldmann [/bib_ref] and GM-CSF. By targeting this cytokine signaling, the hyper-inflammation can be reduced [bib_ref] TH17 responses in cytokine storm of COVID-19: An emerging target of JAK2..., Wu [/bib_ref].
Remdesivir is a broad-spectrum antiviral prodrug, which is a nucleotide analog and is metabolized in the cell to adenosine triphosphate analog, which inhibits the viral RNA polymerases. Remdesivir is the drug of choice against several viral families, including Ebola, SARS-CoV, and MERS-CoV. Remdesivir is being used for prophylactic and therapeutic purposes and has shown remarkable efficacy in the non-clinical model of coronaviruses [bib_ref] Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of..., De Wit [/bib_ref] [bib_ref] Comparative Antiviral Activity of Remdesivir and Anti-HIV Nucleoside Analogs against Human Coronavirus..., Parang [/bib_ref]. It has also shown promising activity against SARS-CoV-2 [bib_ref] Coronavirus infections-More than just the common cold, Paules [/bib_ref] [bib_ref] Influenza A reinfection in sequential human challenge: Implications for protective immunity and..., Memoli [/bib_ref]. A large clinical trial of remdesivir is currently underway for patients with mild to moderate symptoms. The critically ill patients treated with amicable use of remdesivir showed better efficacy, and the clinical condition of the patients was improved [bib_ref] Compassionate Use of Remdesivir for Patients with Severe Covid-19, Grein [/bib_ref]. On 1 May 2020, the Food and Drug Administration (FDA) of the USA authorized the emergency use of remdesivir for the treatment of COVID-19. The recent version of treatment guidelines by NHC recommends the use of lopinavir/ritonavir and IFN-α. The first version of treatment guidelines was issued on 15 January 2020, later revised five times, and the 6th edition is the most recent one that was issued on 18 February 2020. Lopinavir/ritonavir, ribavirin, and IFN-α were added in the 5th edition of treatment guidelines for combating COVID-19. Later, arbidol and chloroquine phosphate have also been added in the 6th edition, as these drugs show better clinical efficacy. Potential drugs included in the 6th edition of treatment guidelines by NHC of China, as shown in [fig_ref] Table 4: Antivirals included in the treatment guidelines issued by NHC of China for... [/fig_ref] , presented promising therapeutic effects [bib_ref] Discovering drugs to treat coronavirus disease 2019 (COVID-19), Dong [/bib_ref]. Although the recent study was an open-label, individually randomized, controlled trial representing patients admitted in hospitals, treated with lopinavir/ritonavir (400 mg/100 mg), no clinical improvement was observed by this treatment beyond standard care [bib_ref] A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19, Cao [/bib_ref]. On the basis of the guidelines, ritonavir is used as a booster to enhance the efficacy of lopinavir. These medications are prescribed to be taken for a short period of time (<2 weeks), suggesting their acute response. Hydroxychloroquine, lopinavir/ritonavir, chloroquine, and darunavir/cobicistat are being used for patients with severe conditions. Interferon-beta B1 is extensively used for critical patients [bib_ref] Therapeutic Management of COVID-19 Patients: A systematic review, Tobaiqy [/bib_ref]. Another drug, oseltamivir, is being used in China by the medical personnel for suspected patients, but there is a lack of evidence on the specific activity of oseltamivir against COVID-19. A 54-year-old female patient from Wuhan city presented with fever for the past two days. Two tests were conducted, the first test was not positive, but the second test confirmed the disease. Meanwhile, this woman was treated with oseltamivir for three days, and her chest CT indicated reminiscent improvement in the density of GGO [bib_ref] Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in..., Wang [/bib_ref].
Patients with vigorous immune reactions can be treated with glucocorticoids such as methylprednisolone, which is used at 1-2 mg/kg/day dose in children and 25-150 mg/day in adults. It is believed to reduce the prognosis of the disease in patients with impaired oxygen index, that is, less than 300 mm Hg, limited to 5 days [bib_ref] Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province, Kui [/bib_ref]. For patients with premature disease stage, without hypoxia, use of corticosteroids should be avoided. However, in critical situations where mechanical ventilation is mandatory, anti-inflammatory therapy is possibly useful and can be employed [bib_ref] COVID-19 illness in native and immunosuppressed states: A clinical-therapeutic staging proposal, Siddiqi [/bib_ref]. A glucocorticoids-based drug dexamethasone may be employed against COVID-19. Dexamethasone is a synthetic corticosteroid approved by the FDA in 1958 as a broad-spectrum immunosuppressor, which is about 30 times as active as cortisone and possesses a longer duration of action (2-3 days). Dexamethasone may not only limit the production and damaging effect of the cytokines, but may also inhibit the protective function of T cells and reduce the ability of B cells to synthesize antibodies [bib_ref] Dexamethasone for COVID-19? Not so fast, Theoharides [/bib_ref]. Dexamethasone may be a useful agent for short-term therapy in severe intubated COVID-19 patients [bib_ref] Dexamethasone in the management of covid-19, Johnson [/bib_ref]. A recent controlled open-label, randomized trial of dexamethasone in hospitalized patients, concluded that the use of dexamethasone with the aid of mechanical ventilation or oxygen alone resulted in lower mortality at the dose of 6 mg once daily for 10 days. However, dexamethasone was not effective in those patients who were not receiving respiratory support [bib_ref] Dexamethasone in Hospitalized Patients with Covid-19-Preliminary Report, Recovery Collaborative Group [/bib_ref]. Dexamethasone is on an essential medicines list recommended by the World Health Organization and is readily accessible worldwide at a low cost. Guidelines issued by the U.K.'s chief medical officers and by the National Institutes of Health in the United States have already been updated to recommend the use of glucocorticoids such as dexamethasone in hospitalized patients with COVID-19.
As discussed earlier, a variety of drugs are recommended and tested in non-clinical models; among them, penciclovir, ribavirin, and favipiravir require high doses to decrease the infection and are supposed to be less effective [bib_ref] Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in..., Wang [/bib_ref]. Chloroquine and its derivative hydroxychloroquine are considered to be effective against COVID-19 and are currently used in Chinese patients to reduce viral infection. Potential drug therapy for COVID-19 is listed in [fig_ref] Table 4: Antivirals included in the treatment guidelines issued by NHC of China for... [/fig_ref] to gain a better understanding of treatment [bib_ref] Hydroxychloroquine and azithromycin as a treatment of COVID-19: Results of an open-label..., Gautret [/bib_ref]. An open-label, non-randomized clinical trial showed that hydroxychloroquine (600 mg/day) treatment is magnificently related to a decreased rate of viral replication in patients, and its effectiveness is reinforced by azithromycin (500 mg/day) [bib_ref] Hydroxychloroquine and azithromycin as a treatment of COVID-19: Results of an open-label..., Gautret [/bib_ref]. Hydroxychloroquine showed superior sensitivity over chloroquine against COVID-19 [bib_ref] In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine..., Yao [/bib_ref]. Contrary to the previous results, at the time of publication, an open-label, randomized controlled clinical trial showed that the use of hydroxychloroquine alone or with azithromycin did not improve the clinical status of hospitalized patients with mild to moderate COVID-19 as compared with standard care [bib_ref] Hydroxychloroquine with or without Azithromycin in Mild-to-Moderate Covid-19, Cavalcanti [/bib_ref].
According to many reports, the COVID-19 patients are at high risk for venous thromboembolism, a term that associates deep vein thrombosis (DVT) [bib_ref] Incidence of thrombotic complications in critically ill ICU patients with COVID-19, Klok [/bib_ref]. In this context, patients are treated prophylactically by giving low molecular weight heparin (LMWH), when multiple risk factors exist. Hence, hospitalized COVID-19 patients are generally treated with higher doses of LMWH than recommended for thromboprophylaxis [bib_ref] Pulmonary embolism or pulmonary thrombosis in COVID-19? Is the recommendation to use..., Cattaneo [/bib_ref]. A recent document by the Italian Drug Agency (AIFA) suggested the use of 80 to 100 mg enoxaparin daily, instead of the usual 40 mg, while in some hospitals, even higher, up to full anticoagulant doses of LMWH or unfractionated heparin are used [bib_ref] ISTH interim guidance on recognition and management of coagulopathy in COVID-19: A..., Barrett [/bib_ref].
SARS-CoV-2 targets the ACE2 receptor via spike-like protein, so it is an efficient target for the drugs, including chloroquine, promazine, and emodine, to prevent the viral attachment with the surface of the host cell [bib_ref] Therapeutic opportunities to manage COVID-19/SARS-CoV-2 infection: Present and future, Shetty [/bib_ref]. S protein priming is the next precarious phase of viral entry into the host cell, where it is required to be cleaved by intracellular proteases including TMPRSS2, cathepsins, and furin for the fusion of viral membrane with the cell membrane of the host. S-protein priming by TMPRSS2 is the most crucial phase for the prognosis of infection into the host, so the viral infection can be controlled with the use of protease inhibitors, including camostat and nafamostat, which target TMPRSS2, and thereby inhibit the viral fusion [bib_ref] Nafamostat mesylate blocks activation of SARS-CoV-2: New treatment option for COVID-19, Hoffmann [/bib_ref]. The immune system represents the defensive mechanism in attacking the virus; major immune cells include T-lymphocytes and natural killer cells [bib_ref] Therapeutic opportunities to manage COVID-19/SARS-CoV-2 infection: Present and future, Shetty [/bib_ref]. Apart from antiviral therapy, the use of antibiotics can be used for the associated co-infections, community-acquired, or hospital-acquired pneumonia, and it depends on the clinical results of the patients. These broad-spectrum antibiotics, including azithromycin and fluoroquinolones, can be given as empirical therapy to cover all attainable pathogens, continuing the therapy until all pathogenic bacteria are cleared from the body. The combination therapy of azithromycin with hydroxychloroquine showed significant activity against COVID-19 infection [bib_ref] No evidence of rapid antiviral clearance or clinical benefit with the combination..., Molina [/bib_ref]. [fig_ref] Table 5: Potential therapeutics against SARS-CoV-2 [/fig_ref] delineates the potential therapeutics against COVID-19. Some researchers recommend the use of vitamin C supplements that may help to treat the clinical symptoms of the patient by potentially activating the immune system. These supplements help to reduce the severity of pneumonia; however, this requires more clinical evaluation [bib_ref] Clinical considerations for patients with diabetes in times of COVID-19 epidemic, Gupta [/bib_ref]. Supplements of vitamin D 3 can also be useful for combating the infection. While vitamin D can activate the innate immune system, its decreased value indicates enhanced autoimmunity and susceptibility towards infection. Grant et al. indicated the use of vitamin D in decreasing the probability of respiratory infections caused by COVID-19. As vitamin D induces the antimicrobial peptides such as defensins and cathelicidins that eventually interfere with the viral growth and pro-inflammatory cytokines, more evaluation is needed by randomized controlled trials on a large number of individuals to reach to a conclusion [bib_ref] Evidence that vitamin D supplementation could reduce risk of influenza and COVID-19..., Grant [/bib_ref].
Presently, no vaccine exists against COVID-19; however, several clinical trials are underway, as discussed in [fig_ref] Table 6: Development of vaccines for COVID-19 that are under clinical trials [/fig_ref]. Scientific research is ongoing by various medical institutes and companies to produce effective vaccines all over the world that can combat COVID-19.
A most recent study published in Lancet claims the production of a vaccine named PittCoVacc developed by the University of Pittsburgh School of Medicine. PittCoVacc uses S-protein, which provides protection by the induction of specific antibodies in the body. Researchers are conducting different analyses on the vaccine, and they are planning a series of tests in humans in the coming months [bib_ref] COVID-19 in Italy: An overview from the first case to date, Santacroce [/bib_ref]. Another vaccine named mRNA-1273 is being developed by the National Institute of Allergy and Infectious Disease researchers combined with biotechnology institution Moderna. In this case, mRNA is enclosed in lipid nanoparticles that encode the S-protein of the virus, and then it takes the antigen into the body. In response to antigen, specific antibodies are produced that provide protection against SARS-CoV-2. The clinical trial of the mRNA vaccine is underway, while scientists are working together to evaluate the pathogenesis and clinical features for combating the disease by approaching a series of therapeutic options and development of other vaccines [bib_ref] The COVID-19 vaccine development landscape, Le [/bib_ref].
University of Oxford, United Kingdom, conducted a randomized clinical trial of the COVID-19 vaccine named ChAdOx1 nCoV-19 vaccine. The estimated participants in this study were about 10,260, and this vaccine was administered intramuscularly. Fortunately, this vaccine appeared to be most effective in participants showing an effective response and has already entered into second and third clinical trials (clinicaltrials.gov).
Finally, there is an emerging concern that patients recovering from COVID-19 may be at risk of reinfection. To address this concern, Bao et al. investigated acquired immunity to SARS-CoV-2 in rhesus macaques. This study published recently suggests that immunity acquired following primary infection with SARS-CoV-2 may protect upon subsequent exposure to the virus [bib_ref] Will we see protection or reinfection in COVID-19?, Ota [/bib_ref]. However, more studies are needed to reach a definitive conclusion.
## Prevention and control
Several methods have been implemented based on actual infection control and segregation to control the transmission of COVID-19. WHO and CDC published guidelines for the prevention and control of the infection. For instance, individuals should wash their hands with alcohol-based sanitizers on a routine basis. Touching of nose, mouth, and eyes should be avoided as they are the critical part for holding the infection. Moreover, a distance of approximately of 1 m from those who are sneezing and coughing should be maintained. People should uphold respiratory hygiene, such as covering nose and mouth during coughing. Individuals should wear a mask while meeting with other persons or going outside. Individuals are supposed to sanitize their workplaces. Medical teams are supposed to cover their nose and mouth by licensed N95 masks. In some areas, the availability of the N95 mask is limited; in this case, the use of surgical masks while treating suspected and confirmed patients should be ensured. Proper use and disposal of the surgical mask is necessary to minimize the risk of transmission [bib_ref] Supporting the health care workforce during the COVID-19 global epidemic, Adams [/bib_ref] [bib_ref] Suggestions for safety and protection control in Department of Nuclear Medicine during..., Zhang [/bib_ref]. As there are clinical trials of vaccines underway, for now, the most effective prevention for the entire population is to minimize the exposure to the virus. Center for Disease Control, USA, WHO, and the Chinese CDC have provided proper guidelines to enhance awareness among the population about the prevention of COVID-19. The main emphasis of the guidelines includes awareness on how to wear a face mask, appropriate hand washing techniques, preventive measures at home and workplaces, and disinfection procedures and protocols. Furthermore, the guidelines are available on the treatment, prevention, and control of COVID-19. The guidelines also endorse ways of minimizing the fear of COVID-19 among the overall population [bib_ref] Noval coronavirus disease (COVID-19): A pandemic (Epidemiology, Pathogenesis and potential therapeutics), Hamid [/bib_ref]. Considering all the nonpharmaceutical measures assist the maintenance of the public outcomes of the pandemic to be in control, because of several financial reasons, the numbers are again elevating. There are multiple steps involved in preparation for personal protection equipment (PPE), including identifying PPE and to ensure a proper gown size, performing hand hygiene using hand sanitizer, wearing proper masks, put on face shield or goggles, and gloves in areas that could be necessary. The guidelines could be different based on the profession, location, and severity of the disease.
# Conclusions
This review provides insight into various aspects of COVID-19, including disease pathophysiology, its clinical representation, mechanism of drugs actions, and impacted population. However, the major concentration of this report was to emphasize potential therapeutics and provide all essential information related to it in one document. On the basis of the class of drugs, multiple classes of drugs have been shown to work primarily to stabilize the patient and, secondly, target the virus for treatment.
The first class of drugs is anti-inflammatory ones that deal with cytokines signaling through targeting various receptors such as IL-6. The major outcome of the treatment is to reduce the inflammation during the therapy. The second group of medications is classic orally administered drugs that have been used for antiviral or anti-parasite ones such as chloroquine phosphate and lopinavir/ritonavir. Among them, remdesivir has been approved by FDA for the emergency room treatment of COVID-19, suggesting its promising potency. In addition to all the mentioned medications, corticosteroids are used to assist with reducing the pro-inflammatory effect. However, owing to their unfavorable side effect profile, their use should be limited to a short period of time in critical patients.
It needs to be emphasized that, among all tested drugs, antiviral compounds such as remdesivir inhibit RNA transcription, leading to reduced viral load. This class of drugs is potentially the most promising tool with which to treat COVID-19 patients. Thus, although there is no solid evidence available to prove their long-term clinical outcomes as of this day, drugs targeting viral RNA are the most promising tools in this battle. Further investigations are required to test a library of synthesized antiviral agents to obtain the most potent candidate to be used in clinics. The ongoing trials of the vaccine will be a hope to provide a vaccine displaying efficacy without side effects to overcome the COVID-19 pandemic to humankind.
## Conflicts of interest:
The authors declare no conflict of interest.
## Abbreviations
[fig] Figure 1: The life cycle of a coronavirus, modified, and used from Zhang et al. [/fig]
[fig] Figure 2: Int. J. Environ. Res. Public Health 2020, 17, Inflammatory responses during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. SARS-CoV-2 attaches to the ACE2 receptor, and the spike protein is cleaved by the type II transmembrane serine protease (TMPRSS2), resulting in viral replication. Mature viruses are released from the cell by exocytosis. In the dysfunctional immune response, cells may undergo pyroptosis and release adenosine triphosphate (ATP), nucleic acids, and ASC oligomer. These impaired-associated molecular forms can be detected by the neighboring epithelial cells and other alveolar macrophages. In response, the pro-inflammatory cytokines are released along with chemokines, including IL-6, IP-10, macrophage inflammatory protein 1α (MIP1β), MCP1, and MIP1α. These released proteins draw the monocytes, T cells, and lymphocytes to the inflammatory site, causing exaggerated inflammation at the site of action. This exaggerated response damages the lung's infrastructure. This cytokine storm can move to other organs and may damages the other organs as well. [/fig]
[fig] Figure 3: Atypical CT imaging of a 56-year-old female presented with a history of 3 days of persistent fever. Laboratory findings: reduced total protein level (54.0 g/L), reduced albumin value (35.5 g/L), low value of globulin (18.5 g/L), white blood cells (WBCs) (4.87 × 10 9 /L), lymphocytes (0.49 × 10 9 / L), and decreased level of eosinophil (0 × 10 9 /L). Imaging inspection: (A) A lateral section of the center of right lung lobe covered GGO. (B) Patchy GGO in the upper part of the right lung with sporadic consolidated lesions. (C) GGO in both lungs with sporadic consolidation lesions. (D) Uneven GGO in the middle and dorsal section of the right lung. Adopted from Kui et al. through open-access license[69]. [/fig]
[fig] Author: Contributions: The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. M.M. and M.I.S. contributed equally. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research received no external funding and the APC was funded by College of Pharmacy, Marshall B. Ketchum University, Fullerton, CA, USA and Chapman University School of Pharmacy, Irvine, CA, USA. Acknowledgments: Authors acknowledge the support from Chapman University School of Pharmacy, Irvine, CA, USA, College of Pharmacy, Marshall B. Ketchum University, Fullerton, CA, USA and University of Central Punjab, Lahore, Pakistan. [/fig]
[table] Table 1: Case fatality ratio of Coronavirus Disease 2019 (COVID-19) in major countries of the world *. CFR, case fatality ratio. [/table]
[table] Table 3: Potential therapeutic targets associated with macrophage activation under clinical trials[93][94][95]. [/table]
[table] Table 4: Antivirals included in the treatment guidelines issued by NHC of China for COVID-19, version 6 [107,109]. NHC, National Health Commission; t.i.d, three times a day; b.i.d, two times a day. [/table]
[table] Table 5: Potential therapeutics against SARS-CoV-2 (COVID-19)[68,105,112,116,[125][126][127][128][129][130]. IFNs, interferons. [/table]
[table] Table 6: Development of vaccines for COVID-19 that are under clinical trials. Controlled, Phase I/II Clinical Trial, to Evaluate the Safety and Immunogenicity of the SARS-CoV-2 Inactivated Vaccine in Healthy Adults Observer-Blind, Placebo-Controlled Trial to Evaluate the Safety, Tolerability, and Immunogenicity of the bacTRL-Spike Oral Candidate Vaccine for the Prevention of COVID-19 in Healthy Adults Placebo-controlled, Phase Ia/IIa Trial of an Inactivated SARS-CoV-2 vaccine in Healthy People Aged 18 to 59 Years Multiple sites in China NCT04412538 BCG, Bacillus Calmette-Guérin; a clinical trials not yet recruiting at the time of publication; data from clinical trials.gov. [/table]
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Efficacy of phosphodiesterase type 5 inhibitors for the treatment of distal ureteral calculi: A systematic review and meta-analysis
Urolithiasis is one of the most common disorders of the urinary tract and affects approximately 5% to 10% of the population. There has been a progressive increase in prevalence in recent decades that has reached close to 20% Purpose: To determine the efficacy of phosphodiesterase type 5 inhibitors (PDE5i) as medical expulsive therapy (MET) for the treatment of distal ureteral calculi. Materials and Methods: A search strategy was conducted in the MEDLINE, CENTRAL, and Embase databases. Searches were also conducted in other databases and unpublished literature. Clinical trials were included without language restrictions. The risk of bias was evaluated with the Cochrane Collaboration's tool. An analysis of random effects due to statistical heterogeneity was conducted. The primary outcome was the expulsion rate of the distal ureteral calculus in 28 days. The secondary outcomes were the time to expulsion, side effects of treatment, and amount (mg) of nonopioid analgesia. The measure of the effect was the risk difference (RD) with a 95% confidence interval (CI). The planned interventions were PDE5i vs. placebo, tadalafil vs. placebo, and tadalafil vs. tamsulosin. Results: Four articles were included in the qualitative and quantitative analysis. Records of 580 patients were found among the four studies. A low risk of bias was shown for the majority of the study items. The calculi expulsion rate had an RD of 0.26 (95% CI, 0.15-0.37) and a less prolonged expulsion as a secondary outcome with a mean difference of -4.39 days (95% CI, -6.69 to -2.09) in favor of PDE5i compared with the placebo. No significant difference was found for these outcomes when comparing tadalafil with tamsulosin. Conclusions: Compared with a placebo, PDE5i could be effective as MET for the treatment of distal ureter calculi.
# Introduction
PDE5i for the treatment of distal ureteral calculi the complications are equally frequent . The objective of current treatment is to resolve the ureteral obstruction with minimal complications. Many descriptive studies have reported spontaneous expulsion rates of up to 50% for all calculi and up to 85% for calculi <5 mm in size between 28 and 40 days [bib_ref] Time to stone passage for observed ureteral calculi: a guide for patient..., Miller [/bib_ref] [bib_ref] Randomized trial of the efficacy of tamsulosin, nifedipine and phloroglucinol in medical..., Dellabella [/bib_ref]. When spontaneous expulsion does not occur, it is possible to use minimally invasive therapeutic modalities, which are highly effective but not innocuous procedures that are responsible for a large part of the cost overrun for the health system. Thus, the therapeutic spectrum has shifted towards improving medical expulsive therapy (MET) by seeking more accurate indications and more effective medications to reduce the complications associated with both the surgical intervention and the circumstance of inappropriately postponing it.
Selective alpha-blockers, such as tamsulosin and silodosin, and calcium antagonists, such as nifedipine, have traditionally been the treatment of choice, with proven efficacy in multiple clinical trials. Additionally, in the last decade, the possibility of using phosphodiesterase type 5 inhibitors (PDE5i) in MET has been studied owing to their direct effects on the relaxation of the ureteral smooth muscle in both animal and human models. This effect impacts the frequency of the peristaltic waves and the muscle tone of the ureter along its entire extent. Vardenafil appears to play a leading role in the in vitro models, followed by sildenafil and tadalafil, with potent effects on the formation of the derived second messengers cyclic guanosine monophosphate and cyclic adenosine monophosphate [bib_ref] In vitro effects of PDE5 inhibitors sildenafil, vardenafil and tadalafil on isolated..., Gratzke [/bib_ref]. However, the independent mechanisms of action of nitric oxide formation have been questioned because this pathway alone accounts for between 20% and 30% of the detected effect. Some authors have suggested that the inhibition caused by the influence of ionic calcium by 2 routes would intervene in ureteral smooth muscle contractions [bib_ref] In vitro effects of PDE5 inhibitors sildenafil, vardenafil and tadalafil on isolated..., Gratzke [/bib_ref] [bib_ref] Vardenafil effect on ureteric smooth muscle: in vitro study in porcine model, Al-Aown [/bib_ref]. Owing to the aforementioned considerations, the possibility of studying the efficacy of PDE5i in the MET of patients with distal ureterolithiasis has been proposed.
The objective of this review was to determine the efficacy of PDE5i as monotherapy in MET of distal ureteral calculi of less than 10 mm.
# Materials and methods
This study was conducted according to the recommendations of the Cochrane Collaboration following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement. The protocol was registered in the international prospective register of systematic reviews (PROSPERO; https://www.crd.york.ac.uk/PROSPERO/) under number CDR42016038858.
## Selection criteria 1) studies
Parallel randomized clinical trials performed between January 1980 and May 2016 were included. Open and closed trials and studies with simultaneous interventions were excluded. No language restriction was imposed.
## 2) participants
Women and men over 18 years of age who were diagnosed with single, unilateral symptomatic distal ureterolithiasis with a ureteral calculus of 10 mm or less in its largest dimension were included. Studies that included patients with acute renal injury secondary to the ureteral obstruction, monorenal patients, or patients with associated urinary sepsis, bilateral or multiple ureterolithiasis, or concomitant treatment with PDE5i were excluded.
## 3) interventions
The planned interventions were PDE5i vs. placebo, PDE5i vs. nonintervention, and PDE5i vs. other medical intervention. The PDE5i were administered daily for a minimum period of 14 days without restrictions on the dose supplied.
## 4) outcomes
The primary outcome was the calculus expulsion rate in 28 days. The secondary outcomes were time to expulsion, side effects associated with treatment, episodes of ureteral colic, and the need for nonopioid analgesia.
## Information sources and search strategy
## Study selection
Two investigators reviewed the titles and abstracts independently and blinded to determine the potential usefulness of the articles within the systematic review. The eligibility criteria were applied during the review of the full text of potentially eligible articles for the final selection. Discrepancies were resolved by consensus of the 2 researchers.
## Data collection process
Relevant data were collected in duplicate by using a standardized data extraction sheet that contained the study design, participants, interventions and comparators, and final outcome details. The reviewers confirmed all data entries and checked for completeness and accuracy at least twice. If some information was missing, we contacted the authors to obtain complete data.
Risk of bias in and across individual studies was assessed independently by 2 researchers using the Cochrane Collaboration risk of bias tool. We solved disagreements by consensus.
The "Risk of bias table" was edited by using Review Manager Software ver. 5.3 (RevMan; Cochrane Collaboration, Copenhagen, Denmark) to illustrate the judgments for each study. A risk of bias summary was made to show the judgments in a cross-tabulation of study by entry.
## Summary measures
Analyses were performed in RevMan 5.3 and Stata 13 (StataCorp., College Station, TX, USA) as needed. The risk ratio and the risk difference (RD) were the effects measured for the primary and secondary outcomes with 95% confidence intervals (95% CIs). We performed a randomeffects meta-analysis to assess heterogeneity (clinical and statistical) in the included clinical trials.
Heterogeneity between trials was assessed through the I 2 statistic. An I 2 value greater than or equal to 50% could represent heterogeneity according to Higgins et al.. The results were reported as forest plots showing the effect size of all included studies with 95% CIs.
## Additional analyses
No meta-regression was performed owing to the number of included studies. We undertook the sensitivity analysis based on the exclusion of each one of the trials, but no differences were found [bib_ref] Meta-analysis in clinical trials, Dersimonian [/bib_ref]. Analysis of publication bias was not performed owing to the number of studies found (less than 10 studies) according to Higgins et al..
# Results
Study selection: A total of 27 records were found with the search design strategies. Among them was a phase III clinical trial in progress that had recently completed the recruitment period and thus had no results available. Finally, four studies were included in the qualitative and quantitative analysis [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref].
Characteristics of the included studies: The selected studies included 580 patients with a median of 145 patients per study (range, 60-270). The 4 studies evaluated the calculus expulsion rate at 28 days as a primary outcome [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref]. All studies reported the time to expulsion as one of the secondary outcomes and provided information regarding the response to pain and the side effects during treatment, with the exception of the study by Abhishek et al.. Three studies used comparisons with a placebo [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] , 1 study used sildenafil , and 3 studies used tadalafil as the PDE5i [fig_ref] Table 1: Characteristics of the included studies [/fig_ref] [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref].
Characteristics of the excluded studies: The reasons for exclusion were indications other than MET for the distal [bib_ref] Characterization of cyclic nucleotide phosphodiesterase isoenzymes in the human ureter and their..., Taher [/bib_ref] PDE5i for the treatment of distal ureteral calculi calculi, no clinical trials, interventions in combination with other medications, duplicates, and no relationship to the topic . Risk of bias in the studies: An evaluation of the risk of bias for the expulsion rate and the time to expulsion was performed owing to their close relationship in the evaluation of these outcomes; thus, the same information was required for their presentation. Kumar et al. [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref] and Shokeir et al. showed a low risk for the majority of the items, whereas Abhishek et al.showed no clear risk for the first 4 evaluation items and a low risk for the rest .
## Pde5i vs. placebo: results according to the outcome 1) expulsion rate of the ureteral calculus
Three studies [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] included 256 participants (129 in the PDE5i group and 127 in the placebo group). According to our analysis, no heterogeneity among the studies was found; therefore, a fixed effects model was used for the analysis. The PDE5i were associated with a significantly higher expulsion rate than the placebo (79% vs. 53% [RD, 0.26; 95% CI, 0. 15 to 0.37]).
## 2) time to expulsion
Two studies [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] included 160 participants (80 in the PDE5i group and 80 in the placebo group). The PDE5i were associated with a significantly lower time to expulsion than the placebo (RD, -4.39; 95% CI, -6.69 to -2.09). Significant heterogeneity was found with an I 2 index of 75%; therefore, a random effects model was used for the analysis of the data.
## 3) use of analgesia
Two studies evaluated the use of analgesia during the course of the MET. However, the studies could not be included in the quantitative analysis because they differed widely in follow-up times and the analgesic used as the standard therapy. Abhishek et al.described the use of minor oral diclofenac in the tadalafil group compared with a placebo during 28-day MET (132.93 mg vs. 331 mg, respectively, p<0.0001), whereas Hasan et al. [bib_ref] The role of tadalafil in lower ureteric stone expulsion, Hasan [/bib_ref] described the use of minor oral indomethacin in the tadalafil group compared with a placebo during 14-day MET (133 mg vs. 790 mg, respectively, p<0.0001). Similarly, both studies described a decrease in the number of ureteral colic episodes and a lower average pain score according to the visual analogue scale during MET.
## Pde5i for the treatment of distal ureteral calculi
## 4) side effects associated with met
Only one study described the side effects associated with treatment. Shokeir et al. reported a frequency of uncomplicated cephalalgia of 4% in the intervention group and 0% in the placebo group in their study of sildenafil vs. placebo, although the difference was not significant (p=0.0579). Other effects of the treatment were not reported in this study.
## Tadalafil vs. tamsulosin: results according to the outcome 1) expulsion rate of the ureteral calculus
Two studies [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref] included 280 participants (140 in the tadalafil group and 140 in the tamsulosin group). According to our analysis, no heterogeneity was found among the studies; therefore, a fixed effects model was used. The efficacy of tadalafil was comparable with that of tamsulosin, with a nonsignificant risk difference of 4% in favor of tadalafil (RD, 0.04; 95% CI, -0.07 to 0. 14) .
## 2) time to expulsion
Only one study reported time to expulsion as a secondary outcome. Kumar et al. [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref] reported no significant differences when comparing tadalafil vs. tamsulosin in terms of time to expulsion of the calculus (16.2±4.2 vs. 16.5±4.6, respectively, p=0.648).
## 3) use of analgesia
A single study reported the comparison between tadalafil and tamsulosin in terms of the use of analgesia. Kumar et al. [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref] reported no significant differences when comparing tadalafil vs. tamsulosin, with an average use of diclofenac of 215±12.4 vs. 220±10.8, respectively (p=0.8).
## 4) side effects associated with met
Only one study described the side effects associated with treatment. Kumar et al. [bib_ref] Role of tamsulosin, tadalafil, and silodosin as the medical expulsive therapy in..., Kumar [/bib_ref] reported multiple side effects associated with treatment, including cephalalgia, dizziness, back pain, orthostatic hypotension, and abnormal ejaculation. The frequencies varied between 3% and 15.5%, with cephalalgia (10%-15.5%) and dizziness (10%-15.5%) being more frequent. No significant differences were found for the frequency of each symptom mentioned when comparing tadalafil and tamsulosin (p≥0.05).
# Discussion
Ureteral colic is primarily due to ureterolithiasis, which represents approximately 1% to 2% of admissions to emergency services. The natural course of lithiasic disease must be balanced with the risks of surgical intervention. Therefore, MET has recently emerged as a therapeutic alternative for the initial treatment of distal ureterolithiasis in selected patients [bib_ref] Medical expulsive therapy in adults with ureteric colic: a multicentre, randomised, placebo-controlled..., Pickard [/bib_ref]. Some of the therapeutic approaches in MET include alpha-blockers, calcium antagonists, and corticosteroids. A decrease of peristalsis, reduction of inflammation, and relaxation of the ureteral smooth muscle are the basis for the use of these medications. However, the true indication for MET is confined to distal ureterolithiasis with a calculus diameter of between 5 and 10 mm. There is clear and consistent evidence of the high probability of expulsion of minor calculi up to 5 mm, which in some reports is close to 85% [bib_ref] Time to stone passage for observed ureteral calculi: a guide for patient..., Miller [/bib_ref] [bib_ref] Randomized trial of the efficacy of tamsulosin, nifedipine and phloroglucinol in medical..., Dellabella [/bib_ref]. This issue has been a source of constant errors in the methodological designs of many studies that attempt to elucidate the efficacy of MET, because they include this group of participants in whom the probability of expulsion, per se, without intervention is quite high. Moreover, participants with calculi of the middle and even proximal ureter have been included, which contrarily may dilute the differences in favor of MET, leading to unexpected conclusions, as Pickard et al. stated in a more recent prospective study [bib_ref] Medical expulsive therapy in adults with ureteric colic: a multicentre, randomised, placebo-controlled..., Pickard [/bib_ref] [bib_ref] Silodosin to facilitate passage of ureteral stones: a multiinstitutional, randomized, double-blinded, placebo-controlled..., Sur [/bib_ref].
Recently, the multiple mechanisms of action of PDE5i have increased the number of therapeutic indications for this pharmacological group. Taher et al. reported the presence of PDE1, 2, 4, and 5 isoenzymes in cytosolic solutions of human ureteral tissue, which suggested nonadrenergic ureteral motility mechanisms and even mechanisms independent of nitric oxide [bib_ref] Characterization of cyclic nucleotide phosphodiesterase isoenzymes in the human ureter and their..., Taher [/bib_ref] the same principle and on the in vitro effects on ureteral smooth muscle [bib_ref] In vitro effects of PDE5 inhibitors sildenafil, vardenafil and tadalafil on isolated..., Gratzke [/bib_ref] [bib_ref] Vardenafil effect on ureteric smooth muscle: in vitro study in porcine model, Al-Aown [/bib_ref] , PDE5i have been proposed as an option for MET.
This study is the first controlled systematic review and meta-analysis of clinical trials published to date. We attempted to determine the efficacy of PDE5i in MET. In summary, PDE5i are superior to placebos as MET, achieving a 26% increase in the probability of expulsion and decreasing the time to expulsion of the ureteral calculus by 4.3 days. The results of the latter outcome are subject to the presence of significant heterogeneity in the measurement of the effect (I 2 index of 75%), which is principally explained by 2 factors: (1) 1 of the 2 studies included only calculi of the juxtavesical portion, which theoretically would reduce the time of expulsion and possibly the expulsion rate proportionally, and (2) the time of MET was 14 days instead of 28 days, which was in contrast to the majority of the studies on this topic.
Apparently, PDE5i could also improve the painf ul experiences of the patients during treatment by reducing the episodes of ureteral colic and the amount of analgesia required for their relief. However, there is insufficient information available to summarize the evidence in a quantitative analysis.
No differences were found for any of the outcomes evaluated in the comparisons of tadalafil vs. tamsulosin. This finding may be consistent with the pharmacodynamics of these 2 groups of medications and suggests the possibility of a synergistic combination that could increase the ef fectiveness of each of the drugs when administered separately. This hypothesis was proposed by Kumar et al. [bib_ref] Comparative efficacy of tamsulosin versus tamsulosin with tadalafil in combination with prednisolone..., Kumar [/bib_ref] and Jayant et al. [bib_ref] Tamsulosin versus tamsulosin plus tadalafil as medical expulsive therapy for lower ureteric..., Jayant [/bib_ref] in 2 clinical trials, in which the expulsion rate for tadalafil plus tamsulosin ranged between 83% and 84%.
One of the strengths of our review was the quality of the search strategies designed for each database, which were highly sensitive and specific for the detection of records related to the question of the systematic review. Additionally, the characteristics of the included studies are highlighted because they included only patients with calculi between 5 and 10 mm, which is a group that has a real indication for MET as mentioned previously. Supporting these inclusion criteria, there is an ongoing randomized clinical trial from Mansoura University in Egypt (NCT02519153) comparing the efficacy of sildenafil versus placebo that has the same kind of participants in terms of calculi size, location, and following time. That trial supports the finding of this systematic review that MET was well-indicated in all included studies. The fact that new trials are focusing on this group of patients will add homogeneity to future updates of this topic.
The limitations of the systematic review and metaanalysis primarily included the small number of available studies and the quality of reporting of the clinical trials that composed them. This limitation does not necessarily indicate bad or good methodological quality. However, this variable could incur underestimation of the quality because the best tool available to evaluate quality is the Cochrane Collaboration tool, which requires an appropriate report to properly weigh the items. Nonetheless, the studies generally had a low risk of bias for the tool items.
Finally, this review was conducted to answer a question in relation to a highly prevalent urological pathology based on standardized recommendations. Therefore, although we recommend this intervention for various institutions worldwide, we suggest conducting trials with larger sample sizes and better methodological quality to improve the recommendations derived from this systematic review and meta-analysis.
# Conclusions
PDE5i may be an effective treatment for distal ureter calculi compared with placebo in MET in terms of the expulsion rate and the time to expulsion of the calculus. However, more high-quality trials are needed to lend support to this conclusion and recommendation.
[fig] Figure 3: (A) Phosphodiesterase type 5 inhibitors (PDE5i) versus placebo: calculus expulsion rate. (B) PDE5i versus placebo: time to expulsion. df, degrees of freedom; CI, confidence interval. [/fig]
[table] Table 1: Characteristics of the included studies [/table]
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Distill: a suite of web servers for the prediction of one-, two- and three-dimensional structural features of proteins
Background:We describe Distill, a suite of servers for the prediction of protein structural features: secondary structure; relative solvent accessibility; contact density; backbone structural motifs; residue contact maps at 6, 8 and 12 Angstrom; coarse protein topology. The servers are based on large-scale ensembles of recursive neural networks and trained on large, up-to-date, nonredundant subsets of the Protein Data Bank. Together with structural feature predictions, Distill includes a server for prediction of C α traces for short proteins (up to 200 amino acids).Results:The servers are state-of-the-art, with secondary structure predicted correctly for nearly 80% of residues (currently the top performance on EVA), 2-class solvent accessibility nearly 80% correct, and contact maps exceeding 50% precision on the top non-diagonal contacts. A preliminary implementation of the predictor of protein C α traces featured among the top 20 Novel Fold predictors at the last CASP6 experiment as group Distill (ID 0348). The majority of the servers, including the C α trace predictor, now take into account homology information from the PDB, when available, resulting in greatly improved reliability.Conclusion:All predictions are freely available through a simple joint web interface and the results are returned by email. In a single submission the user can send protein sequences for a total of up to 32k residues to all or a selection of the servers. Distill is accessible at the address
# Background
De novo prediction of protein three-dimensional structure from the primary sequence remains a fundamental and extraordinarily challenging problem. Many onedimensional and two-dimensional structural features, i.e. structural properties of individual residues or of couples of residues in a protein, have long been identified as useful intermediate representations between the primary sequence and the full three-dimensional structure, which can be adopted as a stage towards the prediction of protein structure and function. For instance accurate secondary structure and solvent accessibility information have been shown to improve the sensitivity of fold recognition methods (e.g. [bib_ref] GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences, Jones [/bib_ref] [bib_ref] Improving fold recognition without folds, Przybylski [/bib_ref] and are at the core of most ab initio methods (e.g. see [bib_ref] Rosetta predictions in CASP5: Successes, failures, and prospects for complete automation, Bradley [/bib_ref] for the prediction of protein structure.
We have developed a number of predictors of structural features of proteins. Some of the features predicted are novel and highly informative (e.g. protein contact density [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] , local structural motifs [bib_ref] Protein Structural Motif Prediction in Multidimensional φ -ψ Space leads to improved..., Mooney [/bib_ref] , multi-class coarse contact maps [bib_ref] Modular DAG-RNN Architectures for Assembling Coarse Protein Structures, Pollastri [/bib_ref] , others are well established (secondary structure, solvent accessibility, residue contact maps) but predicted at state-of-the-art accuracy levels [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] [bib_ref] Porter: a new, accurate server for protein secondary structure prediction, Pollastri [/bib_ref].
All methods are freely available through simple web interfaces, which allow the processing of medium-to largescale jobs by any selection of the servers with only a small number of manual submissions.
## Implementation
## Algorithms
All one-and two-dimensional structural feature predictors are based on single-or dual-layer Recursive Neural Network architectures for Directed Acyclic Graphs (DAG RNNs) [bib_ref] The Principled Design of Large-Scale Recursive Neural Network Architectures -DAG-RNNs and the..., Baldi [/bib_ref]. One-dimensional feature predictors (i.e. those mapping the primary sequence into a sequence of the same length) are based on 1D DAG RNNs [bib_ref] Exploiting the past and the future in protein secondary structure prediction, Baldi [/bib_ref] , while twodimensional feature predictors (where a property of pairs of residues or secondary structure elements is predicted) are based on 2D DAG RNNs [bib_ref] The Principled Design of Large-Scale Recursive Neural Network Architectures -DAG-RNNs and the..., Baldi [/bib_ref] [bib_ref] Prediction of Contact Maps by Recurrent Neural Network Architectures and Hidden Context..., Pollastri [/bib_ref] , or combinations of the two [bib_ref] Modular DAG-RNN Architectures for Assembling Coarse Protein Structures, Pollastri [/bib_ref]. All RNNs have shortcut connections to cut the length of paths between different inputs. This facilitates the transmission of long-range information, which is relevant to determine non-local structural properties such as the formation of β-sheets. In dual-layer RNNs, the second layer, or filter, incorporates long-range information directly (e.g. predicted secondary structure and solvent accessibility composition, averaged over multiple contiguous windows). In this way, information from up to 225 residues is taken into account when a final prediction is made. For a more detailed description of the models and training algorithms, see [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] [bib_ref] Protein Structural Motif Prediction in Multidimensional φ -ψ Space leads to improved..., Mooney [/bib_ref] [bib_ref] Modular DAG-RNN Architectures for Assembling Coarse Protein Structures, Pollastri [/bib_ref] [bib_ref] Porter: a new, accurate server for protein secondary structure prediction, Pollastri [/bib_ref] [bib_ref] World Scientific in press. on the S258 set (see text for details), Pollastri [/bib_ref]. All systems adopt largescale ensembles of predictors (40 or more models for each architecture), trained on large, non-redundant datasets extracted from the PDB [bib_ref] The Protein Data Bank, Berman [/bib_ref].
The predictor of C α traces relies on a simple optimisation procedure, similar to that in, guided by a potential or pseudo-energy based on one-and two-dimensional feature predictions. The target of the optimisation is realising the C α trace which enforces the predicted features (e.g. presence of helices, presence or absence of contacts between residues) "best", while preserving some trivial properties such as realistic distances between neighbouring C α s and absence of clashes. The optimisation algorithm and potential are described in detail in [bib_ref] World Scientific in press. on the S258 set (see text for details), Pollastri [/bib_ref].
## Data sets
All predictors of one-dimensional and two-dimensional features are trained on datasets extracted from the December 2003 25% pdb_select set [bib_ref] Enlarged representative set of protein structures, Hobohm [/bib_ref]. We use the DSSP pro-gram [bib_ref] Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, Kabsch [/bib_ref] to assign target structural features and remove sequences for which DSSP does not produce an output due, for instance, to missing entries or format errors. After processing by DSSP, the set contains 2171 protein and 344,653 amino acids (set S2171).
Multiple sequence alignments for S2171 are extracted from the NR database as available on March 3 2004 containing over 1.4 million sequences. The database is first redundancy reduced at a 98% threshold, leading to a final 1.05 million sequences. The alignments are generated by three runs of PSI-BLAST [bib_ref] Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Altschul [/bib_ref]. Multiple alignment generation in online server operation is handled transparently and does not require user intervention.
The predictor of C α traces is benchmarked on a subset of the PDB available on April 5 2005 generated as follows: all proteins showing more than 22% sequence similarity to S2171 or shorter than 30 residues were excluded; the remaining set was redundancy reduced at a maximum 22% sequence similarity threshold; proteins longer than 200 residues were excluded. The final set contains 258 proteins (S258).
## Servers
The Distill suite of servers currently contains 7 predictors: 4 of one-dimensional features (Porter, Porter+, Pale Ale, BrownAle); 2 of two-dimensional features (XStout and XXStout); the predictor of C α traces (3Distill). The chart in [fig_ref] Figure 2: Distill's flowchart [/fig_ref] represent the flow of information between the servers. Details about each server are provided below.
## Secondary structure
Porter [bib_ref] Porter: a new, accurate server for protein secondary structure prediction, Pollastri [/bib_ref] is a system for protein secondary structure prediction based on an ensemble of 1D DAG-RNNs. Porter is an evolution of the SSpro [bib_ref] Improving the prediction of protein secondary structure in three and eight classes..., Pollastri [/bib_ref] server. Porter's improvements include: rich input coding (each residue is coded as a letter out of an alphabet of 25); output filtering and incorporation of predicted long-range information (225 residues are considered to yield the final predictions); large training sets (S2171); large-scale ensembling (45 models). Moreover, when this is available, homology information to structures in the PDB is provided as a further input to the system(see results section).
## Structural motifs
Porter+ [bib_ref] Protein Structural Motif Prediction in Multidimensional φ -ψ Space leads to improved..., Mooney [/bib_ref] classifies each residue into one out of 14 local structural motifs. The motifs are built by applying multidimensional scaling and clustering to pair-wise angular distances between quadruplets of Φ -Ψ dihedral angle pairs collected from high-resolution protein structures [bib_ref] Protein conformational space in higher order ψ-φ maps, Sims [/bib_ref]. Structural motif predictions are highly informative and provide a finer-resolution picture of a protein backbone than (and may be used to improve [bib_ref] Protein Structural Motif Prediction in Multidimensional φ -ψ Space leads to improved..., Mooney [/bib_ref] traditional 3-class secondary structure. The definition and one-letter code for the 14 structural motifs are provided in the help web page.
## Relative solvent accessibility
PaleAle, a novel component of Distill, classifies each residue as being in one of 4 (approximately equally frequent) classes of solvent accessibility: B = completely buried (0-4% exposed); b = partly buried (4-25% exposed); e = partly exposed (25-50% exposed); E = completely exposed (more than 50% exposed). The architecture of PaleAle's classifier is an exact copy of Porter's (described above and in [bib_ref] Porter: a new, accurate server for protein secondary structure prediction, Pollastri [/bib_ref]. As in the case of Porter, when available, homology information to structures in the PDB is provided as a further input, yielding more accurate predictions(see results section).
## Contact density
BrownAle [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] is a system for the prediction of protein Contact Density. We define Contact Density as the Principal Eigenvector (PE) of a protein's residue contact map at 8Å, multiplied by the principal eigenvalue. Let λ(C) = {λ : Cx = λx} be the spectrum of C (where C is a protein's contact map, for whose definition see below), λ = {x : Cx = λx} the corresponding eigenspace and = max{λ ∈ λ(C)} the largest eigenvalue of C. The principal eigenvector of C, , is the eigenvector corresponding to , i.e.
such that C = . Thus, we define a protein's contact density as .
BrownAle predicts Contact Density in 4 classes. The class thresholds are assigned so that the classes are approximately equally numerous, as follows: N = very low contact density (0,0.04); n = medium-low contact density (0.04,0.18); c = medium-high contact density (0.18,0.54); C = very high contact density (greater than 0.54). BrownAle's architecture is an exact copy of Porter's (described above). Secondary Structure (by Porter) is fed as input into BrownAle, beside the primary sequence. Predicted Contact Density contributes significantly to improved residue contact map predictions [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] , especially for long-ranged contacts.
Coarse contact maps and topologies XStout [6] is a system for the prediction of protein coarse topologies. A protein is represented by a set of rigid rods associated with its secondary structure elements (α-helices and β-strands, as predicted by Porter). 4-class distance maps and 3-class angle maps between secondary structure elements are first predicted based on the primary sequence and on predicted secondary structure and solvent accessibility (by Porter and PaleAle), and coarse 3D folds of proteins are then assembled starting from these maps. 3D reconstruction is carried out by minimising a cost function taking the form of a purely geometrical potential. Coarse folds are only predicted when C α trace predictions are not available (for proteins longer than 200 residues), or not requested by the user.
## Residue contact maps
XXStout [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] is a system for the prediction of protein residue contact maps. The contact map of a protein with N amino acids is a symmetric N × N matrix C, with elements C ij defined as:
We define two amino acids as being in contact if the distance between their C α is less than a given threshold.
XXStout predicts contacts at three different thresholds: 6 Å, 8 Å and 12 Å. Contact maps are predicted as follows: protein secondary structure, solvent accessibility and contact density are predicted from the sequence using, respectively, Porter, PaleAle and BrownAle; ensembles of twodimensional Recursive Neural Networks [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref] [bib_ref] The Principled Design of Large-Scale Recursive Neural Network Architectures -DAG-RNNs and the..., Baldi [/bib_ref] [bib_ref] Prediction of Contact Maps by Recurrent Neural Network Architectures and Hidden Context..., Pollastri [/bib_ref] predict the contact maps based on the sequence, a 2-dimensional profile of amino-acid frequencies obtained from PSI-BLAST alignments of the sequence against the NR, and predicted secondary structure, solvent accessibility and contact density. XXStout is trained and tested in cross-validation on a sample of the S2171 dataset containing only sequences of length at most 200 residues (1602 proteins in total). C α traces 3Distill is a server for the prediction of protein C α traces. 3Distill relies on a fast optimisation algorithm guided by a potential built on the 6 classes of structural features predicted by the other systems. A preliminary implementation of 3Distill (group Distill, ID 0348) [bib_ref] World Scientific in press. on the S258 set (see text for details), Pollastri [/bib_ref] , was ranked with model 1 (only one model was submitted) 9-th out of 181 predictors for GDT_TS on Novel Fold hard targets, and 20-th for Z-score for all Novel Fold and Near Novel Fold targets (13-th for NF-hard) at the CASP6 competition. The online version currently available is a substantial improvement of the CASP6 one, based on more accurate structural feature predictions, and on a refined search algorithm. Homology information to structures in the PDB is also now exploited, when available. This is achieved by two means: secondary structure, solvent accessibility and contact maps are predicted by specialised systems which incorporate homology information as a further input; structural similarity to homologues is directly used in the optimisation. Not surprisingly, the availability of homology information results in greatly increased accuracy of the models (see results section).
[formula] λ x λ x λ x λ x C ij = ⎧ ⎨ ⎩ ( ) 1 0 [/formula]
1 if amino acid i and j are in contact otherwise
## Input format
Input into the servers is handled by two simple HTML forms: one for submissions of single queries (see ; the other for submitting multiple queries. In both forms the user must provide an email address, to which the response will be sent. In the single-query form the user has also the option of providing a query name, that will be reported in the server response. In the single-query form the user needs to provide the query sequence in plain 1-letter code, with no headers. New-line, tab and space characters are ignored. In the multi-query form the user may provide the queries in FASTA format, with each query name preceded by the > symbol on a line and the corresponding protein sequence on the following line or lines (space and tab characters are ignored). If the user adopts the multi-query form, a separate email will be sent for each query. The query name quoted in the response will be the query name as parsed from the FASTA format. In both forms the user can select any combination of the servers via a set of tick-boxes. Submissions of up to 32,768 characters (roughly 100 average proteins) are accepted by the multi-query form, making medium-to large-scale predictions possible with only a small number of manual submissions.
## Output format
Server responses are sent by email. One-dimensional feature predictions (secondary structure, structural motifs, solvent accessibility, contact density) are sent in plain text attached to the email. A detailed description of the codes, of their precise definitions, and an example of server response, are provided in the help web page. The following is an example of server's response to a query sequence of length 106: All one-dimensional features come predicted in 1-letter codes. The predictions are split into groups of lines of length 60. The first line in each group is the 1-letter code for the primary sequence (always present). The second line is the secondary structure predicted by Porter (always present). The symbols have the following meaning:
- H = helix: DSSP's H (α-helix) or G (3-10 helix) or I (πhelix) classes.
- E = strand : DSSP's E (extended strand) or B (β-bridge) classes.
- C = the rest : DSSP's T (turn) or S (bend) or . (the rest).
The third line represents predictions by Porter+. The line is present if the user asked for Porter+ predictions. Each residue is mapped into one of 14 possible structural motifs coded as 1-letter symbols -the 1-letter code for the motifs is devised to be mnemonically related to secondary structure (e.g. motifs 'H' and 'h' are frequent in helices, 'E' and 'e' in strands, etc.). The online help page provides a complete description of the codes representing Porter+ outputs.
The fourth line represents relative solvent accessibility predicted by PaleAle: B = completely buried (0-4% exposed), b = partly buried (4-25% exposed), e = partly exposed (25-50% exposed), E = completely exposed (50% exposed or more). The line is present if the user requested a PaleAle prediction. Two dimensional feature predictions (residue and coarse contact maps) come as files attached to the email. XStout's outputs come as 6 attachments:
- Attachment 1 (number.xstout4c, where number is a 5 digit code for the submission) : the distance map. The segments (as predicted by Porter -only helices and strand of length at least two are considered) are listed first, followed by one line for each pair of segments, indicating predicted distance range between the two contacts, followed by a reliability index. Distance ranges predicted are [0Å,10Å), [10Å,18Å), [18Å,29Å) and [29Å, + ∞). An example of XStout output is provided in the online help of the servers.
- Attachments 2-6 (number.x.topo.pdb, with x = 1 ... 5): 5 coarse reconstructions, in PDB format. Points represented are the termini of all Helices and Strands of length 2 or greater -there will be 2N such points in a protein with N segments.
Residue contact maps defined at 6, 8 and 12 Å (files number.xxstout06, number.xxstout08 and number.xxstoutl2) are formatted as an N × N matrix of real numbers, where the j-th number on row i represents the estimated probability of contact between residues in positions i and j. PNG images containing grey-scale representations of residue contact maps are also automatically generated, if the user ticks the appropriate box. shows the PNG image of the contact map (thresholded at 12Å) for the previous example. Probabilities of contacts are represented as levels of grey ranging from pure black XXStout PNG example output XXStout PNG example output. The 12Å contact map predicted for the example given in the "output format" section.
(certainty of contact) to pure white (certainty of non-contact).
If 3Distill is selected, 5 models for the C α trace are provided, if the query is at most 200 residues long. In case it is longer, fold predictions by XStout are automatically sent instead. The models come attached to the email in PDB format, and readable directly by common PDB viewers such as Rasmol [bib_ref] RasMol: Biomolecular graphics for all, Sayle [/bib_ref]. An index of reliability of the model is provided in the remark fields of the PDB files. The index is an estimate of the TM score [bib_ref] Scoring function for automated assessment of protein structure template quality, Zhang [/bib_ref] of the model against the true structure, based on: the degree to which the model enforces the various predicted constraints; the size and estimated secondary structure composition of the query; the absence or presence (and, in the latter case, degree) of sequence similarity between the query and entries in the PDB. The reliability index, a novel component of Distill, is estimated by an artificial neural network trained in 5-fold cross-validation on 25800 C α trace reconstructions from the S258 set.
If homology information from the PDB is detected and used, this is indicated in the text of the response (alongside the percentage of sequence similarity to the best PDB template used) and in the remarks of the PDB files when these are present.
# Results
Porter, tested by a rigorous 5-fold cross validation procedure on S2171, achieves 79% correct classification on the "hard" CASP 3-class assignment (DSSP H, G, I → helix; E, B → strand; S, T, . → coil) [bib_ref] Porter: a new, accurate server for protein secondary structure prediction, Pollastri [/bib_ref] , and currently has the highest performance (approximately 80%) of all servers tested by assessor EVA [bib_ref] EVA: continuous automatic evaluation od protein structure prediction servers, Eyrich [/bib_ref]. When homology information from the PDB is available, Porter's predictions are more reliable, up to over 90% correct (in the sense of matching DSSP assignments) when templates with over 90% sequence similarity are available, and about 88% for all residues for which any template information is available. This result is not surprising by itself because, although different programs for assigning secondary structure from the experimental structure often differ by up to 20%, once a semantics is chosen (e.g. DSSP over STRIDE or DEFINE) it is possible to classify secondary structure almost perfectly. That is, it is true that there is some ambiguity in the assignment of secondary structures, but this is in large part due to the different definitions of secondary structures by different automated assignment programs, and only by a smaller amount to actual uncertainties as to what the structure may be.
PaleAle's accuracy, measured on the same large, nonredundant set adopted to train Porter (S2171) exceeds 53% correct 4-class classification, and roughly 80% 2class classification (Buried vs Exposed, at 25% threshold). As in the case of Porter, predictive accuracy improves significantly when homology information is available, up to 70% correct prediction for the 4-class case and 87% for the 2-class one, when templates with over 90% sequence similarity are available, and roughly 65% for residues for which any kind of template information is available.
The accuracy of BrownAle, measured on S2171, is 46.5% for the 4-class problem, and roughly 73% if the 4 classes are mapped into 2 (dense vs. non dense). In both cases the classification performance of BrownAle is 16% above a base-line statistical predictor [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref].
Tables 1 and 2 summarise the performances of XXStout measured on one fifth of the S2171 set after the exclusion of proteins longer than 200 residues (327 chains in total). Performances are given for the protein length/5 and protein length/2 contacts with the highest probability, for sequence separations of at least 6, at least 12, and at least 24, in CASP style [bib_ref] Critical assessment of methods of protein structure prediction (CASP)-round V, Moult [/bib_ref]. These performances compare favourably with the best predictors at the latest CASP competition [bib_ref] A two-stage approach for improved prediction of residue contact maps, Vullo [/bib_ref]. shows the expected accuracy of the C α trace reconstructor, as a function of sequence similarity to the closest homologue in the PDB, measured on the S258 set. The accuracy is measured as the TM score to the C α trace of the experimental structure. For sequence similarity above 30% the predictions' TM score is on average slightly above 0.7 indicating high reliability, is approximately 0.45 in the 20-30% interval, and 0.27 in the region below 20%. If reconstruction performances are measured on the S258 set without allowing homology information at any stage (pure ab initio predictions) the average TM score is 0.27, with 43 of the 258 structures above a TM score of 0.4 [bib_ref] World Scientific in press. on the S258 set (see text for details), Pollastri [/bib_ref]. The reliability score reported in the remarks field of the Top protein length/2 contacts classification performance as: precision%(recall%) PDB file has an average correlation of 0.7 with the TM score against the true structure, and thus provides a good estimate of the quality of the prediction.
# Conclusion
The servers we designed allow the annotation of protein sequences with a number of structural features which are at least partially orthogonal. Given the speed of the underlying methods, large-or even genomic-scale predictions can be handled by our servers in response of users' queries -with up to 32,768 residues handled in a single submission. Up to 20,000 queries per day can be processed by the servers based on their current implementation (a 40 CPU cluster), and nearly 30,000 tasks from 63 national or supernational domains have been served to date.
Our servers provide fast, reliable prediction of protein structural features for the ab initio case, and allow fast, reliable, large-scale predictions of protein structures for the case in which some homology to the PDB is detectable.
We are in the process of: extending the use of homology information to all prediction stages; building a parallel pipeline to Distill, for the case in which marginal similarity templates exist in the PDB for a query. Training of the systems for the latter case is completed and we expect that the new pipeline will be running over the next few months, complementing the current system with highthroughput fold recognition facilities.
## Availability and requirements
The servers are freely available for academic users at the address http://distill.ucd.ie/distill/. Linux and Windows binaries for all the servers are freely available for academic users upon request. The sets used for training, testing and benchmarking the servers (S2171 and S258) are available upon request.
[fig] Figure 2: Distill's flowchart. Information flows from the top of the chart to the bottom. ! " # Distill's single-query interface Figure 1Distill's single-query interface. The multi-query interface is identical, except that the query name box is missing (names are extracted directly from the FASTA format). [/fig]
[table] Table 2: XXStout performance. [/table]
[table] Table 1: XXStout performance. [/table]
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Preoperative MRI findings and functional outcome after selective dorsal rhizotomy in children with bilateral spasticity
Purpose To identify MRI characteristics that may predict the functional effect of selective dorsal rhizotomy (SDR) in children with bilateral spastic paresis. Methods We performed SDR in a group of 36 patients. The gross motor functioning measure-66 (GMFM-66) was applied before and after SDR. Available cerebral MRIs were retrospectively classified into three diagnostic groups: periventricular leucomalacia (PVL; n=10), hydrocephalus (n=2), and normal (n=6). In patients with PVL, we scored the severity of the MR abnormalities. We compared the changes in the GMFM-66 after SDR in the diagnostic groups. In patients with PVL, we correlated the severity of the MR abnormalities with the changes in the GMFM-66.ResultsThe mean follow-up period was 5 years and 4 months (range, 1 year and 1 month to 9 years). The best improvement in gross motor function was observed in patients with normal MRI, and the slightest improvement was observed in patients with hydrocephalus. The severity of the PVL did correlate with the GMFM-66 score before SDR but not with the functional effect of SDR. Conclusion We conclude that with respect to gross motor skills, the improvements after SDR are good in patients with no MRI abnormalities. In the patients with hydrocephalus, the improvements after SDR were insignificant. In patients with PVL, the improvements were intermediate and did not correlate with the degree of PVL.
# Introduction
Spasticity is defined as a velocity-dependent increase in the tonic stretch response with excessive tendon jerk reflexes [bib_ref] Symposium synopsis, Lance [/bib_ref] and is caused by the reduction of inhibitory impulses on lower motor neurons. Spasticity can cause pain, muscle shortening, and orthopedic malformations, and it can severely interfere with functional abilities and gait pattern. Cerebral palsy (CP) is the most frequent cause of spasticity in children. Cerebral palsy is a group of disorders of movement and posture due to a nonprogressive lesion of the developing brain [bib_ref] A report: the definition and classification of cerebral palsy, Rosenbaum [/bib_ref]. As a consequence of this definition, its causes are diverse.
Different treatment modalities are used to treat spasticity in children. Selective dorsal rhizotomy (SDR) is a neurosurgical treatment that is mainly performed at lumbar level in patients with bilateral spastic paresis. By incomplete transection of the (sensory) posterior lumbosacral rootlets, SDR reduces the excitatory input from the lower limbs that enters the spinal cord. In children with spastic CP, SDR leads to a more normal gait pattern [bib_ref] Biomechanical changes in gait following selective dorsal rhizotomy, Abel [/bib_ref] [bib_ref] A prospective gait analysis study in patients with diplegic cerebral palsy 20..., Langerak [/bib_ref] [bib_ref] Comprehensive short-term outcome assessment of selective dorsal rhizotomy, Trost [/bib_ref] [bib_ref] Outcomes after selective dorsal rhizotomy for spastic cerebral palsy, Steinbok [/bib_ref] , better performance of the activities of daily life [bib_ref] Long-term outcomes five years after selective dorsal rhizotomy, Nordmark [/bib_ref] [bib_ref] Selective dorsal rhizotomy in cerebral palsy to improve functional abilities: evaluation of..., Van Schie [/bib_ref] , and improvement in gross motor function [bib_ref] Outcomes after selective dorsal rhizotomy for spastic cerebral palsy, Steinbok [/bib_ref] [bib_ref] Long-term outcomes five years after selective dorsal rhizotomy, Nordmark [/bib_ref] [bib_ref] Selective dorsal rhizotomy in cerebral palsy to improve functional abilities: evaluation of..., Van Schie [/bib_ref] [bib_ref] Selective dorsal rhizotomy: efficacy and safety in an investigator-masked randomized clinical trial, Mclaughlin [/bib_ref] [bib_ref] Selective dorsal rhizotomy: meta-analysis of three randomized controlled trials, Mclaughlin [/bib_ref] [bib_ref] Evaluation of selective dorsal rhizotomy for the reduction of spasticity in cerebral..., Wright [/bib_ref] [bib_ref] A randomized clinical trial to compare selective posterior rhizotomy plus physiotherapy with..., Steinbok [/bib_ref] [bib_ref] Longterm functional outcome after selective posterior rhizotomy, Mittal [/bib_ref]. Data on functional outcome after SDR in patients with other conditions than spastic CP are limited, though promising [bib_ref] Effectiveness of selective dorsal rhizotomy in two patients with progressive spasticity due..., Grunt [/bib_ref]. Detailed information about the clinical characteristics of the selected children, other than spasticity alone, is often lacking.
Medical history and clinical examination does not always provide sufficient evidence to establish the diagnosis of the underlying disorder in patients with CP. The Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society both advise magnetic resonance imaging (MRI) of the brain for all CP patients [bib_ref] Practice parameter: diagnostic assessment of the child with cerebral palsy: report of..., Ashwal [/bib_ref]. In patients with bilateral spastic CP, the most common MRI abnormality is periventricular leucomalacia (PVL), which is characterized by a damage of the periventricular white matter [bib_ref] Clinical and MRI correlates of cerebral palsy: the European cerebral palsy study, Bax [/bib_ref] [bib_ref] The role of magnetic resonance imaging in elucidating the pathogenesis of cerebral..., Krageloh-Mann [/bib_ref] [bib_ref] Magnetic resonance imaging findings in a population-based cohort of children with cerebral..., Robinson [/bib_ref] [bib_ref] A systematic review of neuroimaging for cerebral palsy, Korzeniewski [/bib_ref]. Gray matter lesions, early developmental abnormalities of the central nervous system, and abnormalities of the cerebrospinal fluid space, such as hydrocephalus, are less commonly found [bib_ref] The role of magnetic resonance imaging in elucidating the pathogenesis of cerebral..., Krageloh-Mann [/bib_ref] [bib_ref] Magnetic resonance imaging findings in a population-based cohort of children with cerebral..., Robinson [/bib_ref] [bib_ref] A systematic review of neuroimaging for cerebral palsy, Korzeniewski [/bib_ref] , and in some children with bilateral spastic CP no abnormalities are found with MRI [bib_ref] Clinical and MRI correlates of cerebral palsy: the European cerebral palsy study, Bax [/bib_ref] [bib_ref] The role of magnetic resonance imaging in elucidating the pathogenesis of cerebral..., Krageloh-Mann [/bib_ref] [bib_ref] Magnetic resonance imaging findings in a population-based cohort of children with cerebral..., Robinson [/bib_ref] [bib_ref] A systematic review of neuroimaging for cerebral palsy, Korzeniewski [/bib_ref]. In patients with normal MRI findings and bilateral spastic paresis, genetic causes or spinal cord involvement should be considered.
The goal of this study was to identify possible relationships between the MRI findings with the level of gross motor function of the patient before SDR and the change in functioning after SDR. For this purpose, we retrospectively analyzed the MRIs of the patients who underwent SDR in our clinic.
# Methods and materials
## Subjects
This study was part of the project: "Investigation of longterm effects of SDR in children with spastic diplegia" and was approved by the medical ethical committee of the VU University Medical Center, Amsterdam, The Netherlands (project number 2006/105). Informed consent was given by the parents of all participants. The study population consisted of all the patients who underwent SDR in our clinic between January 1998 and December 2007. The first nine patients have been described in a previous report [bib_ref] Selective dorsal rhizotomy in cerebral palsy to improve functional abilities: evaluation of..., Van Schie [/bib_ref]. The patients were selected for SDR according to the criteria shown in . In our previous study, we only included patients with bilateral spastic CP and documented PVL [bib_ref] Selective dorsal rhizotomy in cerebral palsy to improve functional abilities: evaluation of..., Van Schie [/bib_ref].
In the present study, we included patients with nonprogressive spasticity due to different diagnoses. In our analysis, we included data on patients who underwent pre-and postoperative gross motor function measure (GMFM-66) analysis with a follow-up period of at least 12 months and of whom MRI was available for review.
## Procedure
Selective dorsal rhizotomy was performed by the same neurosurgeon (WVO) in all children. Dorsal roots L2-S1 were exposed and separated into different rootlets after laminotomy L2-L5 and opening of the dura. Transection of Selection criteria for selective dorsal rhizotomy in the VU University Medical Center Criteria for selective dorsal rhizotomy 1.) Bilateral spasticity of the lower extremities interfering with walking performance 2.) Presence of spasticity (defined as velocity-dependent resistance to passive stretch) in at least six muscle groups of the lower limbs 3.) Sufficient force in the quadriceps femoris muscle (squatting at least seven times) and the hip extensors (kneel with extended hips, support for balance allowed)
4.) Absence of structural orthopedic deformities or contractures at hip, knee, or ankle 5.) Presence of moderate to good selective motor control in the lower limbs 6.) Gross motor function classification system (GMFCS) level I, II, or III 7.) Good support from parents and rehabilitation setting the rootlets was performed after electro-stimulation, according to palpable muscle contraction and EMG response. At most, 50% of the rootlets were transected on one level. To prevent sexual and bladder disturbances, rootlets/fascicles showing electrical response after stimulation of the penis/ the clitoris were spared. Postoperative rehabilitation included intensive physical therapy for 12 months.
## Neuroimaging
The MRIs were assessed at two different points in time by two investigators-one neuropediatrician (8 years of experience in the assessment of MR neuroimaging; RJV) and one pediatrician with specialization in pediatric neurorehabilitation (2 years of experience in the assessment of MR neuroimaging; SG). Imaging was classified into three diagnostic groups according to the following criteria: periventricular leucomalacia (PVL; increased signal intensity of the periventricular white matter in T2 weighted imaging and/or FLAIR; other abnormalities optional); hydrocephalus (signs of ventricular dilatation and increased intracranial pressure); normal MRI (see [fig_ref] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR [/fig_ref]. In one patient, the MR abnormalities could not be classified into one of the three diagnostic groups and he was excluded from further data analysis (patient number 19, [fig_ref] Table 2: Summary of the characteristics of the patients included in the study F... [/fig_ref] ; his MRI showed delayed myelinisation and slightly enlarged ventricular size but no other white matter abnormalities and there were no signs of increased intracranial pressure). In the patients with PVL, we graded the severity of their MRI abnormalities based on the work of Cioni et al. [bib_ref] MRI findings and sensorimotor development in infants with bilateral spastic cerebral palsy, Cioni [/bib_ref]. The following items were assessed: ventricular size, evidence and extension of white matter signal intensity, evidence and extension of white matter loss, thinning of the corpus callosum, dimension (size) of subarachnoidal space, evidence and size of cysts, and presence of gray matter abnormalities. We also investigated whether white matter loss was occipitally and/or frontally present. The items were scored on a three-point scale, with a score of three indicating the most severe MRI abnormalities. The scores were summed to obtain a total score for each child (minimal score, 7 and maximal score, 21). Intra-and interobserver agreement was assessed for all items. If the two investigators did not agree, the classification and/or scoring were determined by a consensus.
## Outcome measure
All patients had a detailed pre-and postoperative clinical evaluation, including spasticity assessment, range of motion of single joints, gait analysis, and the GMFM. In this study, we only report the results of the GMFM. The GMFM is a criterion-referenced observational measure that was developed to assess children with CP. The validity, reliability, and responsiveness of the GMFM have been demonstrated in a population of patients similar to the participants in the present study [bib_ref] The gross motor function measure: a means to evaluate the effects of..., Russell [/bib_ref]. For our data analysis, we used the GMFM-66 version, which assesses 66 items covering five gross motor dimensions (lying and rolling/crawling and kneeling/standing and walking/running/jumping) and is elaborated to a numerical scale ranging from 0 to 100.
As outcome parameters we used the mean of all postoperative GMFM-66 scores from 1 year after surgery. The changes after SDR were expressed as the difference between GMFM-66 score and the preoperative GMFM-66 score (Delta-GMFM-66).
# Statistical analysis
For the reliability analysis of the MRI scoring, we used Kappa statistics. Agreement strengths for the Kappa values were classified according to Landis and Koch [bib_ref] The measurement of observer agreement for categorical data, Landis [/bib_ref]. For the comparison of the outcome in patients with different MRI characteristics, we used nonparametric tests (Kruskal-Walis test for group comparison; post hoc analysis was performed using the Mann-Whitney test when the level of significance was reached). Correlations were assessed with the Spearman's rank correlation coefficient. For the statistical analysis, we used SPSS® version 14.0 for Windows. Level of significance was set at 0.05.
# Results
## Clinical characteristics and level of functioning
Thirty-six patients underwent SDR of whom 32 patients had a brain MRI performed before SDR, and 26 MRIs were available for review. Of the patients with available MRI, four did not have any preoperative GMFM-66 assessments. Three patients had a GMFM-66 assessment before SDR but with a follow-up period of less than 12 months. The detailed patient characteristics of the remaining 19 patients are summarized in [fig_ref] Table 2: Summary of the characteristics of the patients included in the study F... [/fig_ref]. The mean number of GMFM assessments per patient was 4.9 (standard deviation (SD) 3.5, range 1 to 12), and the mean follow-up period was 5 years and 4 months (SD 2 years and 9 months, range 1 year and 1 month to 9 years). The mean age at the time of the operation was 6 years and 10 months (SD 1 year and 6 months, range 5 years and 9 months to 10 years and1 month). The age at the time of SDR neither correlate with the preoperative GMFM-66 (rho, 0.328; p=0.184) nor with the postoperative GMFM-66 scores (rho, 0.336; p= 0.173) and the improvement after SDR (rho, −0.049; p= 0.847). We found no difference in the Delta-GMFM-66 score between boys and girls. [fig_ref] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR [/fig_ref] , bilaterally increased periventricular white matter signal intensity [fig_ref] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR [/fig_ref] , a slight ventricular enlargement, and a loss of the occipital white matter [fig_ref] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR [/fig_ref]. g-i MR images classified as hydrocephalus. The lateral ventricles and the third and fourth ventricles [fig_ref] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR [/fig_ref] are enlarged Neuroimaging We diagnosed PVL in ten patients, hydrocephalus in two patients, and no MRI abnormalities in six patients. The classification of MRIs did not differ between the two observers. With respect to the grading of the MR abnormalities, the intrarater agreement was perfect for the frontal and occipital white matter loss, cysts, thinning of the corpus callosum, subarachnoidal space, and gray matter abnormalities (Kappa, 1.0) and almost perfect for the ventricular size (Kappa, 0.90) and the white matter signal intensities (Kappa, 0.87). The interrater agreement was considerably less than the intrarater agreement. It was perfect for the subarachnoidal space and the gray matter abnormalities (Kappa, 1.0), almost perfect for the ventricular size (Kappa, 0.90), substantial for the occipital white matter loss (Kappa, 0.74), and moderate for the frontal white matter loss (Kappa, 0.45). Kappa statistics [fig_ref] Table 3: Differences of gross motor outcome in with patients with different MRI classification [/fig_ref] and in [fig_ref] Figure 2: Box plots of the preoperative GMFM-66 [/fig_ref]. The preoperative GMFM-66 scores were significantly higher in patients with normal MRI than in patients with PVL (p= 0.001). Patients with hydrocephalus had intermediate scores and did not differ significantly from the other groups. In the follow-up measurements, the mean GMFM-66 was the highest in patients with a normal MRI (group difference, p= 0.003; difference between PVL and normal, p=0.002). The patients with normal MRI also made the best postoperative improvements. Almost no improvement was observed in the patients with hydrocephalus (group difference p=0.030; difference between PVL and hydrocephalus, p=0.032; difference between hydrocephalus and normal MRI, p= 0.046). There was a significant group difference in age at the time of the operation (p=0.028). Patients with normal MRI and patients with hydrocephalus were older at the time of the operation than patients with PVL-for the patients with normal MRI this difference was significant (p=0.044).
The scoring of the MRI abnormalities in the patients with PVL is summarized in. Two patients with PVL did not show ventricular enlargement, and eight patients had moderate ventricular enlargement. All patients had moderate white matter signal intensity. Nine patients had moderate occipital white matter loss, and one patient had moderate frontal white matter loss. Small cysts were found in one patient, and large cysts were found in two. Four patients had no thinning of the corpus callosum, and six patients had moderate thinning of the corpus callosum. None of the patients had gray matter abnormalities or an enlargement of the subarachnoidal space. The mean total score was 10.8 (SD, 1.1; range, 8 to 12). The total score did not correlate with any of the outcome parameters. The ventricular size showed a significant correlation with the preoperative GMFM-66 score (rho, −0.696; p=0.025). However, none of the items in the scoring correlated with the postoperative changes of the GMFM-66.
# Discussion
The aim of this study was to identify possible relationships between MR characteristics and the level of functioning before and after SDR in patients with bilateral spastic paresis. We classified the MRIs in three MRI categories: PVL, hydrocephalus, and normal MRI and compared the postoperative improvement in the GMFM in the three groups. The best results were found in the group with normal MRI, and the poorest results were found in patients with hydrocephalus. The poor outcome of patients with hydrocephalus could be a consequence of persistently elevated intracranial pressure, which can be observed in the absence of characteristic symptoms in children with CP [bib_ref] Occult hydrocephalus in children with cerebral palsy, Albright [/bib_ref]. In the present study, both patients with hydrocephalus (patient 11 and patient 12) underwent surgical correction. Patient 11 had a ventriculo peritoneal shunt placed early during childhood. Patient 12 was diagnosed with hydrocephalus a few months before SDR, and he underwent third ventriculostomy. Unfortunately, there was no follow-up imaging available for either of these patients, and although neither of them showed any clinical evidence of elevated intracranial pressure, it cannot be ruled out definitively. Previous studies found greater postoperative improvements after SDR in children with less motor impairment [bib_ref] Long-term outcomes five years after selective dorsal rhizotomy, Nordmark [/bib_ref] [bib_ref] Longterm functional outcome after selective posterior rhizotomy, Mittal [/bib_ref].
In contrast, in the three randomized controlled trials comparing functional outcome after SDR, the poorest results were found in the study which included children with the best preoperative gross motor skills [bib_ref] Selective dorsal rhizotomy: efficacy and safety in an investigator-masked randomized clinical trial, Mclaughlin [/bib_ref] [bib_ref] Selective dorsal rhizotomy: meta-analysis of three randomized controlled trials, Mclaughlin [/bib_ref]. In the present study, the patients with normal MRI had much better preoperative gross motor skills than the patients with PVL and hydrocephalus. We found no correlation between the preoperative GMFM score and its improvement postoperatively. McLaughlin et al. found an inverse correlation between the age at the time of the operation and the postoperative changes in the GMFM [bib_ref] Selective dorsal rhizotomy: efficacy and safety in an investigator-masked randomized clinical trial, Mclaughlin [/bib_ref]. This could be explained as a consequence of faster spontaneous motor development in early childhood. However, in our study, not only the children with hydrocephalus (who showed the poorest outcome), but also the children with normal MRI (who showed the best outcome) were older than the patients with PVL when SDR was performed. There was no correlation between the age at the time of SDR and outcome.
In the patients with PVL, we graded the severity of the MR abnormalities and correlated them with the gross motor abilities. For this purpose, we used a grading scale that has previously been used to describe MR abnormalities in patients with spastic CP [bib_ref] MRI findings and sensorimotor development in infants with bilateral spastic cerebral palsy, Cioni [/bib_ref] [bib_ref] Spastic cerebral palsy: clinical magnetic resonance imaging correlation of 129 children, Kułak [/bib_ref]. We performed a reliability analysis of the various items included in this grading scale. The agreement between investigators was substantially worse than in the Cioni et al. study in which two experienced raters scored the MRIs [bib_ref] MRI findings and sensorimotor development in infants with bilateral spastic cerebral palsy, Cioni [/bib_ref]. In the present study, the interobserver reliability was notably poor for the assessment of the cysts. Several explanations should be considered: it was difficult to detect cysts in an area with extensive gliosis, there is a difference between the level of experience with brain MRI in the two investigators (2 years vs 8 years). Only the amount of ventricular enlargement correlated with the preoperative gross motor abilities. Correlations between ventricular size and gross motor abilities have been described previously in patients with spastic CP [bib_ref] Periventricular leukomalacia: relationship between lateral ventricular volume on brain MR images and..., Melhem [/bib_ref]. In contrast to the findings of previous studies [bib_ref] MRI findings and sensorimotor development in infants with bilateral spastic cerebral palsy, Cioni [/bib_ref] [bib_ref] Spastic cerebral palsy: clinical magnetic resonance imaging correlation of 129 children, Kułak [/bib_ref] [bib_ref] Corpus callosum size in children with spastic cerebral palsy: relationship to clinical..., Kulak [/bib_ref] , we did not find a correlation between gross motor skills and the total MRI score/the thinning of the corpus callosum. However, the present study was limited to ambulatory patients with GMFCS levels I to III, and patients with more severe motor handicaps where more severe brain abnormalities could be expected on MRI have not been studied. No correlations were found between the severity of the brain anomalies and the outcome after SDR.
The outcome assessment in the present study consisted in the GMFM-66 which-according to the International Classification of Functioning, Disability, and Healths (ICF)-assesses the domain of activity. However, the other two domains of the ICF-body structure and participationhave not been evaluated. One other limitation of the present study was the small sample size. Unfortunately, we did not have access to all MRI scans and not all patients had a preoperative GMFM assessment. We could only include data on a small number of patients in our analysis. Notably, the group with hydrocephalus was small and comprised only two patients. Therefore, the association between the type of brain lesion and the outcome after SDR needs to be proved in future studies with larger study samples, including the other ICF domains-body structure and participation.
# Conclusion
We found significant differences in the post-operative changes in the GMFM-66 in patients with different brain MRI abnormalities. The largest postoperative improvement was observed in patients with normal MRI, and the poorest outcome was observed in patients with hydrocephalus. In patients with PVL, we could not detect any relation between MRI abnormalities and the postoperative improvement in of gross motor function after SDR. We conclude that MRI of the brain can provide additional information for the selection of patients for SDR. However, the degree of PVL does not provide information about the degree of improvement in gross motor function after SDR.
[fig] Figure 1 a: -i MR imaging of three patients with bilateral spastic paresis undergoing SDR. a, d, g Midsagittal T1 weighted images. b, e, h Transversal T2 weighted images at the level of the centrum semiovale. g, h, i Transversal T2 weighted imaging at the level of the basal ganglia. a-c MR images classified as "normal." d-f MR images classified as periventricular leucomalacia showing thinning of the corpus callosum involving the total body [/fig]
[fig] Figure 2: Box plots of the preoperative GMFM-66 (white boxes) and the mean values of all postoperative GMFM- [/fig]
[table] Table 2: Summary of the characteristics of the patients included in the study F female, M male, GA gestational age, BW birth weight, SDR selective dorsal rhizotomy, GMFCS gross motor function classification system, GMFM gross motor function measure, PVL periventricular leucomalacia, g gram, mts months [/table]
[table] Table 3: Differences of gross motor outcome in with patients with different MRI classification [/table]
[table] 66: measurements (dashed boxes) in patients with different MRI classification. Note that the largest improvements after SDR were observed in patients with normal MRI and that the patients with hydrocephalus did not improve after SDR [/table]
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Aberrant caspase-activated DNase (CAD) transcripts in human hepatoma cells
The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.
Apoptosis is a programmed cell death process that removes toxic or useless cells during mammalian development [bib_ref] Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Wyllie [/bib_ref]. The biological hallmark of apoptosis is the cleavage of chromosomal DNA into nucleosomal fragments [bib_ref] Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Wyllie [/bib_ref]. Human caspase-activated DNase (CAD, also called DFF40/CPAN) is the key enzyme for nucleosome cleavage during cell apoptosis [bib_ref] DFF, a heterodimeic protein that functions downstream of caspase-3 to trigger DNA..., Liu [/bib_ref] [bib_ref] caspase-activated DNase that degrades DNA during apoptosis and its inhibitor ICAD, Enari [/bib_ref] [bib_ref] CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45, Halenbeck [/bib_ref] [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. CAD contains two different functional domains: the Nterminal regulatory domain (CIDE-N or CAD domain) and the Cterminal catalytic domain. The CAD domain shares sequence homology with the N-terminal of the inhibitor of CAD (ICAD) [bib_ref] DFF, a heterodimeic protein that functions downstream of caspase-3 to trigger DNA..., Liu [/bib_ref] [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. Structural and mutational analyses of the CAD domains revealed that they account for the ability of CAD and ICAD to form a heterodimer [bib_ref] Identification of regulatory and catalytic domains in the apoptosis nuclease DFF40/CAD, Inohara [/bib_ref] [bib_ref] Solution structure of the CIDE-N domain of CIDE-B and a model for..., Lugovskoy [/bib_ref] [bib_ref] Structure of the heterodimeric complex between CAD domains of CAD and ICAD, Otomo [/bib_ref] [bib_ref] Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone..., Zhou [/bib_ref]. ICAD inhibits CAD activity by binding to CAD. During the apoptotic process, CAD is released from the ICAD/CAD complex via proteocleavage of the ICAD by caspase 3 or caspase 7. CAD then degrades chromosomal DNA. Moreover, ICAD also works as a chaperon to help the correct folding of the CAD protein [bib_ref] Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA..., Zhang [/bib_ref] [bib_ref] Functional differences of two forms of the inhibitor of caspase-activated DNase, ICAD-L,..., Sakahira [/bib_ref]. The active CAD can be synthesised only in the presence of ICAD both in vitro and in vivo. The CAD synthesised in vitro without ICAD does not have DNase activity at all. Inactivation of CAD has been shown to render cells resistant to undergoing apoptosis and may cause cell transformation. Recently, human CAD gene was mapped at chromosome 1p36.3 [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. Human chromosome 1p36 is frequently associated with hemizygous deletions in several types of cancers, particularly in hepatocellular carcinoma (HCC), indicating the presence of one or more tumour suppressor genes in the region [bib_ref] Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma, Buetow [/bib_ref] [bib_ref] Abnormalities of chromosome 1 and loss of heterozygosity on 1p in primary..., Simon [/bib_ref] [bib_ref] Frequent genetic alternations at the distal region of chromosome 1p inhuman hepatocellular..., Yeh [/bib_ref]. It is, therefore, tempting to speculate that CAD might be one of the tumour suppressors for hepatocellular carcinogenesis. To address this hypothesis, we examined the CAD transcripts in human HCC cells.
# Materials and methods
## Hepatoma cell lines, liver tissue and peripheral leukocytes
Human hepatoma cell lines, HepG2, Hep3B, Huh7, Sk-Hep1 [bib_ref] One hundred and twenty-seven cultured human tumor cell lines producing tumors in..., Fogh [/bib_ref] [bib_ref] Regulation of HIV-1 LTR transactivating activities in two different human hepatocellular carcinoma..., Chang [/bib_ref] , and Mahlavu were obtained from American Type Culture Collection (Manassas, VA, USA), while J328 was kindly provided by Dr CS Yang . All media were supplemented with 10% (v/v) foetal bovine serum and 2 mM L-glutamine. One non-HCC liver sample, obtained from a patient who received liver resection on account of a benign focal nodular hyperplasia of the liver, and peripheral blood leukocyte (PBL) samples obtained from three healthy individuals were included in the study as non-HCC controls. The liver sample was frozen immediately in liquid nitrogen after resection and stored at À801C until processing. The Ethics and Science Committee of Chang Gung Memorial Hospital approved specimen collection procedures and informed consent was obtained from each subject or subject's guardian.
## Rna preparation and rt -pcr
Total cellular RNA was extracted from HCC cell lines, liver tissue and peripheral blood leukocytes using the single-step acid -phenol extraction method as described before [bib_ref] Genome-wide hypomethylation in hepatocellular carcinogenesis, Lin [/bib_ref]. Total RNA (2 mg) was converted to cDNA via a random-primer method as described previously [bib_ref] Cloning and characterization of the extreme 5'-terminal sequences of the RNA genomes..., Hsieh [/bib_ref]. The whole reading-frame of the CAD gene was cloned using nested PCR . In brief, 1/50th of the cDNA was subjected to amplification for the first round of PCR in a total volume of 50 ml, which contained 4 pmol of each primer, 200 nM of each dNTP, and 2 U of Taq DNA polymerase in a buffer containing 40 mM Tricine-KOH (pH 8.7), 3.5 mM MgCl 2 , 15mM KOAc, 0.005% Tween-20 and 0.005% Nonidet-P40 (Advantage 2 PCR kit, CLONTECH, Palo Alto, CA, USA). Amplification was performed using 30 cycles of 951C for 30 s, 651C for 4 min. The first-round PCR products were diluted 1/50 and 1 ml of the dilute was subjected to the second round of PCR with the inner primer pairs using conditions similar to that of the first-round PCR. After gel-electrophoresis analysis, the second-round PCR products were directly cloned using a PCR-TA cloning kit (pCRII TOPO, Invitrogen, Carlsbad, CA, USA). At least 10 clones derived from each HCC cell line and the liver sample, and at least five clones from each peripheral blood leukocyte samples were randomly selected for sequencing analysis. Sequencing analysis, using the two primers encoded in the vector and the two CAD-specific primers as illustrated in , was conducted as described before [bib_ref] Identity of a novel swine hepatitis E virus in Taiwan forming a..., Hsieh [/bib_ref].
The primer sequences for the nested PCR were as follows:
First-round PCR: 5 0 -CCCAGAGGGCTTGAGGACATCTGCAA-3 0 5 0 -ACCAGGACGTGGTGTGTACGTGTCA-3 0 Second-round PCR: 5 0 -GAGGACATCTGCAATGCTCCAGAAGC-3 0 5 0 -TCACTGGCGTTTCCGCACAGGCTG-3 0
The sequences of the primers for sequencing are:
T7 primer: 5 0 -GTAATACGACTCGCTATAGGG-3 0 SP6 primer: 5 0 -TATTTAGGTGACACTATAG-3 0 CAD sense: 5 0 -GTGGTTTGAACGCTTGCAGTCCCCGA-3 0 CAD antisense: 5 0 -GACCGGAGCCTCTGGCACGTGGA-3 0
The nucleotide numbering used in this report is according to the numbering system by [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref] (GenBank accession numer: AB013918).
## Uv irradiation and dna fragmentation
At 24 h after plating (2 Â 10 6 /100 mm dish), cells were irradiated with 50 -200 mJ cm À2 of UV and harvested for DNA fragmentation at 60, 90, 120, 150, 180 and 210 min thereafter, respectively. PBSwashed cells were suspended in DNA extraction buffer containing 50 mM Tris (pH 8.0), 10 mM EDTA, 0.5% N-laurosarcosine and 1 mg ml À1 proteinase K, and incubated for 1 h at 501C in a water bath. Then DNase-free RNase was added at a final concentration of 250 mg ml À1 , at 501C for another hour followed by ethanol precipitation of DNA. The DNA pellets were resuspended in TE buffer (10 mM Tris, pH 8.0/1 mM EDTA). DNA (1 mg) was end labelled with a 32 P-dATP in a solution containing 1 mg DNA, 0.5 mCi a 32 P-dATP, 5 U Klenow polymerase, 10 mM Tris, pH 7.5, 5 mM MgCl 2 for 30 min at room temperature. The reaction was terminated by adding an equal volume of 10 mM EDTA. The labelled DNA was purified via a Sephadex G-50 spin-column (Pharmacia Biotech, Quarry Bay, Hong Kong), and analysed by electrophoresis on a 1% agarose gel and then by autoradiography. For quantification of DNA fragmentation upon UV-induced apoptosis in each HCC cell line, we used a cellular DNA fragmentation ELISA kit in accordance with the manufacturer's instruction (Roche Diagnostics GmbH, Mannheim, Germany). In brief, cells were metabolically labelled with 10 mM of thymidine analogue BrdU for overnight. At 4 h after UV irradiation with 0, 100 or 200 mJ cm À2 , cell membrane was lysed for 30 min at 201C in a solution containing 10 mM EDTA and 0.1% Tween-20. Following centrifugation for 10 min at 2500 g to remove cellular debris and nucleus component, the cytoplasmic lysate was subjected to an ELISA to detect fragmented DNA in cytoplasm.
# Results
To study abnormalities in the CAD transcripts in HCC, we reverse transcribed mRNA and amplified the complete CAD coding sequences by nested PCR . As illustrated in [fig_ref] Figure 2: Expression of the CAD gene by nested RT-PCR [/fig_ref] , the 1-kb PCR products, which represented the full-length CAD transcripts, were detected in all the examined samples. Small PCR products, about 0.34 kb, representing sub-full-length CAD transcripts were found in the non-HCC liver sample and in PBL from the three normal individuals [fig_ref] Figure 2: Expression of the CAD gene by nested RT-PCR [/fig_ref] , whereas 0.45-kb and 0.55-kb PCR products for sub-full-length CAD transcripts were detected in Mahlavu and SK-Hep1 cells, respectively [fig_ref] Figure 2: Expression of the CAD gene by nested RT-PCR [/fig_ref] , lanes 5 and 6). The PCR products were directly cloned. At least 10 clones derived from each hepatoma cell line and non-HCC liver sample and at least five clones derived from each PBL sample were randomly selected for sequencing analysis. The sequencing results have been deposited in the GenBank and are summarised in [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref].
The prototype transcripts were found in HepG2, Huh7 cells, non-HCC liver sample and the three PBL samples [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref]. The full-length transcripts with single-nucleotide polymorphism (SNP) without change of amino acid were identified in J328 cells (wild type, in terms of amino-acid sequences). In addition, the fulllength transcripts with SNP leading to amino-acid changes (missense mutations) were noted in all of the 6 HCC cell lines [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref]. All these mis-sense mutations corresponding to the CAD readingframe are illustrated in . No hotspot for these mis-sense mutations in CAD was found, although most of them were located in the catalytic domain of CAD.
Sub-full-length transcripts were found in all of the six hepatoma cell lines [fig_ref] Figure 4: Scheme of the aberrant CAD transcripts and their predicted proteins [/fig_ref] , even though sub-full-length transcripts were detected by gel electrophoresis only in Mahlavu and SK-Hep1 among the six examined HCC cell lines. In HepG2 cells, a minor sub-full-length transcript (one out of the 10 selected clones) with a deletion from nt 367 to nt 702 was identified. Of note, a fournucleotide identity, CTGC, was noted exactly at both of the 5 0 -and 3 0 -deletion ends (nucleotides 367 -370 and 703 -706), suggesting Schematic representation of the strategies for cloning and sequencing of the CAD cDNA. The CAD reading frame was cloned using nested RT -PCR method with two rounds of PCR following reverse transcription. Two primer pairs for amplification of the nested PCR are shown (arrows in both ends of the CAD reading frame). After examination with gel electrophoresis, the PCR products were cloned directly and then subjected to sequencing analysis using two vector primers (stippled arrows) flanking the cloning site and two CAD-specific primers (stippled arrows inside the reading frame).
involvement of a recombination deletion. In Hep3B cells, an aberrant CAD transcript (three out of the 10 clones) corresponded to replacement of exon 3 with a 121-base sequence, which was homologous to part of the human transposon, Alu repeat. In both Huh7 and J328 cells, there were minor small transcripts (two and one out of the 10 clones, respectively) with the absence of exons 2 -6 resulting in conjunction of exon 1 to exon 7, suggestive of transcripts derived from an alternative splicing process. Similar alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were also noted in the non-HCC liver tissue and the three PBL samples as well [fig_ref] Figure 2: Expression of the CAD gene by nested RT-PCR [/fig_ref] and [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref]. In Mahlavu and SK-Hep1 cells, relatively abundant alternatively spliced transcripts (three and seven out of the 10 clones, respectively) coincided with the absence of exons 3 to 6, or to 5 leading to fusion of exon 2 to exon 7 or 6, respectively [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref] and [fig_ref] Figure 4: Scheme of the aberrant CAD transcripts and their predicted proteins [/fig_ref]. The predicted amino-acid sequences for these aberrant transcripts are aligned to that of the prototype CAD and shown in [fig_ref] Figure 4: Scheme of the aberrant CAD transcripts and their predicted proteins [/fig_ref]. Of note, the amino-acid sequences for CAD domain (aa 1 -80) were preserved in the aberrant CAD transcripts identified in HepG2, Hep3B, Mahlavu and SK-Hep1 cells.
To inspect the nucleosome cleavage activity of CAD during apoptosis in these HCC cell lines, DNA fragmentation assay was conducted after UV induction of apoptosis. As shown in , the relative efficiency of DNA fragmentation upon UV irradiation in HepG2, Hep3B, Huh7, J328 and Mahlavu cells was 0.56-, 1.26-, 1.60-, 1.81-and 0.87-fold, respectively, as compared to that in HepG2
[formula] ' ' ' ' ' ' ' Prototype * ' ' ' * ' ' G33S/S225 ' ' | | ' n367-702 Hep3B ' ' ' ' * ' ' R196K ' ' A ' ' ' ' Alu repeat replacement Huh7 ' ' ' ' ' ' ' Prototype ' * ' ' ' ' ' L89P ' ' Exons 1-7 J328 ' ' ' ' ' ' ' SNP ACG177ACA ' ' * * ' ' ' E110Q/Q150R ' ' Exons 1-7 Mahlavu ' ' ' ' * ' ' V171A ' ' ' Exons 1-2-7 SK-Hep1 ' ' ' ' * ' * E184L/K264T ' ' ' ' Exons 1-6-7 Non-HCC liver ' ' ' ' ' ' ' Prototype ' ' Exons 1-7 PBL ' ' ' ' ' ' ' Prototype ' ' ' ' ' ' Skipping exon 3 ' ' Exons 1-3 PBL ' ' ' ' ' ' ' Prototype ' ' ' ' ' ' Skipping exon 3 ' ' Exons 1-7 PBL ' ' ' ' ' ' ' Prototype ' ' [/formula]
Exons 1-7 *: mis-sense mutation; A: the Alu repeat; n: deletion with CTGC repeats at both ends; 1: incomplete exon sequences.
# Discussion
Herein we report the identity of aberrant CAD transcripts in human HCC cells. Two classes of human CAD cDNA have been cloned by [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. One encompasses the entire coding region, whereas the other contains many deletions, insertions and point mutations in the corresponding coding region. The latter is thought to be derived from a pseudogene [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. Obviously, the subfull-length CAD cDNAs reported herein were not derived from pseudogenes. Instead, most of them were more likely generated via aberrant RNA processing mechanisms. The minor CAD transcript (representing one out of the 10 isolated CAD cDNA clones) in HepG2 cells with a deletion from nucleotides 367 to 702 possibly resulted from a recombination processing, because of a four-nucleotide identity, CTGC, noted exactly in both the deletion ends (nucleotides 367 -370 and 702 -705, respectively). Two different species of full-length CAD transcripts, a prototype and a transcript with mis-sense mutations, were identified, suggesting that the recombination occurs more Scheme of mapping the mis-sense mutations to the corresponding CAD reading frame. Amino acids 1 -80 is the CIDE-N or CAD domain that directs interaction with ICAD; the C-terminal is the catalytic domain containing nuclease activity. [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. The box labelled with 'Alu' in Hep3B cells represents a sequence homologous to a human Alu repeat. (B) Alignment of the predicted amino-acid sequences for the aberrant CAD transcripts to the prototype CAD amino-acid sequences [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref]. Note that CAD domain (aa 1 -80) is preserved in the aberrant transcripts from HepG2, Hep3B, Mahlavu and SK-Hep1 cells.
## Prototype
likely during mRNA processing than at the chromosomal DNA level [fig_ref] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue... [/fig_ref]. The sub-full-length CAD transcript (representing three out of the 10 isolated CAD cDNA clones) corresponding to replacement of exon 3 with a truncated human transposon, Alu repeat, was isolated in Hep3B cells. Alu elements are a specific human family of interspersed repetitive sequences. It was estimated to be over 1 000 000 copies per genome. Their dispersion and plethora is attributable to transposition via a mechanism that has not been fully understood yet [bib_ref] Reverse transcriptase encoded by a human transposable element, Mathias [/bib_ref]. It was likely that the CAD transcript with replacement of exon 3 with the Alu element reported herein was generated via an Alu-mediated genomic mutation mechanism. One possible mechanism is that a de novo Alu-insertion mutation occurs in one of the two alleles of the CAD gene. In theory, mobilization of Alu elements requires a cellular source of reverse transcriptase that is generally believed to be provided by a concurrently activated retrotransposon, such as LINE-1 elements [bib_ref] Reverse transcriptase encoded by a human transposable element, Mathias [/bib_ref]. Indeed, global DNA demethylation has been generally found in human cancers including HCC [bib_ref] Genome-wide hypomethylation in hepatocellular carcinogenesis, Lin [/bib_ref] , which in turn leads to activation of transposable elements, such as LINE-1 and Alu repeats. It is, therefore, conceivable to speculate that mutations secondary to transposition of transposons in human cancers may not be rare events. Indeed, de novo Alu insertion leading to human diseases, particularly in tumours, has been reported before [bib_ref] Mobile elements and disease, Kazazian [/bib_ref]. For examples, the insertions cause heamophilia B (factor IX gene) [bib_ref] Hemophilia B due to a de novo insertion of a human-specific Alu..., Vidaud [/bib_ref] , neurofibromatosis (NF-1 gene) [bib_ref] A de novo Alu insertion results in neurofibromatosis type 1, Walllace [/bib_ref] , Apert syndrome (FGFR2 gene), acholinesterasemia (ChE gene) [bib_ref] Inactivation of the cholinesterase gene by Alu insertion: possible mechanism for human..., Muratani [/bib_ref] , desmoid tumors (APC gene) and breast cancer (see the review by [bib_ref] Mobile elements and disease, Kazazian [/bib_ref]. Alternatively, this Alu-containing CAD transcript might result from an Alu/Alu homologous recombination/deletion, which led to exon 3 deletion and activation of a cryptic splicing acceptor site in Alu elements. Experiments to elucidate the roles of Alu-elements in genomic mutations for the CAD gene are in progress.
On the other hand, the sub-full-length CAD transcripts found in Huh7 and J328 cells exhibited loss of exons 2 -6 and resulted in fusion of exons 1 and 7. Since the fusion junctions just coincided with the splice sites, they must be generated via alternative splicing processes. Similar alternative spliced transcripts with fusion of exons 1 -7 were also found in the liver tissue from a non-HCC subject and in the PBL from three healthy individuals. The CAD reading frame encoded by these small transcripts was so greatly disrupted that they might not have any biological significance.
By contrast, the sub-full-length CAD transcripts found in poorly differentiated HCC cells, Mahlavu and SK-Hep1, corresponded to the absence of exons 3 to 6 or 5 and created a junction between exon 2 to exon 7 or 6, respectively. Apparently, they were generated via aberrant splicing mechanisms. These transcripts are of great interest because not only were they cancer specific (only found in poorly differentiated Mahlavu and SK-Hep1 cells) and relatively abundant (30 and 70% of the isolated CAD cDNA clones, respectively), but they also preserved the CAD domain (aa 1 -80) directing CAD interaction with its inhibitor (ICAD) [bib_ref] Molecular cloning and characterization of human caspaseactivated DNase, Mukae [/bib_ref] [bib_ref] Structure of the heterodimeric complex between CAD domains of CAD and ICAD, Otomo [/bib_ref]. Since ICAD functions as a chaperon for the regulation of CAD activity [bib_ref] Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis, Sakahira [/bib_ref] [bib_ref] Specific chaperone-like activity of inhibitor of caspase-activated DNase for caspase-activated DNase, Sakahira [/bib_ref] , these predicted aberrant CAD products might sequester ICAD from interacting with the wild-type CAD, thereby preventing the correct folding of CAD and subsequently blocking the nuclease activity of the wild-type CAD in the cells. Interference of the CAD activity renders cells more resistant to cell death, and in turn may award cells with advantages for survival and, possibly, for carcinogenesis . It is, therefore, intriguing to speculate that these aberrant CAD products encoded by the aberrant CAD transcripts in SK-Hep1 and Mahlavu cells (and those in HepG2 and Hep3B cells, [fig_ref] Figure 4: Scheme of the aberrant CAD transcripts and their predicted proteins [/fig_ref] and B) play significant roles in cell apoptosis and in hepatocarcinogenesis. Indeed, in the experiments for DNA laddering under UVinduced apoptosis, the phenomenon of DNA laddering was remarkably impaired in Mahlavu and SK-Hep1 cells, and partially impaired in HepG2 and Hep3B cells, as compared to that in HeLa, Huh7 and J328 cells. Studies to further dissect the effects of these C 0 -truncated CAD on nucleosome cleavage activities of the wildtype CAD are currently ongoing.
Alternative splicing has been regarded as an important mechanism that contributes to genetic diversity by generating multiple protein isomers from a single gene [bib_ref] ISIS, the intron information system, reveals the high frequency of alternative splicing..., Croft [/bib_ref]. However, alternative splicing can also lead to human disease [bib_ref] Exonic splicing enhancers: mechanism of action, diversity and role in human genetic..., Blencowe [/bib_ref] [bib_ref] RNA processing and human disease, Philips [/bib_ref]. Alterations in alternative splicing of certain pre-mRNA have recently been correlated to either neoplastic transformation and/or acquisition of metastatic potential [bib_ref] Alternative splicing in fibroblast growth factor receptor 2 is associated with induced..., Savagner [/bib_ref] [bib_ref] To metastasize or not? Selection of CD44 splice sites, Cooper [/bib_ref] [bib_ref] Androgen receptor mutations in prostate cancer, Barrack [/bib_ref] [bib_ref] Estrogen receptor mRNA variants. Do they have a physiological role?, Pfeffer [/bib_ref] [bib_ref] RNA processing and clinical variability in neurofibromatosis type 1 (NF1), Skuse [/bib_ref] [bib_ref] Genetic and epigenetic alterations of the estrogen receptor gene and hormone independence..., Iwase [/bib_ref] [bib_ref] RNA processing and human disease, Philips [/bib_ref]. Most of these altered splicing patterns are because of either changes in trans-acting factors or mutations within the canonical sequences at the intro/exon border that are required for splicing. However, mutations in an auxiliary element required for pre-mRNA splicing within exons were found more recently. It was reported that in human neuroblastomas, ataxia telangiectasia and breast cancer some nonsense mutations caused skipping of exons, a phenomenon known as nonsense-associated altered splicing [bib_ref] Slicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences, Teraoka [/bib_ref] [bib_ref] Mutations affecting mRNA splicing are the most common molecular defects in patients..., Ars [/bib_ref] [bib_ref] Pre-mRNA splicing in the new millennium, Hastings [/bib_ref] [bib_ref] A mechanism for exon skipping caused by nonsense or missense mutations in..., Liu [/bib_ref]. The latter was attributable to result from non-sense mutations in exon leading to disrupting a splicing enhancer in exon, which contains recognition sequences for splicing factors, such as SF2/ASF [bib_ref] Identification of a new class of exonic splicing enhancers by in vivo..., Coulter [/bib_ref] [bib_ref] Pre-mRNA splicing in the new millennium, Hastings [/bib_ref] [bib_ref] A mechanism for exon skipping caused by nonsense or missense mutations in..., Liu [/bib_ref]. The exon-skipping phenotype has been reproduced in vitro and was shown not to result from disruption of the translational reading frame [bib_ref] Exonic splicing enhancers: mechanism of action, diversity and role in human genetic..., Blencowe [/bib_ref]. The canonical sequences at the intron-2/exon-3 junction and the coding sequences in exon 3 for the CAD genes in Hep3B, Mahlavu and SK-Hep1 cells have been examined. However, neither mutations to disrupt the intro/exon sequence nor non-sense mutations in exon 3 was found (data not shown). Further studies to elucidate the underlying molecular mechanism ELISA for cellular DNA fragmentation. After 0, 100 or 200 mJ cm À2 of UV irradiation, cytoplasmic lysate of cells containing DNA metabolically prelabelled with BrdU was subjected to an ELISA with anti-DNA antibody binding to fragmented DNA in cytoplasm followed by quantification with a peroxidase-conjugated anti-BrdU antibody (Roche Diagnostics GmbH, Mannheim, Germany). The representative results were derived from experiments conducted in a manner of duplication for each sample in every assay in a total of three assays. The averages and ranges of the ratio of DNA fragmentation before vs after UV irradiation for each cell line are illustrated. The relative amount of DNA fragmentation for each HCC cell line was determined via comparison to that for HeLa cells.
for altered alternative splicing for the CAD gene in HCC are warranted.
In summary, we report the identity of aberrant transcripts and frequent mutations for the CAD gene in human HCC cells, suggesting a significant role of CAD in human hepatocarcinogenesis.
[fig] Figure 2: Expression of the CAD gene by nested RT-PCR. After reverse transcription, the samples derived from six HCC cell lines were subjected to two rounds of PCR as described in the legend ofFigure 1. (A) Lanes 1 -8 represent the size marker, the PCR products derived from HepG2, Hep3B, Huh7, J328, Mahlavu, SK-Hep1 cells and the non-HCC liver sample, respectively. Of note, HepG2 and Hep3B are well-differentiated HCC cells, Huh7 and J328 are moderately differentiated HCC cells, and Mahlavu and SK-Hep1 are poorly differentiated HCC cells. (B) Lanes 1 -5 represent the PCR products derived from the non-HCC liver sample and from the three PBL samples and a negative control for PCR respectively. [/fig]
[fig] Figure 4: Scheme of the aberrant CAD transcripts and their predicted proteins. (A) The coding exons 1 -7 are demonstrated as open squares. The heavy lines in the transcripts in HepG2 cells represent a four-nucleotide identity at the two skipping ends (nucleotides 367 -370 and 702 -705, in accordance with the numbering system by [/fig]
[table] Table 1: Variants of CAD cDNA in human HCC cell lines, non-HCC liver tissue and PBL [/table]
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A Narrative Review of the Launch and the Deployment of Telemedicine in Italy during the COVID-19 Pandemic
# Introduction
Telemedicine as a diagnostic, monitoring, and rehabilitation treatment tool is showing great potential during the pandemic, as highlighted by Bahsnur et al.. We believe that the current spread of telemedicine, compared with previous pandemic situations, can be explained by the simultaneous occurrence of unprecedented conditions of technological availability and exceptional medical circumstances. In fact, one aspect that drove this boom was the vastness of the pandemic, the most terrible of the past century; however, the real engine of the boom of telemedicine during the epidemic for the SARS-CoV-2 virus of 2019, has been mobile technology based on smartphones; these are capable of hosting telemedicine applications, for example, based on wearable sensors, which in the past needed proper technological solutions [bib_ref] Wearable sensors/systems and their impact on biomedical engineering, Bonato [/bib_ref]. During each wave of the pandemic's evolution, telemedicine has shown exponential development. In reference to the Italian situation, despite the available innovative solutions, cultural barriers and organization limits did not allow an easy introduction of these solutions to the health domain. The introduction happened with lights and shadows [bib_ref] Instant paper from the field" on rehabilitation answers to the COVID-19 emergency, Negrini [/bib_ref]. We can identify two periods of maturation of the use of telemedicine during the pandemic. During the first period, around the first lockdown
## -
The drafting of national guidelinesfor the provision of telemedicine services by the Ministry of Health, to avoid the patchy use of telemedicine and make telemedicine health services officially recognized health services that will have the same value as those in existence. It is understood that the doctor will always decide whether to use them or not. - Regional initiatives aimed at the standardization and homologation of telemedicine services such as the one of the Lazio region, as a non-exhaustive example.
## Purpose of the study
The purpose of the study was to address the launch and deployment of telemedicine in Italy through a narrative review, addressing both the promotion initiatives at national level and the experiences of application also at the level of acceptance. All of this facilitates both answering the important question of "how telemedicine started in Italy during the pandemic and how it is going", and making a point about the use of telemedicine in Italy.
# Methods
This narrative review faced two points of view: (a) the first was aimed at analysing institutional government initiatives designed to promote the use of telemedicine and to raise awareness among stakeholders; (b) the second was aimed at analysing the evolution of scientific literature in the sector, through a Pubmed overview, with reference to the Italian situation. We followed both a narrative checklist and an eligibility approach, based on a scoring system (with different parameters and a score with five levels) applied by two qualified experts, to include each reference found in the second point of view.
We followed the narrative checklist reported in. The manuscript was developed in accordance with this checklist, which requires compliance with some qualifying points in the development of the document, starting from the title and ending with the conclusions. [fig_ref] Table 1: Parameters used for the eligibility [/fig_ref] shows the parameters used for the qualification process applied, before inclusion, on the references found in the second point of view.
We assigned a score to these parameters, ranging from a minimum score of one (very poor) up to a maximum of five (outstanding). As far as the "added contribution to the field" parameter is concerned, we used weighing.
Both to consider the criticality of the first phases of the pandemic and to relativize the reference to the importance of the first period, the assigned vote was multiplied by:
## -
A factor × 1.3 (for studies published in the first three months of the pandemic).
- A factor × 1.15 (for studies published in the period ranging from 1 to 6 months of the pandemic). The study was excluded if, regardless of the score, there were critical issues of conflict of interest (for example, if it was conducted without guarantees of objectivity by the system manufacturer). The reference was included in the review if all parameters after weighing showed a score higher than three in AND logic.
The keys reported in [fig_ref] Table 2: The keys applied in the search [/fig_ref] were applied in the second point of view.
# Results
## An overview of italian national recommendations and indications
In Italy, an important role in fighting the pandemic was played by the Istituto Superiore di Sanità (ISS). The ISS defined various working groupswith ISS researchers and experts on the various strategic issues related to the fight against the pandemic. The groups also worked in synergy with each other. Important products from the working groups were the ISS COVID-19 Reports containing guidelines and recommendations for all the insiders in the health domain. The COVID-19 Reports provide essential and urgent information for emergency management and are subject to updates. These reports were produced in the national language] and translated into English or other languagesto share/export the knowledge. We accessed the online archive and focused on content dedicated to telemedicine. [fig_ref] Table 3: ISS reports dealing with the telemedicine [/fig_ref] shows the COVID-19 reports that dealt directly or indirectly with telemedicine. Interim guidelines for the appropriate support of children with adrenal insufficiency also using telemedicine
The first documentprovided support for the realization of services in Telemedicine during a COVID-19 emergency, offering indications, identifying operational problems, and proposing solutions supported by evidence, but that are also easily dispensable in practice. The indications aimed to be used in various combinations to provide health services and psychological support; they also aimed to proactively monitor the health conditions of people in quarantine, in isolation, after discharge from the hospital, or of those who were isolated at home due to the rules of social distancing but were in need of continuity of care, even if they were not COVID-19 infected. The second documentprovided the scientific indications to support the implementation of telemedicine health services for pediatric patients, both in early childhood and in developmental age, and during the different phases of the COVID-19 pandemic. It described how telemedicine can solve operational problems in managing the doctor-patient-family relationship in the pediatric field. It also provided concrete elements for the definition of specific characteristics, and the eligibility and exclusion criteria of the pediatric patient, also affected by rare or common chronic diseases.
The third documentillustrated the results of the development and submission of a survey (in September 2020) proposed by the National Centre for Innovative Technologies in Public Health and the National Centre for Rare Diseases of the ISS; its aim was to investigate the state of use of technologies (also based on telemedicine) by people with frailty, disabilities, and rare diseases. The document was intended to report evidence to stakeholders through the survey tool which played a sensor role. The fourth documentreported guidelines for the appropriate support of children with adrenal insufficiency during the current SARSCoV-2 pandemic emergency. Contact with reference centers to ensure advice from specialists was highly recommended, also using telemedicine systems.
All four of the documentsalso highlighted particular attention to rare diseases and the frailty towards which telemedicine can play an important supporting role.
## An overview of italian national scientific literature production
The eligibility process applied to the selected references, after the elimination of the duplicated ones, returned 39 works, including a review focused on the relationship between telemedicine and radiotherapy [bib_ref] Management of differentiated thyroid cancer through nuclear medicine facilities during COVID-19 emergency:..., Klain [/bib_ref].
A total of 17 studies were published in 2021, and the remaining 22 in 2020. The eligibility process also showed that the selected papers did not show critical issues regarding conflict of interest. [fig_ref] Figure 1: Output from the qualification process [/fig_ref] reports the average scores assigned by the two experts after the weighing process, both for each parameter and averaged for all the parameters. duplicated ones, returned 39 works, including a review focused on the relationship between telemedicine and radiotherapy [bib_ref] Management of differentiated thyroid cancer through nuclear medicine facilities during COVID-19 emergency:..., Klain [/bib_ref].
A total of 17 studies were published in 2021, and the remaining 22 in 2020. The eligibility process also showed that the selected papers did not show critical issues regarding conflict of interest [fig_ref] Figure 1: Output from the qualification process [/fig_ref] reports the average scores assigned by the two experts after the weighing process, both for each parameter and averaged for all the parameters. [fig_ref] Table 4: Cont. [/fig_ref] reports the references of the studies selected in the narrative review using the eligibility process with a summary of their focus.
The studies show:
- An initial disorientation [bib_ref] Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and..., Ferorelli [/bib_ref] in the use of telemedicine, during the implementation period of the guidelines/recommendationsand before the standardization initiatives; -
The scientific reflections regarding telemedicine usefollowing the immediate period after the first Italian national lock-down; -
The differentiated applications; - An expansion of the telemedicine boundaries; -
A high acceptance, as tested through specific questionnaires in some studies. [fig_ref] Table 4: Cont. [/fig_ref] reports the references of the studies selected in the narrative review using the eligibility process with a summary of their focus. [fig_ref] Table 4: Cont. [/fig_ref]. Cited articles with a summary of the focus.
## Cited article brief description of the focus
Caponnetto, V., et al.. The COVID-19 Pandemic as an Opportunity to Improve Health Care Through a Nurse-Coordinated Multidisciplinary Model in a Headache Specialist Center: The Implementation of a Telemedicine Protocol.
The contribution described the implementation of a structured telemedicine protocol during the COVID-19 pandemic. The study performed a quality improvement study in a Headache Specialist Center. A total of 207 telemedicine visits involving 100 patients was performed. Telemedicine-facilitated follow-ups, ensuring multidisciplinary care and high patient satisfaction, justifying its wider adoption in headache care.
Lazzeroni, P. et al. [bib_ref] Improvement in glycaemic control in paediatric and young adult type 1 diabetes..., Lazzeroni [/bib_ref]. Improvement in glycaemic control in paediatric and young adult type 1 diabetes.
The aim of the work was to assess metabolic control before and after lockdown in the cohort of type 1 diabetes patients, followed-up by telemedicine. A total of 139 patients were enrolled. Results showed a global improvement in mean HbA1c, with a stronger result for patients with a previous non-satisfactory control. No worsening of metabolic control was shown for patients.
van Ooijen, L.T., et al. [bib_ref] A trans-national examination of the impact of the COVID-19 pandemic on abortion..., Van Ooijen [/bib_ref]. A trans-national examination of the impact of the COVID-19 pandemic on abortion requests through a telemedicine service.
This contribution is logically connected to the next, giving a transnational overview of the topic.
Brandell, et al. [bib_ref] Telemedicine as an alternative way to access abortion in Italy and characteristics..., Brandell [/bib_ref]. Telemedicine as an alternative way to access abortion in Italy and characteristics of requests during the COVID-19 pandemic.
Induced abortion is legal in Italy, but with restrictions. The online abortion provider Women on Web serves as an alternative way to access abortion. The study highlighted an increase in requests during the COVID-19 pandemic compared with the previous year (12% in the first 9 months). The most common reasons for requesting a telemedicine abortion through WoW were privacy-related (40.9%); however, this shifted to COVID-19-specific (50.3%) reasons during the pandemic.
Scalise, A., et al. [bib_ref] What COVID-19 taught us: New opportunities and pathways from telemedicine and novel..., Scalise [/bib_ref]. What COVID-19 taught us: New opportunities and pathways from telemedicine and novel antiseptics in wound healing.
The aim of this multidisciplinary work was to highlight the importance of a new pathway of wound care with a patient-based therapeutic approach, tailored treatments based on the characteristics of the wound, and fast tracks focused on outpatient management, reserving hospital assessment only for patients with complicated or complex wounds.
## Cited article brief description of the focus
Bizot, A., et al. [bib_ref] Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine..., Bizot [/bib_ref]. Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine visits during the COVID-19 pandemic.
The study examined the satisfaction of 1299 patients with breast cancer who underwent teleconsultations during this period. Standardized questionnaires were electronically proposed. Patients were satisfied with oncology teleconsultations during the COVID-19 pandemic. Teleconsultation may be an acceptable alternative follow-up modality in specific circumstances.
Maietti, E., et al. [bib_ref] The experience of patients with diabetes with the use of telemedicine and..., Maietti [/bib_ref]. The experience of patients with diabetes with the use of telemedicine and teleassistance services during the COVID-19 pandemic in Italy: Factors associated with perceived quality and willingness to continue.
The purpose of this study was to investigate the individual and contextual determinants of the perceived quality of the telemedicine and teleassistance services, and the willingness to continue with them, among patients with diabetes. The study identified several determinants of perceived quality and willingness to continue. These socio-demographic and related factors should be considered in the implementation of care pathways integrating in-person visits with telemedicine.
Tornese, et al. [bib_ref] The effect of the COVID-19 pandemic on telemedicine in pediatric diabetes centers..., Tornese [/bib_ref]. The effect of the COVID-19 pandemic on telemedicine in pediatric diabetes centers in Italy: Results from a longitudinal survey.
The study investigated the increase in the use of telemedicine in two diabetes centers during the evolution of the pandemic. Eighty-two percent of responder centers reported an increase in the use of telemedicine, with tele visits by video calling implemented in over half of the centers. There was a significant increase in the number of centers formally tracking telemedicine use and obtaining reimbursement from the national health service (42% vs. 29% and 62% vs. 32%; p < 0.001, respectively). No reimbursement was provided to centers not using televisits. The study highlighted that telemedicine from a procedure with a lack of traceability has become a new structured reality that may help our pediatric patients beyond this pandemic.
Gallo, G., et al. [bib_ref] E-consensus on telemedicine in colorectal surgery: A RAND/UCLA-modified study, Gallo [/bib_ref]. Telemedicine in Colorectal Surgery Italian Working Group, Grossi U. E-consensus on telemedicine in colorectal surgery: a RAND/UCLA-modified study.
The aim of the study was to reach consensus among experts on the possible applications of telemedicine in colorectal surgery. A panel of experts was defined. The panel voted against the use of telemedicine for a first consultation. Consensus was achieved in all but one statement concerning the cost of a teleconsultation. There was strong agreement on the usefulness of teleconsultation during the follow-up of patients with diverticular disease after an in-person visit.
Pardolesi, A., et al. [bib_ref] Telemedicine for management of patients with lung cancer during COVID-19 in an..., Pardolesi [/bib_ref]. Telemedicine for management of patients with lung cancer during COVID-19 in an Italian cancer institute: SmartDoc Project.
The study reported the outcome of a project on lung cancer monitoring. A total of 83 patients participated in the SmartDoc project and received a teleconsultation. A survey was proposed to the participants. A "complete satisfaction" score (5 out of 5 points) was reported in 70.59% of all the respondents; most patients (76.5%) preferred video-consulting and defined it as better than or comparable to an in-person visit.
Gava, G., et al. [bib_ref] Mental Health and Endocrine Telemedicine Consultations in Transgender Subjects during the COVID-19..., Gava [/bib_ref]. Mental Health and Endocrine Telemedicine Consultations in Transgender Subjects During the COVID-19 Outbreak in Italy: A Cross-Sectional Web-Based Survey.
The study evaluated the impact of the pandemic and the access to health care services during the COVID-19 pandemic on the mental health of transgender people living in Italy. An anonymous web-based survey was conducted among transgender people living in Italy. It highlighted how telemedicine services may serve to mitigate negative psychological effects.
Luzi, L., et al. [bib_ref] Telemedicine and urban diabetes during COVID-19 pandemic in Milano, Italy during lock-down:..., Luzi [/bib_ref]. Telemedicine and urban diabetes during COVID-19 pandemic in Milano, Italy during lock-down: epidemiological and sociodemographic picture.
A pilot study was conducted to assess the feasibility and efficacy of telemonitoring of glucose control in a cohort of diabetic patients. The study demonstrated a reduction in glycated hemoglobin at 3 months follow-up during the lock-down period, indicating glucose monitoring and remote control as a potential methodology for diabetes management. During the lockdown period, 181 in-person and 99 telemedicine consultations were provided by a stoma center. A questionnaire was used to assess the acceptance. Of the 65 patients who completed the questionnaire, 82% indicated being extremely satisfied. The reorganization of stoma care services, including the availability of telemedicine, did not result in a decrease in the number of consultations provided. The results suggest that stoma care services using telemedicine may provide valid support for patients with an ostomy in the future.
Miceli, L., et al. [bib_ref] Through a Telemedicine Co-production and Co-learning Journey, Miceli [/bib_ref]. Doctor@Home: Through a Telemedicine Co-production and Co-learning Journey.
The National Cancer Institute of Aviano, Italy, has recently launched a program called "Doctor @ Home" (D@H). The pillars of the program were described in the contribution.
Predieri, B., et al.. Control Improvement in Italian Children and Adolescents With Type 1 Diabetes Followed Through Telemedicine During Lockdown due to the COVID-19 Pandemic.
Sixty-two children and adolescents with type 1 diabetes were enrolled in a study. Overall, in the children and adolescents, control improved during lockdown. Despite patients being confined to their homes and limited to exercise, the data suggest that the use of real-time measurement of glucose, continuous parental management, and telemedicine can result in beneficial effects.
Checcucci, E., et al. [bib_ref] Implementing telemedicine for the management of benign urologic conditions: A single centre..., Checcucci [/bib_ref]. Uro-technology and SoMe Working Group of the Young Academic Urologists Working Party of the European Association of Urology. Implementing telemedicine for the management of benign urologic conditions: a single centre experience in Italy.
The use of telemedicine with phone-call visits, as a practical tool to follow-up with patients affected by urological benign diseases, was investigated on 607 patients. Telemedicine was shown to limit the number of instances of unnecessary access to medical facilities, and represented an important tool for the limitation of the risk of transmission of infectious diseases, such as COVID-19.
Ferorelli, D., et al. [bib_ref] Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and..., Ferorelli [/bib_ref]. Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and Focus on Care Services During the COVID-19 Health Emergency.
The paper discussed of the legal problems on the telemedicine delivery ranging, from the profiles on the subject of authorization and accreditation to those concerning the protection of patient confidentiality.
Ceccato, F., et al. [bib_ref] Tele-medicine versus face-to-face consultation in Endocrine Outpatients Clinic during COVID-19 outbreak: A..., Ceccato [/bib_ref]. Telemedicine versus face-to-face consultation in Endocrine Outpatients Clinic duringCOVID-19
outbreak: a single-center experience during the lockdown period.
The study aimed to assess the efficacy of the emergency plan to continue the follow-up of outpatients in tele-endocrinology The study showed a similar outcome both in young and aged patients with endocrine diseases.
Di Franco, R., et al. [bib_ref] COVID-19 and radiotherapy: Potential new strategies for patients management with hypofractionation and..., Franco [/bib_ref]. COVID-19 and radiotherapy: potential new strategies for patients' management with hypofractionation and telemedicine.
Cancer patients are at higher risk of COVID-19 infection because of their immunosuppressive state caused by both the tumor itself and the anticancer therapy adopted. In this setting, the radiation therapy clinical decision-making process was partly reconsidered; thus, to reduce treatment duration and minimize infection risk during a pandemic, hypofractionated regimens were revised. This review aimed to point out the importance of hypofractionated radio therapy and telemedicine in cancer patient management in the COVID-19 era.
Molinari, G., et al. [bib_ref] Impact of 2020 SARS-CoV-2 outbreak on telemedicine management of cardiovascular disease in..., Molinari [/bib_ref]. Impact of 2020 SARS-CoV-2 outbreak on telemedicine management of cardiovascular disease in Italy.
The study analyzed data from three telemedicine dispatch centers focused in heart care. Records from the time interval March 1 2020 and April 1 2020 were compared with the corresponding periods in 2019. The comparative analysis of data showed a significant reduction in telemedicine electrocardiogram transmission.
## Cited article brief description of the focus
Zingone, F., et al. [bib_ref] Perception of the COVID-19 Pandemic among Patients with Inflammatory Bowel Disease in..., Zingone [/bib_ref]. Perception of the COVID-19 Pandemic Among Patients With Inflammatory Bowel Disease in the Time of Telemedicine: Cross-Sectional Questionnaire Study.
The study, based on a survey, demonstrated that lockdown had a significant impact on the psychological aspects of patients with IBD and suggest the need to increase communication with patients with inflammatory bowel disease (e.g., through telemedicine) to ensure that patients receive adequate health care, correct information, and proper psychological support.
Runfola, M., et al. [bib_ref] Telemedicine Implementation on a Bariatric Outpatient Clinic during COVID-19 Pandemic in Italy:..., Runfola [/bib_ref]. Telemedicine Implementation on a Bariatric Outpatient Clinic During COVID-19 Pandemic in Italy:
an Unexpected Hill-Start.
This paper aimed to evaluate the impact of teleconsulting technology in a single bariatric center on 33 booked participants. A total of 19 (57.6%) participated in the telemedicine program. No significant differences were found between participants and non-participants in terms of age and gender ratio. A total of 52.6% completed a survey reporting levels of satisfaction ranging from high to very high.
Klain, M., et al. [bib_ref] Management of differentiated thyroid cancer through nuclear medicine facilities during COVID-19 emergency:..., Klain [/bib_ref]. Management of differentiated thyroidcancer through nuclear medicine facilities during COVID-19 emergency: the telemedicine challenge.
Th study investigated whether a telemedicine service carried out during the COVID-19 pandemic impacted the management of patients with differentiated thyroid cancer. The number of outpatient visits performed during the pandemic (n = 445) and by in-ward access in the corresponding period of 2019 (n = 525) was comparable. The findings demonstrated the utility of telemedicine tools to avoid the potential negative impact of interruption or postponement of diagnostic and/or therapeutic procedures.
Peretto, G., et al. [bib_ref] Telemedicine in myocarditis: Evolution of a mutidisciplinary "disease unit" at the time..., Peretto [/bib_ref]. Telemedicine in myocarditis: Evolution of a mutidisciplinary "disease unit" at the time of COVID-19 pandemic.
More than 300 patients coming from the whole Country are currently followed up at a specialized multidisciplinary outpatient clinic. Following the pandemic outbreak of the SARS-CoV-2 infection in Italy, the authors presented how the multidisciplinary output clinic rapidly evolved to a "telemultidisciplinary output clinic", via a dedicated multitasking digital health platform.
Longo, M., et al. [bib_ref] Glycemic control in people with type 1 diabetes using a hybrid closed..., Longo [/bib_ref]. Glycemic control in people with type 1 diabetes using a hybrid closed loop system and followed by telemedicine during the COVID-19 pandemic in Italy.
The study was aimed at evaluating the metrics of glycemic control in people with type 1 diabetes using the hybrid closed loop (HCL) system during the COVID-19 lockdown. Adults with type 1 diabetes using HCL showed a significant improvement in most of the metrics of glucose control during the COVID-19 lockdown.
Guarino, M., et al. [bib_ref] Use of Telemedicine for Chronic Liver Disease at a Single Care Center..., Guarino [/bib_ref]. Use of Telemedicine for Chronic Liver Disease at a Single Care Center During the COVID-19 Pandemic: Prospective Observational Study.
The aim of this study was to analyze the benefits of using telemedicine services for patients with chronic liver disease at a tertiary care center in Italy during the COVID-19-mandated lockdown. During the lockdown in Italy, almost 400 visits were conducted using telemedicine. It was shown to be a useful tool for following up patients with chronic liver disease and for reducing the impact of the COVID-19 pandemic.
Negrini, S., et al. [bib_ref] Feasibility and Acceptability of Telemedicine to Substitute Outpatient Rehabilitation Services in the..., Negrini [/bib_ref]. Acceptability of Telemedicine to Substitute Outpatient Rehabilitation Services in the COVID-19 Emergency in Italy: An Observational Everyday Clinical-Life Study.
The study investigated the feasibility and acceptability of telemedicine as a substitute for outpatient services in emergency situations. Telemedicine services included teleconsultations and telephysiotherapy. Continuous quality improvement questionnaires were also evaluated. A total of 325 teleconsulations and 882 telephysiotherapy sessions were provided in 15 days. Patients' satisfaction with telemedicine was very high (2.8 out of 3).
Cilia, R., et al.. Telemedicine for parkinsonism: A two-step model based on the COVID-19 experience in Milan, Italy.
During the COVID-19 crisis, a telemedicine program for patients with parkinsonism was boosted in Milan, Italy. This two-step model integrated a telenursing forward triage followed by video-consultations by experienced neurologists.
## Cited article brief description of the focus
Capozzo, R., et al. [bib_ref] Telemedicine for Delivery of Care in Frontotemporal Lobar Degeneration during COVID-19 Pandemic:..., Capozzo [/bib_ref]. Telemedicine for Delivery of Care in Frontotemporal Lobar Degeneration During COVID-19 Pandemic: Results from Southern Italy ct.
The study evaluated the multidisciplinary assessment of patients with frontotemporal lobar dementia using telehealth during the COVID-19 pandemic. The study indicated that telemedicine is a valid tool to triage patients with frontotemporal lobar dementia to increase practice outreach and efficiency.
Giansanti, D.. The Italian Fight Against the COVID-19 Pandemic in the Second Phase: The Renewed Opportunity of Telemedicine.
The letter discussed the importance of telemedicine after the lock down as a means of continuity of care, maintaining "social distancing".
Capozzo, R., et al. [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref]. Telemedicine is a useful tool to deliver care to patients with Amyotrophic Lateral Sclerosis during COVID-19 pandemic: results from Southern Italy.
The study evaluated the feasibility of the multidisciplinary assessment of patients with Amyotrophic Lateral Sclerosis using telemedicine during the emergency determined by the COVID-19 pandemic. In a successive survey, most of patients were satisfied with the neurological interview (85%), the possibility to interact directly with the clinician while at home (85%), and the reduction in economic and time costs because they avoided unnecessary travel to the clinic.
Salzano, A., et al. [bib_ref] Heart failure management during the COVID-19 outbreak in Italy: A telemedicine experience..., Salzano [/bib_ref]. Heart failure management during the COVID-19 outbreak in Italy: a telemedicine experience from a heart failure university tertiary referral centre.
The letter described a telemedicine experience in heart failure management during COVID-19, showing on 103 patients that telemedicine, in most cases, allowed a clinical decision to be reached.
Siniscalchi, M., et al. [bib_ref] COVID-19 pandemic perception in adults with celiac disease: An impulse to implement..., Siniscalchi [/bib_ref]. COVID-19 pandemic perception in adults with celiac disease: an impulse to implement the use of telemedicine.
The authors aimed to evaluate the application perception of the use of a large-scale remote consultation approach-based on a Web surveyi-in 651 Celiac Disease patients who require a lifelong gluten-free diet as therapy. The remote tool allowed assessment of their psychological perceptions.
Tolone, S., et al. [bib_ref] Telephonic triage before surgical ward admission and telemedicine during COVID-19 outbreak in..., Tolone [/bib_ref]. Telephonic triage before surgical ward admission and telemedicine during COVID-19 outbreak in Italy. Effective and easy procedures to reduce in-hospital positivity.
The comment described the telephonic triage before surgical ward admission and telemedicine during the COVID-19 outbreak in Italy. It described effective and easy procedures to reduce in-hospital positivity.
Omboni, S.. Telemedicine During the COVID-19 in Italy: A Missed Opportunity?
The letter stated that Italy was found unprepared to manage lockdown patients with chronic diseases, due to limited availability and the diffusion of large-scale telemedicine solutions; it stated that the epidemic should help to promote better use and a larger integration of telemedicine services in the armamentarium of health care services.
Ohannessian, R., et al.. A Global Telemedicine Implementation and Integration Within Health Systems to Fight the COVID-19 Pandemic: A Call to Action.
The contribution highlighted that Italy did not include telemedicine in the essential levels of care granted to all citizens within the National Health Service, while other nations authorized, reimbursed, and actively promoted the use of telemedicine. The authors highlighted the challenges remaining for the global use and integration of telemedicine into the public health response to COVID-19 and future outbreaks.
Sossai, P., et al. [bib_ref] Telemedicine and the 2019 coronavirus (SARS-CoV-2), Sossai [/bib_ref]. Telemedicine and the 2019 coronavirus (SARS-CoV-2).
The contribution reported the experience of telemedicine conducted by hepatologists in a tertiary-care Center for Liver Disease of a University Hospital in Northern Italy, for a 2-week period during the COVID-19 pandemic, on 138 patients. The study emphasized the usefulness of telemedicine for maintaining continuity of care among patients with autoimmune liver diseases during the pandemic.
Rigamonti, C., et al. [bib_ref] Rates of Symptomatic SARS-CoV-2 Infection in Patients with Autoimmune Liver Diseases in..., Rigamonti [/bib_ref]. Rates of Symptomatic SARS-CoV-2 Infection in Patients With Autoimmune Liver Diseases in Northern Italy: A Telemedicine Study.
The contribution reported a project that used an online platform between general practitioners and patients, in order to reduce moving infected individuals and to perform diagnosis and treatment early on. The studies show:
- An initial disorientation [bib_ref] Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and..., Ferorelli [/bib_ref] in the use of telemedicine, during the implementation period of the guidelines/recommendationsand before the standardization initiatives; -
The scientific reflections regarding telemedicine usefollowing the immediate period after the first Italian national lock-down; -
The differentiated applications; - An expansion of the telemedicine boundaries; -
A high acceptance, as tested through specific questionnaires in some studies.
## Disorientation in telemedicine applications emerging in some studies
The study reported inexpressed dissatisfaction with the lost opportunity to widely spread telemedicine during the lockdown, wherein it is reported, and quoted verbatim: "Italy was found unprepared to manage lockdown patients with chronic diseases, due to limited availability and diffusion of large-scale telemedicine solutions." Among the specific causes hindering the implementation of effective telemedicine solutions, the author indicates, specifically for long-term patients management: (a) the scattered distribution and heterogeneity of available tools; (b) the lack of integration with the electronic health record of the national health system; (c) the poor interconnection between telemedicine services operating at different levels; (d) the lack of a real multidisciplinary approach to the patient management; and (e) the heavy privacy regulations and lack of clear guidelines, together with the lack of reimbursement.
In addition, the study reported inemphasized that Italy did not include telemedicine in the essential levels of care granted to all citizens within the National Health Service, while other nations authorized, reimbursed, and actively promoted the use of telemedicine. The study stimulated the stakeholders to take action in the direction of telemedicine. The study in [bib_ref] Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and..., Ferorelli [/bib_ref] discussed the legal problems in telemedicine delivery, ranging from profiles on the subject of authorization and accreditation to those concerning the protection of patient confidentiality.
## Reflections emerging after the italian national lockdown
The study inhighlighted that: (a) the Italian lockdown model (in March-May 2020) has been imitated by many other states; (b) Italy was probably not a model in the use of telemedicine. However, there was an opportunity to reflect on this and inspire models that could be useful after the first lock down period. The following sectors on which to focus during the pandemic were detected:
## -
Telemedicine and fragility for multiple chronic diseases; - Certainly, a very important sector where telemedicine must intervene is that of the frail: those subjects suffering from single or multiple chronic pathologies (often elderly, but not always), the frequently disabled, and those with an unstable health status are particularly vulnerable in the case of COVID-19 infection; - Telemedicine and fragility for rare diseases; - As is well known, a rare disease can generate multiple chronicity and disabilities together. A telemedicine application must, in this case, be tailored to the patient; - Television, telecooperation, and teleconsultation; - Traditional telemedicine means that during the pandemic, social distancing and the minimization of the risk of contagion were possible; -
The expansion of telemedicine boundaries; -
The expansion to new applications could be possible due to the pandemic; - New models for pulmonary rehabilitation using telemedicine; - A patient returning home after weeks of intubation needed a properly designed home rehabilitation program, also based on pulmonary stimulation tools suitably integrated into telemedicine.
## Collected evidence of telemedicine use
By analyzing the publications found in Pubmed to date, we can trace a picture of telemedicine use in the health domain regarding monitoring, surveillance, and continuity of care.
We find various applications of telemedicine in diabetology [bib_ref] Improvement in glycaemic control in paediatric and young adult type 1 diabetes..., Lazzeroni [/bib_ref] [bib_ref] The experience of patients with diabetes with the use of telemedicine and..., Maietti [/bib_ref] [bib_ref] The effect of the COVID-19 pandemic on telemedicine in pediatric diabetes centers..., Tornese [/bib_ref] [bib_ref] Telemedicine and urban diabetes during COVID-19 pandemic in Milano, Italy during lock-down:..., Luzi [/bib_ref] [bib_ref] Glycemic control in people with type 1 diabetes using a hybrid closed..., Longo [/bib_ref] and also in children, where we see the use of ICT integration tools with self-assessment devices. Cardiology has also recorded the use of telemedicine. The study in [bib_ref] Heart failure management during the COVID-19 outbreak in Italy: A telemedicine experience..., Salzano [/bib_ref] described a telemedicine experience in heart failure management during COVID-19. The study in [bib_ref] Impact of 2020 SARS-CoV-2 outbreak on telemedicine management of cardiovascular disease in..., Molinari [/bib_ref] analyzed data from three telemedicine centers focusing on heart care. The study in [bib_ref] Telemedicine in myocarditis: Evolution of a mutidisciplinary "disease unit" at the time..., Peretto [/bib_ref] reported the evolution of a multidisciplinary center for myocarditis towards a telemedicine system. Important applications are recorded in oncology, such as in breast cancer [bib_ref] Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine..., Bizot [/bib_ref] , lung cancer [bib_ref] Telemedicine for management of patients with lung cancer during COVID-19 in an..., Pardolesi [/bib_ref] , connections to the stoma centers, the management of patients with differentiated thyroid cancer [bib_ref] Management of differentiated thyroid cancer through nuclear medicine facilities during COVID-19 emergency:..., Klain [/bib_ref] , and related radiotherapy applications [bib_ref] COVID-19 and radiotherapy: Potential new strategies for patients management with hypofractionation and..., Franco [/bib_ref]. The neurology sector has also recorded an important use of applications in Parkinson's disease; in the use of telemedicine for the multidisciplinary assessment of patients with Amyotrophic Lateral Sclerosis [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref] ; for multiple sclerosis [bib_ref] Telemedicine during the Coronavirus Disease (COVID-19) Pandemic: A Multiple Sclerosis (MS) Outpatients..., Corea [/bib_ref] ; for a headache specialist center; and in applications of telephysiotherapy [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref]. The overview also reported the application of telemedicine in other sectors less common in telemedicine solutions, such as: colorectal surgery [bib_ref] E-consensus on telemedicine in colorectal surgery: A RAND/UCLA-modified study, Gallo [/bib_ref] ; wound care [bib_ref] What COVID-19 taught us: New opportunities and pathways from telemedicine and novel..., Scalise [/bib_ref] ; urological benign diseases [bib_ref] Implementing telemedicine for the management of benign urologic conditions: A single centre..., Checcucci [/bib_ref] ; endocrinology [bib_ref] Tele-medicine versus face-to-face consultation in Endocrine Outpatients Clinic during COVID-19 outbreak: A..., Ceccato [/bib_ref] ; inflammatory bowel disease [bib_ref] Perception of the COVID-19 Pandemic among Patients with Inflammatory Bowel Disease in..., Zingone [/bib_ref] ; teleconsulting with a bariatric center; and chronic liver disease [bib_ref] Use of Telemedicine for Chronic Liver Disease at a Single Care Center..., Guarino [/bib_ref]. There has been the use of systems, in some simple cases, such as telephony [bib_ref] Implementing telemedicine for the management of benign urologic conditions: A single centre..., Checcucci [/bib_ref] [bib_ref] Telephonic triage before surgical ward admission and telemedicine during COVID-19 outbreak in..., Tolone [/bib_ref] , and in other more complex cases, such as specialized servers [bib_ref] Rates of Symptomatic SARS-CoV-2 Infection in Patients with Autoimmune Liver Diseases in..., Rigamonti [/bib_ref] , which have allowed the application of telemedicine with success.
## Example of the expansion of the boundaries
The boundaries of the use of telemedicine have been expanded during the pandemic. Three examples are show in transgender mental health monitoring [bib_ref] Mental Health and Endocrine Telemedicine Consultations in Transgender Subjects during the COVID-19..., Gava [/bib_ref] , in the field of the abortion [bib_ref] A trans-national examination of the impact of the COVID-19 pandemic on abortion..., Van Ooijen [/bib_ref] [bib_ref] Telemedicine as an alternative way to access abortion in Italy and characteristics..., Brandell [/bib_ref] , and in the field of the animal-assisted therapy. Transgender people are a vulnerable group with a higher incidence of mental health issues and, during the COVID-19 outbreak, they may have faced psychological, physical, and social obstacles. The study in [bib_ref] Mental Health and Endocrine Telemedicine Consultations in Transgender Subjects during the COVID-19..., Gava [/bib_ref] evaluated the impact of the pandemic and access to health care services during the COVID-19 pandemic on the mental health of transgender people living in Italy. An anonymous web-based survey was conducted among transgender people living in Italy. It highlighted how telemedicine services may serve to mitigate negative psychological effects. The studies in [bib_ref] A trans-national examination of the impact of the COVID-19 pandemic on abortion..., Van Ooijen [/bib_ref] [bib_ref] Telemedicine as an alternative way to access abortion in Italy and characteristics..., Brandell [/bib_ref] focused on the application of telemedicine in the field of abortion. Induced abortion is legal in Italy, but with restrictions. The online abortion provider Women on Web serves as an alternative way to access abortion. The study highlighted an increase in requests during the COVID-19 pandemic compared with the previous years without a pandemic (when its use was not sensibly appreciable). The most common reasons for requesting a telemedicine abortion through the system were privacy-related; however, this shifted to COVID-19-specific reasons during the pandemic. Another example of the expansion of the boundaries of telemedicine in complementary and alternative medicine is reported in. The latter is a survey that was administered remotely to quantify the impact of animal-assisted therapy during the lock down. Through the survey, which also reported as a self-assessment test for anxiety, it was shown that pet owners had lower levels of anxiety.
## Examples on the acceptance of use
Several studies have accompanied the use of telemedicine with the application of questionnaires (in some cases even standardized) to investigate acceptance and satisfaction [bib_ref] Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine..., Bizot [/bib_ref] [bib_ref] The experience of patients with diabetes with the use of telemedicine and..., Maietti [/bib_ref] [bib_ref] Telemedicine for management of patients with lung cancer during COVID-19 in an..., Pardolesi [/bib_ref] [bib_ref] Telemedicine Implementation on a Bariatric Outpatient Clinic during COVID-19 Pandemic in Italy:..., Runfola [/bib_ref] [bib_ref] Feasibility and Acceptability of Telemedicine to Substitute Outpatient Rehabilitation Services in the..., Negrini [/bib_ref] [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref] , from which the desire to continue with telemedicine even in post-pandemic periods have also clearly emerged, directly or indirectly. Telemedicine received a high degree of acceptance, for example, in oncology, where both a study on patients with breast cancer [bib_ref] Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine..., Bizot [/bib_ref] and in the output of a project on lung cancer monitoring [bib_ref] Telemedicine for management of patients with lung cancer during COVID-19 in an..., Pardolesi [/bib_ref] displayed this. The study in [bib_ref] The experience of patients with diabetes with the use of telemedicine and..., Maietti [/bib_ref] investigated the individual and contextual determinants of the perceived quality of telemedicine and teleassistance services, and willingness to continue with them among patients with diabetes. The study showed both a high level of acceptance and several determinants. These socio-demographic and correlated factors should be considered in the implementation of care pathways integrating in-person visits with the telemedicine. In addition, applications in neurology showed a high acceptance, as in the case of a study of the multidisciplinary assessment of patients with amyotrophic lateral sclerosis using telemedicine [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref] , and in a study embedding telephysiotherapy services [bib_ref] Feasibility and Acceptability of Telemedicine to Substitute Outpatient Rehabilitation Services in the..., Negrini [/bib_ref]. Notably, the study in [bib_ref] Telemedicine Implementation on a Bariatric Outpatient Clinic during COVID-19 Pandemic in Italy:..., Runfola [/bib_ref] also assessed the positive impact of a teleconsulting technology in a single bariatric center. Both interesting and innovative for oncology is the study in, which reported a high level of acceptance from patients involved in the experience of telemedicine consultations at a stoma center.
# Discussion
The COVID-19 pandemic, as highlighted by Negrini et al. [bib_ref] Instant paper from the field" on rehabilitation answers to the COVID-19 emergency, Negrini [/bib_ref] , represented an important engine for the development of telemedicine in Italy. Here, we have seen two important phases in the launch of the telemedicine. Many critical issues in the second phase have been addressed, and efforts have been made to improve the usability of telemedicine services, as well as standardization aspects, through institutional and political actions.
In this study we resumed these changes and reported an overview based on two points of view.
The first point of view reported the initiatives for issuing recommendations and indications for the use of telemedicine by the ISS through public reports; these were in the national language and translated, in many cases, into English, and in other cases, into other languages. These reports have been a particular stimulus on the national scene for the use of broad-spectrum telemedicine, particularly in the case of various types of frailties, and also due to rare diseases.
The second point of view reported an analysis of the literature from Pubmed to examine the spread of telemedicine. This analysis highlights:
- An initial disappointment [bib_ref] Medical Legal Aspects of Telemedicine in Italy: Application Fields, Professional Liability and..., Ferorelli [/bib_ref] in relation to the low use of telemedicine due to problems that are not only operational, but also bureaucratic and legislative.
## -
A subsequent broad-spectrum use in traditional applications-such as diabetology, cardiology, oncology, and neurology-but also in original sectors, such as application in bariatric centers, wound care, urological benign diseases, endocrinology, inflammatory bowel disease, and chronic liver disease [fig_ref] Table 3: ISS reports dealing with the telemedicine [/fig_ref].
## -
New emerging applications, such as mental health in transgender people [bib_ref] Mental Health and Endocrine Telemedicine Consultations in Transgender Subjects during the COVID-19..., Gava [/bib_ref] , telemedicine applied to abortion [bib_ref] A trans-national examination of the impact of the COVID-19 pandemic on abortion..., Van Ooijen [/bib_ref] [bib_ref] Telemedicine as an alternative way to access abortion in Italy and characteristics..., Brandell [/bib_ref] , and the assessment of the impact of the animal-assisted therapy. - Studies based on surveys [bib_ref] Multicenter evaluation of breast cancer patients' satisfaction and experience with oncology telemedicine..., Bizot [/bib_ref] [bib_ref] The experience of patients with diabetes with the use of telemedicine and..., Maietti [/bib_ref] [bib_ref] Telemedicine for management of patients with lung cancer during COVID-19 in an..., Pardolesi [/bib_ref] [bib_ref] Telemedicine Implementation on a Bariatric Outpatient Clinic during COVID-19 Pandemic in Italy:..., Runfola [/bib_ref] [bib_ref] Feasibility and Acceptability of Telemedicine to Substitute Outpatient Rehabilitation Services in the..., Negrini [/bib_ref] [bib_ref] Telemedicine is a useful tool to deliver care to patients with Amyotrophic..., Capozzo [/bib_ref] that have shown a high acceptance of telemedicine, the determinants, and a direct or indirect interest in continuing with these solutions in the future.
If we compare the development of telemedicine during the pandemic in Italy with the USA, a nation that showed one of the best telemedical preparedness, we can highlight some important considerations. As we highlighted inin a comment to, the telemedicine boom during the COVID-19 has not been identical across the world, for example it was different between the USA, Italy and Europe. Different regulations, and a less enlightened and more conservative political approach have, in many cases, hampered the spread of telemedicine in the first months of the pandemic. To cite a first example, whereas in the United States, the system based on medical insurance has clearly defined the reimbursement procedures, in Italy and in Europe this has not happened so explicitly. In USA, there were immediately derogations to the law for the use of messaging and video communication systems to be applied to telemedicine. Europe has not clearly made explicit derogations to current regulations in the first phases of the pandemic. However, after an initial disorientation, and some phasing initiatives, as shown in, telemedicine began to be used and stimulated, supported by a public-and equity-based healthcare approach. Then, in the USA-where the health system itself, based on private insurance, had allowed a rapid response to the use of telemedicine-scholars began to question, after a few months, the disparities and inequalities of telemedical treatment based on a private health system.
# Limitations
The study, based on a review of Pubmed and of the ISS online database, has limitations. As regards the publications, it analyzed scientific productions in the English language. It did not analyze publications in other languages (Spanish, French or Italian). It analyzed only the online database Pubmed, which, however, is strategic in the health domain, where the overview is focused. It is not a systematic review, given that the topic (the launch of a technology) required a type of investigation based on polyhedral sources (some nonscientific publications) and specific filtering more suitable for other types of reviews, such as narrative reviews, realistic reviews, hermeneutical reviews, rapid reviews, or simple overview reviews (all reviews admitted in the journal).
# Conclusions
In conclusion, the study highlights that: (a) in Italy, after the first moment of disorientation, telemedicine was used broadly and effectively; (b) new fields of telemedicine application were also explored; (c) dedicated questionnaires showed a high level of acceptance of telemedicine, and a desire to continue using this technology; (d) important suggestions emerged to invest in the use of telemedicine during the pandemic, and in the future after the pandemic.
By comparing the results of this study with other studies focused on other realities based on a different approach to the health system (for example, a private approach), we can highlight how the COVID-19 pandemic has been a stimulus for the development and use of telemedicine, and for the reviewing of regulations and policies in order to improve the use of this service, which can represent an instrument of equity and protection (thanks to social distancing) in this period, and an opportunity for the future.
[fig] Figure 1: Output from the qualification process. [/fig]
[fig] Author: Contributions: Conceptualization, D.G.; methodology, D.G., A.L., M.G., I.A. and G.M.; software, all; validation, all; formal analysis, D.G., A.L., M.G., I.A. and G.M.; investigation, all; resources, all; data curation, all; writing-original draft preparation, D.G.; writing-review and editing, D.G., A.L., M.G., I.A. and G.M.; visualization, all; supervision, D.G.; project administration, D.G. and A.L. All authors have read and agreed to the published version of the manuscript. [/fig]
[table] Table 1: Parameters used for the eligibility. [/table]
[table] Table 2: The keys applied in the search (COVID-19 was also changed with SARS-Cov-2 during the searches). [/table]
[table] Table 3: ISS reports dealing with the telemedicine. [/table]
[table] Table 4: Cont. [/table]
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Recurrent aseptic meningitis in association with Kikuchi-Fujimoto disease: case report and literature review
Background: Kikuchi Fujimoto disease (KFD), or histiocytic necrotising lymphadenitis, is a benign and self-limiting condition characterised by primarily affecting the cervical lymph nodes. Recurrent aseptic meningitis in association with KFD is extremely rare and remains a diagnostic challenge. Case presentation: We report a 28-year-old man who presented 7 episodes of aseptic meningitis associated with KFD over the course of 7 years. Histopathological findings of enlarged lymph nodes led to the diagnosis of KFD. The patient's headache and lymphadenopathy spontaneously resolved without any sequelae. Conclusions: A diagnosis of KFD should be considered when enlarged cervical lymph nodes are observed in patients with recurrent aseptic meningitis. A long-term prognosis remains uncertain, and careful follow-up is preferred.
# Background
Kikuchi-Fujimoto disease (KFD), or histiocytic necrotising lymphadenitis, is recognised as a benign lymphadenopathy that has acute or sub-acute onset and is primarily localised within the cervical lymph nodes. KFD has various extranodal manifestations, including skin lesions, gastrointestinal symptoms or splenomegaly. Neurological complications, including aseptic meningitis, mononeuritis multiplex or acute cerebellar ataxia, are not common [bib_ref] Kinetic tremor and cerebellar ataxia as initial manifestations of Kikuchi-Fujimoto's disease, Moon [/bib_ref] , and a meta-analysis of 244 KFD cases in 181 published case reports demonstrated 11% of incidence of neurological involvements [bib_ref] Kikuchi-Fujimoto disease: analysis of 244 cases, Kucukardali [/bib_ref]. The most common neurological complication is aseptic meningitis, which is observed in 2.8-9.8% of KFD cases [bib_ref] Histiocytic necrotizing lymphadenitis (Kikuchi's disease) with aseptic meningitis, Sato [/bib_ref] [bib_ref] Medical study of 69 cases as Kikuchi's disease, Nakamura [/bib_ref]. KFD usually resolves spontaneously within a few months, and the recurrence rate is 3-4% [bib_ref] Kikuchi's disease (Histiocytic necrotizing lymphadenitis) A clinicopathologic study of 79 cases with..., Kuo [/bib_ref]. Recurrent aseptic meningitis associated with KFD is an extremely rare condition, and only 4 sporadic cases have been reported [bib_ref] A case of necrotizing lymphadenitis associated with aseptic meningitis, Maeda [/bib_ref] [bib_ref] Histiocytic Necrotizing Lymphadenitis (Kikuchi's Disease) with Aseptic Meningitis, Mathew [/bib_ref] [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis 11 years..., Itokawa [/bib_ref] [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis associated with..., Yamashita [/bib_ref]. In this study, we describe a KFD patient who presented with 7 episodes of recurrent meningitis. We also investigate the clinical and laboratory features of 4 patients previously reported in the literature.
## Case presentation
A 28-year-old Japanese man was admitted to our hospital because of headache and remittent fever that had lasted for 12 days after a solar exposure. The patient had a history of atopic dermatitis from age 2 and had 5 previous episodes of aseptic meningitis with an undetermined aetiology between the ages of 21 and 27 years-old . Upon admission, day 12, the patient was alert and had pyrexia of 39°C and severe headache with positive Kernig's Sign. The palpable tender lymph nodes with 10 mm in size were present on the right posterior neck, similar to a recent meningitis episode. No other neurological deficits were noted. Laboratory analysis revealed no abnormalities in the patient's complete blood cell count or liver and thyroid function tests. His serum CRP and IgE levels were elevated, measuring 2.5 mg/dL (normal, <0.3) and 6950 mg/dL (normal, < 295), respectively. The cerebrospinal fluid (CSF) examination showed a crystal clear appearance and pleocytosis of 27 cells/mm 3 (97% mononuclear cells) with a protein concentration of 31 mg/dL (normal, < 45). The CSF glucose/glycaemia ratio was 0.86 with sterile bacterial, tuberculosis and fungal cultures. A CSF polymerase chain reaction (PCR) assay for the herpes simplex virus (HSV) was negative. The patient recovered after the administration of non-steroidal anti-inflammatory drugs as a symptomatic treatment and was discharged in remission on day 26.
Subsequently, the patient was re-admitted on day 37 with headache and remittent fever. He was febrile with re-appearance of Kernig's Sign. The posterior cervical lymph nodes were enlarged and tender bilaterally. Additionally, the patient also had oral aphthae and skin rashes on his trunk along with atopic dermatitis. He was alert, and no focal neurological deficit was noted. A complete blood count revealed leukocytopaenia (2.7 x 10 9 /L) with 1% of atypical lymphocytes. A biochemical examination showed abnormal levels with LDH 729 U/L (normal, <220), ferritin 2660 ng/mL (normal, 25-280) and an erythrocyte sedimentation rate of 59 mm/h (normal, <10). IgE and CRP assays exhibited higher levels than those of previous tests, with values of 10400 mg/dL and CRP 6.5 mg/dL being observed, respectively. Serum anti-nuclear and anti-neutrophil cytoplasmic antibodies and rheumatoid factor were within normal range. The complement 3 level was slightly elevated with 194 mg/dL (normal, 65-135). Human leukocyte antigen B51 was negative. The evidence of other infectious agents, such as human hepatitis viruses B and C, human T-cell lymphoma virus-1, syphilis, HSV-1 and 2, varicella-zoster virus (VZV), cytomegalovirus, Epstein-Barr virus (EBV), human herpes virus type 6 (HHV-6) and toxoplasmosis (Toxoplasma gondii) were not detected. A CSF examination revealed a pleocytosis of 16 cells/mm 3 (97% mononuclear cells) and a protein concentration of 28 mg/dL, as well as a CSF glucose/glycaemia ratio of 0.75 with sterile bacterial, tuberculosis and fungal cultures. CSF PCRs for HSV, HHV-6, VZV and tuberculosis were negative. The patient's CSF IgE level was not elevated (19 IU/ml, IgE index 0.03). Splenomegaly was present upon abdominal echograph. No enlargement of deep lymph nodes was detected using computed tomography. Moreover, brain magnetic resonance imaging showed no structural abnormalities.
An excisional biopsy of the involved posterior cervical lymph nodes was performed. The affected lymph nodes showed focal paracortical necrotic lesions [fig_ref] Figure 2: Histopathological findings of affected lymph node [/fig_ref]. Under a high power field, the lesion had abundant karyorrhectic debris with apoptotic bodies, numerous histiocytes and large lymphoid cells and scattered fibrin [fig_ref] Figure 2: Histopathological findings of affected lymph node [/fig_ref]. However, there were no neutrophils in the lesion. A portion of the phagocytic macrophage had crescent nuclei. Moreover, Giemsa stained sections highlighted the plasmacytoid dendritic cells clusters at the margins of the necrotic foci [fig_ref] Figure 2: Histopathological findings of affected lymph node [/fig_ref]. Immunohistochemical study demonstrated that the histiocytes expressed CD68 and myeloperoxidase [fig_ref] Figure 2: Histopathological findings of affected lymph node [/fig_ref]. Based on these pathological findings, the diagnosis of KFD was made.
The patient did not receive medication during his second admission. Also, his symptoms and CSF parameters spontaneously resolved within two weeks. His enlarged lymph nodes gradually decreased in size. The patient was discharged in remission on day 49, and he remains headache-free after 28 months of follow-up.
# Discussion
Since KFD was first described by both a pathologist and physician independently in 1972 [bib_ref] Lymphadenitis showing focal reticulum cell hyperplasia with nuclear debris and phagocytes, Kikuchi [/bib_ref] [bib_ref] Cervical subacute necrotizing lymphadenitis, Fujimoto [/bib_ref] , the aetiology of KFD remains largely unknown. The histopathological features of affected lymph nodes in KFD are, on occasion, Clinical course of the patient. The patient had his first meningitis episode at 21 years of age. Two resolutions and exacerbations occurred within the following 3 months. He had the fourth to seventh episodes of meningitis between 27 and 28 years of age. In each episode the patient's symptoms and abnormal cerebrospinal fluids were spontaneously resolved. Lymphadenopathy was evident both in the attacks at age 27 and age 28.
notably similar to those of SLE [bib_ref] Enigmatic Kikuchi-Fujimoto disease. A comprehensive review, Bosch [/bib_ref]. Therefore, pathogenic linkage between the two disorders has been proposed [bib_ref] Enigmatic Kikuchi-Fujimoto disease. A comprehensive review, Bosch [/bib_ref]. Infectious agents, including toxoplasmosis, EBV, and HHV-6, have also been considered as possible causal agents, but several studies have failed to confirm their association [bib_ref] Enigmatic Kikuchi-Fujimoto disease. A comprehensive review, Bosch [/bib_ref] [bib_ref] An investigation of the viral pathogenesis of Kikuchi-Fujimoto disease. Lack of evidence..., Hollingsworth [/bib_ref]. There is no specific treatment for KFD because of its unknown aetiology. In general, the patients are treated symptomatically; for example, relief of local and systemic complaints with the use of analgesics, antipyretics and rest [bib_ref] Enigmatic Kikuchi-Fujimoto disease. A comprehensive review, Bosch [/bib_ref]. Furthermore, corticosteroids may be effective in severe cases or for a relapsing condition.
The aseptic meningitis associated with KFD was first reported in 1979 [bib_ref] Histiocytic necrotizing lymphadenitis (Kikuchi's disease) with aseptic meningitis, Sato [/bib_ref]. Today, 18 sporadic case reports have been documented in MEDLINE and Japan Medical Abstracts Society-website, with 4 of them reporting recurrence of meningitis [bib_ref] A case of necrotizing lymphadenitis associated with aseptic meningitis, Maeda [/bib_ref] [bib_ref] Histiocytic Necrotizing Lymphadenitis (Kikuchi's Disease) with Aseptic Meningitis, Mathew [/bib_ref] [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis 11 years..., Itokawa [/bib_ref] [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis associated with..., Yamashita [/bib_ref]. The clinical profiles of our patient and the 4 patients reviewed in the literature are shown in [fig_ref] Table 1: The clinical features of recurrent aseptic meningitis cases withKikuchi-Fujimoto disease [/fig_ref]. In all 5 cases the symptoms resolved within several months. Corticosteroids were administered in 3 out of the 5 patients. All of the 3 did not have early post-treatment relapse after receiving steroids. Steroid treatment may be beneficial for recurrent KFD with aseptic meningitis, although recommendation of steroid administration requires further investigation.
Our patient had concurrent atopic dermatitis, and his serum IgE levels were elevated along with exacerbation from meningitis and lymphadenitis. Because IgE was not elevated in the CSF, his high serum IgE titre did not appear to play a pathogenic role in aseptic meningitis. A prior case with an elevation of serum IgE in recurrent aseptic meningitis with KFD has been reported [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis 11 years..., Itokawa [/bib_ref]. In this study, we speculate that IgE elevation may be reflected the immunostimulatory condition that was activated upon KFD in a patient with atopic dermatitis. A striking histopathological feature of KFD is the clustering of the plasmacytoid dendritic cells at the margins of the necrotic foci of affected lymph node. Plasmacytoid dendritic cells are known to produce type I interferon in response to viral infection and to induce human memory B cells to differentiate into plasma cells and produce immunoglobulin [bib_ref] Plasmacytoid dendritic cells in immunity, Colonna [/bib_ref]. Type I interferon is known as a potential pathogenic agent in the SLE-related neurological involvement [bib_ref] Activation of type 1 interferon in systemic lupus erythematosus: association with distinct..., Karageorgas [/bib_ref]. Moreover, the high titre of antinuclear antibodies had also been observed in another case [bib_ref] A case of subacute necrotizing lymphadenitis with recurrent aseptic meningitis associated with..., Yamashita [/bib_ref]. The progression of recurrent aseptic meningitis with KFD may stem from pathogenic association with SLE or other autoimmune disorders. Further studies are necessary to clarify this hypothesis.
# Conclusions
Recurrent aseptic meningitis with KFD is extremely rare condition. However, awareness of KFD as the differential diagnosis for meningitis might assist with diagnosis of patients presenting with lymphadenopathy. Early excisional lymph node biopsy should be considered to avoid unnecessary treatments. Temporary corticosteroid treatment may be beneficial to patients that present with recurrent meningitis with KFD, although this treatment's long-term efficacy remains uncertain. Because of the association with SLE, patient follow-up visit is necessary upon subsequent development of symptoms.
## Consent
Written informed consent was obtained from the patient for the publication of this case report.
## Ethics approval
The study was approved by the Human Ethics Review Committee of Dokkyo Medical University.
[fig] Figure 2: Histopathological findings of affected lymph node. A: In a low-power field, the affected lymph node showed discreet areas of necrosis paracortex (Hematoxylin-eosin stain, 10X). B: In a high-power field, the lesion demonstrated abundant karyorrhectic debris with apoptosis bodies and numerous histiocytes and large lymphoid cells (arrows). There were no neutrophils in the lesion (Hematoxylin-eosin stain, 100X). C: Giemsa stained section highlighted the plasmacytoid dendritic cells cluster (arrowheads) at the margins of the necrotic foci (may-Giemsa stain, 100X). D: Immunostain demonstrated that many histiocytes in the necrotic area were positive for myeloperoxidase (60X). E: Portions of the hystiocytes were stained with CD68 (60X). The scale bars in each panel indicate 10 μm, except for panel A (50 μm). [/fig]
[table] Table 1: The clinical features of recurrent aseptic meningitis cases withKikuchi-Fujimoto disease [/table]
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Characterization of white matter changes along fibers by automated fiber quantification in the early stages of Alzheimer's disease
A R T I C L E I N F OKeywords:Alzheimer's disease Mild cognitive impairment Automated fiber quantification Diffusion tensor imaging Pointwise comparison A B S T R A C T Brain white matter fiber bundles in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) have abnormalities not usually seen in unaffected subjects. Ideal algorithm of the localization-specific properties in white matter integrity might reveal the changes of tissue properties varying along each tract, while previous studies only detected the mean DTI parameters of each fiber. The aim of this study was to investigate whether these abnormalities of nerve fiber tracts are localized to specific regions of the tracts or spread throughout and to analyze which of the examined fiber tracts are involved in the early stages of Alzheimer's disease. In this study, we utilized VBA, TBSS as well as AFQ together to comprehensively investigate the white matter fiber impairment on 25 CE patients, 29 MCI patients and 34 normal control (NC) subjects. Two tract profiles, fractional anisotropy (FA) and mean diffusivity (MD), were extracted to evaluate the white matter integrity at 100 locations along each of 20 fiber tracts and then we validated the results with 27 CE patients, 21 MCI patients and 22 NC from the ADNI cohort. Also, we compare the AFQ with VBA and TBSS in our cohort. In comparison with NC, AD patients showed widespread FA reduction in 25% (5 /20) and MD increase in 65%(13/20) of the examined fiber tracts. The MCI patients showed a regional FA reduction in 5% (1/20) of the examined fiber tracts (right cingulum cingulate) and MD increase in 5%(1/20) of the examined fiber tracts (left arcuate fasciculus). Among these changed tracts, only the right cingulum cingulate showed widespread disruption of myelin or/and fiber axons in MCI and aggravated deterioration in AD, findings supported by FA/MD changes both by the mean and FA changes by point wise methods and TBSS. And the AFQ findings from ADNI cohort showed some similarity with our cohort, especially in the pointwise comparison of MD profiles between AD vs NC. Furthermore, the pattern of white matter abnormalities was different across neuronal fiber tracts; for example, the MCI and AD patients showed similar FA reduction in the middle part of the right cingulum cingulate, and the anterior part were not damaged. However, the left arcuate fasciculus showed MD elevation located at the temporal part of the fibers in the MCI patients and expanding to the temporal and middle part of the fibers in AD patients. So, the AFQ may be an alternative complementary method of VBA and TBSS, and may provide new insights into white matter degeneration in MCI and its association with AD.
# Introduction
Alzheimer's disease (AD) and its prodromal stage of mild cognitive impairment (MCI) are typical neurodegenerative disorders and the most common forms of dementia in older adults [bib_ref] Global prevalence of dementia: a Delphi consensus study, Ferri [/bib_ref]. A growing number of studies suggest that white matter microstructure differs in patients with AD and MCI compared to healthy controls; therefore, diffusion tensor imaging (DTI) has received increasing attention because it can detect microstructural integrity of white matter fiber bundles [bib_ref] Diffusion tensor imaging of white matter degeneration in Alzheimer's disease and mild..., Amlien [/bib_ref]. Fractional anisotropy (FA) and mean diffusivity (MD) are two frequently used quantitative measures of DTI that detect the anisotropy and overall displacement of water molecule diffusion [bib_ref] Looking into the functional architecture of the brain with diffusion MRI, Bihan [/bib_ref]. Although most researchers suggest that white matter abnormalities exist in AD and MCI, whether the patterns of white matter abnormalities are different across neuronal fiber tracts remains unknown.
Several approaches for DTI analyses, such as region of interest (ROI) analysis, voxel-based analysis (VBA) and tract-based spatial statistics (TBSS) have been applied in AD and MCI studies. For example, Alves reported decreased FA and increased MD in selected brain regions or certain segments of fibers in patients with AD and MCI [bib_ref] Integrating retrogenesis theory to Alzheimer's disease pathology: insight from DTI-TBSS investigation of..., Alves [/bib_ref]. However, this ROI based DTI analysis is hypothesis-dependent, and most of the brain area remains unexamined, making localization difficult. Several other previous studies used VBA and indicated a significant FA reduction in the fornix, the splenium, and several other regions [bib_ref] White matter changes in mild cognitive impairment and AD: a diffusion tensor..., Medina [/bib_ref] [bib_ref] Effectiveness of regional DTI measures in distinguishing Alzheimer's disease, MCI, and normal..., Nir [/bib_ref] [bib_ref] Diffusion Kurtosis Imaging: a possible MRI biomarker for AD diagnosis, Struyfs [/bib_ref] , whereas VBA is not sufficiently precise at the individual level because of the variants in the shape of long-range fiber tracts among subjects [bib_ref] White matter bundle registration and population analysis based on Gaussian processes, Wassermann [/bib_ref] [bib_ref] Anatomical properties of the arcuate fasciculus predict phonological and reading skills in..., Yeatman [/bib_ref]. To improve the specificity of the detection of white matter lesions, tract-based voxel grouping analysis, such as TBSS, has been employed to reveal deterioration in the limbic fibers, the fronto-occipital fasciculi, the inferior longitudinal fasciculi and other [bib_ref] Impact of APOE on the healthy aging brain: a voxel-based MRI and..., Honea [/bib_ref] [bib_ref] Decreased white matter integrity in late-myelinating fiber pathways in Alzheimer's disease supports..., Stricker [/bib_ref] [bib_ref] Regional white matter integrity differentiates between vascular dementia and Alzheimer disease, Zarei [/bib_ref] [bib_ref] Absolute diffusivities define the landscape of white matter degeneration in Alzheimer's disease, Acosta-Cabronero [/bib_ref] [bib_ref] White matter diffusion alterations in normal women at risk of Alzheimer's disease, Smith [/bib_ref] [bib_ref] Diffusion tensor imaging and tract-based spatial statistics in Alzheimer's disease and mild..., Liu [/bib_ref] [bib_ref] Multiple DTI index analysis in normal aging, amnestic MCI and AD. Relationship..., Bosch [/bib_ref]. But, there was only modest agreement between TBSS-based tract definitions and the actual location of a tract in an individual's brain, and voxel-based technique cannot provide the localization-specific properties in white matter integrity along each tract.
In summary, these white matter analysis techniques have merits and demerits respectively, and an ideal algorithm of the localization-specific properties in white matter integrity along each tract may be needed to enrich the white matter studies. In vivo, tissue properties may vary along each tract for the following reasons: different populations of axons enter and exit the tract, and disease can strike at local positions within the tract. Therefore, the diffusion measures along each fiber tract (tract profiles) of whole brain need to be further investigated [bib_ref] Altered microstructure in corticospinal tract in idiopathic normal pressure hydrocephalus: comparison with..., Hattori [/bib_ref]. Automated Fiber Quantification (AFQ) is a new algorithm that automatically identifies white matter tracts in human brains and takes measurements at anatomically equivalent locations along their trajectories [bib_ref] Tract profiles of white matter properties: automating fiber-tract quantification, Beaulieu [/bib_ref]. In this study we performed AFQ to extract FA and MD at 100 locations along 20 fiber tracts to evaluate tract profiles of the white matter integrity across AD and MCI compared with normal controls (NC). These 20 tracts include association tracts, projection tracts, commissural tracts between cortices and fibers in the limbic system. Therefore, in the current study we utilized VBA, TBSS as well as AFQ together to comprehensively investigate the white matter fiber impairment in the MCI and AD patients. We hypothesize that the abnormalities of the fiber tracts are limited to specific regions of tracts in MCI and spread throughout the tracts in AD. Furthermore, certain fibers are involved in the early stages of AD, and white matter impairment spread along fiber tracts in different patterns across all the examined fibers. We hope that as a novel algorithm, the AFQ may be an alternative complementary method of VBA and TBSS, and may provide new insights into white matter degeneration in MCI and its association with AD.
# Materials and methods
## Subjects
Eighty-eight participants were recruited from the Department of Neurology of the Affiliated Drum Tower Hospital at Nanjing University Medical School from July 2013 to December 2015. This study was approved by the ethics committee of the Nanjing Drum Tower Hospital, Nanjing, China. Written informed consent was obtained from each participant.
Each subject underwent a whole brain MRI scan, a general medical examination, and a neuropsychological assessment with the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and clinical dementia rating scale (CDR) score, all performed by neurologists blinded to the results of MR imaging results.
AD was diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable AD [bib_ref] Anxiety disorders in the fourth edition of the classification of mental disorders..., Rabe-Jablonska [/bib_ref]. Twenty-five patients met these criteria. Twenty-nine consecutive patients met the criteria of Petersen et al. [bib_ref] Mild cognitive impairment as a diagnostic entity, Petersen [/bib_ref] for MCI. Thirty-four subjects (NCs) were identified as individuals who (a) had no cognitive complaints, (b) had a normal level of clinical rating scales, and (c) had no evidence of any abnormality determined by conventional MRI.
The exclusion criteria were set as follows: 1) concurrent illnesses (such as diabetes mellitus, addictions or psychiatric diseases other than AD) or treatments interfering with cognitive function; 2) a score higher than 4 on the Hachinski Ischemic Scale; 3) the presence of structural abnormalities that could cause cognitive impairment, or the presence of leukoaraiosis higher than Fazekas' grade I, which were identified by conventional MRI; 4) < 8 years of education; or 5) not right-handed.
At the same time, to validate the findings, we employed 70 patients (27 CE patients, 21 MCI patients and 22 NC) from ADNI with age and gender matched with our cohort.
Part of data used in the preparation of this article was obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (adni. loni.usc.edu). The ADNI was launched in 2003 as a public-private partnership, led by Principal Investigator Michael W. Weiner, MD. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment (MCI) and early Alzheimer's disease (AD).
## Magnetic resonance imaging acquisition
All magnetic resonance imaging scans were performed using a Philips 3.0 T scanner (Achieva 3.0 T TX, Philips Medical Systems, the Netherlands) equipped with an 8-channel phase array head coil. A 3dimensional, high-resolution sagittal T 1 weighted scan with turbo fast echo acquisition was performed with repetition time (TR)/echo time (TE)/inversion time (TI) at 9.8/4.6/900 ms, with a flip angle of 8°and 1.0 mm isotropic resolution for anatomical reference. Diffusion weighted images were collected using a spin-echo echo-planar imaging (EPI) sequence (TR 9154 ms, TE 55 ms, matrix size 112 × 112, FOV 224 × 224 mm, slice thickness 2.5 mm, voxel size 2 × 2 × 2.5 mm 3 ) with both 32-directional diffusion encoding (b = 1000 s/mm 2 for each direction) and no diffusion encoding (b = 0 s/mm 2 ). All scans were conducted by an experienced neurologist with the aim of excluding gross brain abnormalities. The total acquisition time was 13 min and 10s.
## Magnetic resonance image pre-processing
The pre-processing of DTI, voxel-based analysis (VBA) and tractbased spatial statistics (TBSS) were carried out by PANDA [bib_ref] PANDA: a pipeline toolbox for analyzing brain diffusion images, Cui [/bib_ref] by its default pipeline setting. The pre-processing of DTI, automated fiber quantification (AFQ) was performed using FSL 5.0 software (Oxford Centre for Functional Magnetic Resonance Imaging of the Brain, University of Oxford, Oxford, England; http://www.fmrib.ox.ac. uk/fsl/). First, brain extraction was performed to remove non-brain structures, using a brain extraction tool (BET tool of the FSL software). The raw images were corrected for head movements and eddy current by using an affine image registration (FLIRT tool of the FSL software) and eddy current program (command of the FSL software). The diffusion tensor was reconstructed by fitting a diffusion tensor model to each image voxel by using the dtifit program (command of the FSL software), producing fractional anisotropy (FA) and mean diffusivity (MD) images. Subsequently, the diffusion tensor was calculated for each subject.
## Voxel-based analysis (vba) & tract-based spatial statistics (tbss)
First, register individual FA images of native space to the FA template in the MNI space and then apply the resultant warping transformations to write the images of the diffusion metrics into MNI space. Then smooth is needed to alleviate the registration error with an 6 mm full width at Gaussian kernel. Further, resample the volume into a voxel size of 2 × 2 × 2 mm 3 .
The TBSS were carried out as following: To create the mean FA skeleton which represents the centers of all tracts common to the group, the mean FA image was thinned with FA threshold value as 0.25. Then project each subject's FA data onto the mean FA skeleton, a procedure that minimizes misalignment more prevalent in standard registration procedures.
## Automated fiber quantification (afq)
3D-T 1 weighted images were used for registration and segmentation by FSL. The pre-processed DTI images and 3DT 1 weighted images were fed into the AFQ software , which is a MATLAB toolbox and is used to identify and quantify 20 white matter tracts in each subject's brain. The technique details and parameters were exactly same to those described in the original AFQ paper , except that the cingulum was divided into cingulum cingulate and cingulum hippocampus in the new versions. The identification and quantification procedure steps can be summarized as 4 steps: (1) performance of whole-brain tractography for each subject. All fibers are tracked within a white matter mask using deterministic tractography;
(2) segmentation of a whole brain fiber group into fascicles using an automated ROI approach. The ROIs are defined based on a previous study, and the fiber tracts are subsequently refined by comparing each fiber within the fascicle to a probabilistic fiber tract atlas (JHU whitematter tractography atlas); (3) definition of the core of the tract and filtering out of stray fibers that deviate from the core of a fiber group by representing a fiber group as a 3D Gaussian distribution before removing the fibers from the fiber group center; (4) calculation of the diffusion measurements along the trajectory of the fiber group weighing each fiber's contribution to the measurement based on its distance from the tract core. The diffusion measurement along the fiber tract core, which is a vector of 100 values, was defined as the tract profile. In this way, the tract profiles of FA and MD at 100 points along each of the 20 tracts were evaluated for each subject to depict their global white matter status.
The 20 identified tracts included: bilateral anterior thalamic radiation (ATR), corticospinal tract (CST), cingulum cingulate (CCing), cingulum hippocampus (CHippo), inferior fronto-occipital fasciculus (IFOF), inferior longitudinal fasciculus (ILF), superior longitudinal fasciculus (SLF), uncinated fasciculus (UF), arcuate fasciculus (AF), splenium, and genu of the corpus callosum (CC) [bib_ref] Reproducibility of quantitative tractography methods applied to cerebral white matter, Wakana [/bib_ref].
# Statistical analysis
Descriptive analyses were performed to describe the variables in each group. Categorical variables were compared using the Chi-squared test or Fisher's exact test. These analyses were performed using SPSS version 21 (SPSS, Chicago, IL, U.S.A). Continuous variables were compared between the three groups using one-way ANOVA, and then the false discovery rate (FDR) correction was performed in the mean FA and MD comparisons. Then pairwise comparisons among groups were conducted when necessary with the least significance difference (LSD) test.
For the VBA, the ANOVA was used to compare the FA and MD values in a voxel-.
based manner with SPM and the p level was < 0.05. For the TBSS, a nonparametric statistical thresholding with permutation-based inference tool (FSL's "randomize") was used to compare the FA and MD among three group and the p level set at p < .05, corrected for multiple comparisons using threshold-free cluster enhancement.
For the pointwise comparisons, FA and MD at 100 locations along the 20 fiber tracts were fed into a permutation-based statistical analysis with 1000 permutations. For each permutation, all the subjects were redivided into the three groups randomly, while keeping the sample sizes for each group, then the ANOVA and the post hoc test statistics were recalculated for each location of each fiber. After that, for each fiber, the maximum length of the continuous significant locations (for each location, "significant" means this location have a larger t or F statistic that corresponds to a p < .05) for each test was recorded, thus allowing the construction of the permutation distribution of the maximum length of significant locations; those locations with length within the top 5% of such distribution were considered significant at the 0.05 level (thus, familywise error rate corrected) [bib_ref] Nonparametric permutation tests for functional neuroimaging: a primer with examples, Nichols [/bib_ref].
For the comparison among the VBA, TBSS and AFQ, we calculated the percentage of significant voxels/points over the total number of evaluated voxels/points in VBA, TBSS and AFQ. Significant white matter voxels were calculated in the comparisons among groups in VBA, and tract-based voxels are calculated in TBSS. In the AFQ, we calculated the number of points with significant difference in the pointwise comparisons of the tract profiles across the 20 fiber tracts.
# Results
## Demographics and clinical information from our cohort and adni cohort
The full demographic and clinical characteristics of the subjects from our cohort and the ADNI cohort are provided in [fig_ref] Table 1: Comparison of demographic variables between the groups from our cohort and the... [/fig_ref]. In our cohort, for the 25 patients with probable AD (12 of whom were women), the mean age was 73.48 ± 11.83 years, and for the 29 patients with MCI (13 of whom were women), the mean age was 74.07 ± 10.76 years. The 34 normal control subjects (11 of whom were women) had a mean age of 69.03 ± 11.53 years. In the ADNI cohort, for the 27 patients with AD, the diagnosis of which was based on biomarkers, the mean age was 76.00 ± 9.78 years, and for the 21 patients with MCI, the mean age was 72.52 ± 8.71 years. The 22 normal control subjects had a mean age of 73.27 ± 6.05 years. The AD, MCI and NC groups did not differ in age, sex and education both in our cohort and the validation cohort. As expected, the pathological alteration among groups led to significant differences in the scores for the MMSE and MOCA among the patients with AD, MCI, and the NCs. These two cohorts did not differ in age and gender, but showed differences in education.
## Mean diffusion measures of fa and md among groups
AFQ is a new algorithm that can automatically identify white matter tracts in human brains and take measurements at anatomically equivalent locations along their trajectories. But there were some fibers failed tracked using AFQ, especially in the bilateral cingulum hippocampus tracts in this study, this may cause by the threshold setting in the fiber tracking, because the cingulum hippocampus tracts are adjacent to the gray matter. In [fig_ref] Table 2: Comparison of mean FA among the groups from our cohort [/fig_ref] , we listed the successfully traced number of subjects in each fiber (labeled n1: n2: n3) with FDR correction. A group comparison of the mean diffusion measures of each tract between the NC, MCI, and AD groups using a p < .05 threshold showed a significant difference. At the global level, a summary of the mean FA and MD of each identified fiber tract from the normal controls and patients with AD and MCI is shown in Tables 2 and 3.
## Ad patients vs. nc subjects
Compared with the normal controls, two fiber tracts showed a significantly decreased FA in the AD subjects: right CCing and left IFOF. In a group comparison of the mean MD, the AD group showed extensive increased MD relative to the NC group in most of the 20 fiber tracts in addition to the corpus callosum, right ATR, SLF, and AF.
## Mci patients vs. nc subjects
The FA reduction and MD elevation in MCI was not as obvious as in AD subjects when compared with NC. The FA revealed significant reduction in the right CCing. The MD revealed significant elevation in 4 fiber tracts: bilateral CCing, left SLF and right UF.
## Ad patients vs. mci patients
Compared with MCI subjects, the left IFOF showed a significantly decreased FA in AD. The MD values in AD were significantly higher than those in MCI in the following 6 fiber tracts: right CHippo, bilateral IFOF, left ILF, and bilateral UF. 3.3. VBA
## Ad patients vs. mci patients
The AD subjects showed no significant difference in FA and MD after GRF correction compared to MCI (Figs. 1A and 2A).
## Mci patients vs. nc subjects
The MCI subjects showed no significant difference in FA and MD after GRF correction compared to NC (Figs. 1B and 2B).
## Ad patients vs. nc subjects
Compared with the normal controls, the FA reduction and MD elevation in AD involved almost all the white matter region after Gaussian Random Field (GRF) correction (Figs. 1C and 2C). A: comparison between AD and MCI. B: comparison between MCI and NC. C:comparison between AD and N. The blue cluster in white matter region represent the reduction FA in AD compared to NC with Gaussian Random Field (GRF) correction(p < .05). The FA difference in A and B was showed without correction and there were no differences after GRF correction (p < .05). The statistic was displayed with z-values.
## Tbss
## Ad patients vs. mci patients
The MD values in AD were significantly higher than those in MCI in the following fiber tracts: bilateral SLF, ILF, IFOF, ATR, UF, CC, CHippo and left CST. There were no fibers showed FA decreased in AD, compared to MCI [fig_ref] Figure 3: The Tract-based spatial statistics [/fig_ref].
## Mci patients vs. nc subjects
The MD elevation in MCI was not as obvious as in AD subjects when compared with NC. The MD revealed significant elevation in the following fiber tracts: bilateral SLF, ILF, IFOF, Ccing, CST, CC, left UF. There were no fibers showed FA decreased in MCI [fig_ref] Figure 3: The Tract-based spatial statistics [/fig_ref].
## Ad patients vs. nc subjects
Compared with the normal controls, AD showed widespread MD reduction among the white matter tract threshold-free cluster enhancement (TFCE) correction. There were no significant fibers showed FA decreased in AD after TFCE correction [fig_ref] Figure 3: The Tract-based spatial statistics [/fig_ref]. In comparison with the NC subjects, the AD patients showed widespread FA reduction in 25% (5 /20) of examined fiber tracts and MD increase in 65%(13/20) of examined fiber tracts. Relative to the NC subjects, the MCI patients only showed regional FA reduction in 5% (1/
## Ad patients vs. nc subjects
Generally, the tract profiles in the AD group showed a significant difference compared with the NC group.
The FA decrease did not reach a significant level in mean diffusion measures in the bilateral ATR, bilateral CST, bilateral SLF, bilateral ILF, bilateral AF, left CCing, bilateral CHippo, splenium and genu of CC, whereas significant FA reduction at certain points was found in group comparison of FA profiles. The plots of the MD profiles of 20 tracts from AD patients (red), MCI patients (green) and NC subjects (blue) as means (SD) (solid lines for means and shaded areas for SDs). The color bars under MD profiles indicate the regions of significant difference between AD and NC (red), MCI and NC (green) and between MCI and AD (blue). The X-axis represents the location between the beginning and termination waypoint regions of interest. Abbreviations in context: ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus. AF, arcuate fasciculus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) In a pointwise comparison of FA profiles, the AD group showed widespread FA reduction relative to the NC group. Changed locations of fibers were demonstrated as follows: (1) the posterior portion of the left ILF; (2) the frontier portion of the left UF; (3) the middle and posterior component of the right CCing; (4) the left part of the splenium of the CC; (5) the right part of genu of the CC.
The mean diffusion measures failed to show significant elevation in the following fiber tracts: right ATR, right SLF, right AF, splenium, and genu of CC, although they displayed points with significantly increased MD in group comparisons of the MD profiles.
In a pointwise comparison of MD profiles, the AD group showed extensive MD elevation relative to the NC group. Changed locations of fibers were as the follows: (1)
## Mci patients vs. nc subjects
Compared with the NC subjects, the following locations revealed regional decreased FA in the MCI patients: the intermediate component of the right CCing. However, in group comparison of mean diffusion measures, none of the fibers showed reduced FA reached the level of significant after FDR correction.
The MD revealed significant elevations in the temporal lobe portion of left AF. However, in group comparison of mean diffusion measures, none of the fibers showed increased MD reached the level of significant after FDR correction.
## Ad patients vs. mci patients
The AD versus MCI contrast demonstrated significant FA reduction in the AD patients in the intermediate component of right ILF. However, in the group comparison of mean diffusion measures, none of the fibers showed reduced FA reached the level of significant after FDR correction.
The MD values were significantly higher than those of the MCI patients in the following locations: the posterior portion of the left IFOF and the intermediate component of the right UF. The mean diffusion measures failed to show significant MD elevation after FDR correction.
## Number of points with significant differences in tract profiles
To evaluate the percentage of points with white matter lesions for each fiber tract, we calculated the number of points with significant difference in the pointwise comparisons of the tract profiles across the 20 fiber tracts (as described in [fig_ref] Table 4: Number of points with significant difference in pointwise comparison of tract profiles... [/fig_ref].
Tract Profiles of FA and MD among the groups from ADNI cohort. In general, group comparisons of tract profiles between the NC, MCI, and AD groups using a p < .05 threshold (FWE-corrected) identified many points of significant difference. The position of the 20 fiber tracts and tract profiles of 100 locations along the fiber tracts and the results of a pointwise comparison of FA and MD profiles among AD, MCI, and NC groups are illustrated in Figs. 6 and 7.
In comparison with the NC subjects, the AD patients showed FA reduction in 10% (2 /20) of examined fiber tracts and MD increase in 65%(13/20) of examined fiber tracts. Relative to the NC subjects, the MCI patients showed no FA reduction nor MD increase of examined fiber tracts. Compared with the MCI patients, the AD patients showed FA reduction in 10% (2/20) of examined fiber tracts and an MD increase in 15%(3/20) examined fiber tracts.
## Ad patients vs. nc subjects
Generally, the tract profiles in the AD group showed a significant difference compared with the NC group.
In a pointwise comparison of FA profiles, the AD group showed FA reduction relative to the NC group. Changed locations of fibers were demonstrated as follows: (1) the right part of the genu of the CC; (2) the frontier portion of the left UF.
In a pointwise comparison of MD profiles, the AD group showed extensive MD elevation relative to the NC group. Changed locations of fibers were as the follows: (1)
## Mci patients vs. nc subjects
Compared with the NC subjects, none of the fiber locations showed reduced FA nor increased MD reached the level of significant after FWE correction.
## Ad patients vs. mci patients
The AD versus MCI contrast demonstrated significant FA reduction in the AD patients in the frontal component of bilateral UF.
The MD values were significantly higher than those of the MCI patients in the following locations: the posterior and middle portion of the left ILF and the intermediate component of the left UF and CCing.
## Number of points with significant differences in tract profiles
To evaluate the percentage of points with white matter lesions for each fiber tract, we calculated the number of points with significant difference in the pointwise comparisons of the tract profiles across the 20 fiber tracts (as described in [fig_ref] Table 6: Number of points with significant difference in pointwise comparison of tract profiles... [/fig_ref].
## The comparison among the vba, tbss and afq
The quantification FA and MD comparisons among the VBA, TBSS and AFQ were respectively showed in Figs. 8 and 9.
We found that VBA detected the highest percentage of abnormalities either in the FA or MD analysis. The FA decrease and MD elevate percentage in AD was respectively 11.63% and 38.75% in VBA, compared to MCI. TBSS found none FA abnormalities in AD vs MCI, MCI vs NC and AD vs NC, but it showed 29.45% abnormalities in AD, compared to MCI. There were 73.03% abnormalities of MD in AD when compared to NC, and 15.6% in MCI compared to NC. The AFQ detected changes in all the comparisons of FA and MD, mostly in the AD vs NC. When compared to NC, AD showed 7% points among 100 points of 20 fibers respectively FA reduction, and 32.85% in MD elevate. There were 0.95% points showed FA reduction in AD, compared to MCI, and 3.05% points showed MD elevate. The MCI subjects showed 1.55% FA reduction and 1.05% MD elevate when compared to normal control.
## The comparison of the afq findings between adni cohort and our cohort
In a pointwise comparison of FA profiles, the AD group from ADNI cohort showed FA reduction in the genu of the CC and the left UF, which is included in our findings. The AD versus MCI contrast demonstrated significant FA reduction in the AD patients in the bilateral UF, but it showed no similar results in our cohort.
In a pointwise comparison of MD profiles, the AD group from ADNI cohort showed significantly similar fibers changes in MD elevation except the right UF and the splenium of the CC compared to NC. The right portion of the splenium of the CC showed MD elevation in AD patients from ADNI cohort, but there is nothing abnormal in our cohort.
Also, we find the MD elevation in whole right UF, while the fiber showed normal in AD from ADNI cohort. (as described in .
# Discussion
As a relatively new algorithm, AFQ can automatically identify white matter tracts in human brains and anatomically measure at equivalent locations along these fibers. In this study, we performed this method to extract FA and MD at 100 locations along 20 fiber tracts to evaluate tract profiles of the white matter integrity across AD and MCI. In comparison with NC, AD patients showed widespread FA reduction in 25% (5/20) examined fiber tracts and MD increase in 65% (13/20) examined fiber tracts. While MCI subjects showed regional FA reduction in 5% (1/20) examined fiber tracts (right cingulum cingulate) and MD increase in 5% (1/20) of examined fiber tracts (left arcuate fasciculus). Then we validated the findings with ADNI cohort, which showed significantly similar changes in the pointwise comparison of MD in AD compared to NC. In the mean FA and MD comparisons, there were no FA or MD differences reach statistical differences in MCI and NC, which was also similar finding in AD and MCI. In contrast, in the VBA analysis, widespread FA reduction and MD increase were found in AD, when compared to NC. But there was not any difference in the AD and MCI or NC and MCI. In the TBSS analysis, no significant difference was found for FA among all the three groups, while widespread MD increasing was found in AD when compared to NC, which was similar to the VBA results. In summary, using the point wise method of AFQ, we have found the location specific white matter abnormalities, which could not be achieved with the mean FA and MD analyses. Therefore, AFQ is an important supplement in the white matter studies.
In our results of VBA analysis, almost all the white matter voxels showed difference of FA and MD in AD, compared to normal controls, but there is no significant difference between MCI and NC or AD and MCI. Therefore, VBA seems to be very sensitive to those larger differences, like AD vs. NC but have low spatial specificity. When the difference is milder (like MCI and NC or AD and MCI), it showed no Summary of change in mean FA, mean MD, location of points with significant difference in pointwise comparison of tract profiles across 20 fiber tracts among the groups from our cohort.
[formula] ATR_L − − − − − − + − − A/M − − ATR_R − − − − − − − − − − − − CST_L − − − − − − + − − S/M − − CST_R − − − − − − + − − S − − CCing_L − − − − − − + − − − − − CCing_R + − − P/M M − + − − P − − CHippo_L − − − − − − + − − P − − CHippo_R − − − − − − + − − − − − CC Splenium − − − L − − − − − − − − CC Genu − − − R − − − − − − − − IFOF_L + − − − − − + − − P/M − P IFOF_R + − − − − − + − − P/M − − ILF_L − − − P − + − − A/M − − ILF_R − − − − − M + − − A − − SLF_L − − − − − − + − − A/P − − SLF_R − − − − − − − − − − − − UF_L + − − F − + − − M/F − − UF_R + − − − − − + − − W − M AF_L − − − − − − + − − T/M T − AF_R − − − − − − − − − − − − [/formula]
Note: AD, Alzheimer's disease; MCI, mild cognitive impairment; NC, normal control; ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus; AF, arcuate fasciculus. L, left; R, right. +, difference between the groups; −, no difference between the groups; A, difference in anterior part of the fiber; P, difference in posterior part of the fiber; S, difference in superior part of the fiber; M, difference in middle part of the fiber; T, difference in the temporal lobe part of the fiber; W, difference in the entire fiber. FDR correction was used in comparison in mean FA with a p value < .05. FWE correction was used in point wised location changes with a p value < .05. difference either with FA or MD. This "all-or-none" result may because low spatial precisely of the fibers when using morphology based normalization. For TBSS, we also see an "all-or-none" result. MD elevation in inferior longitudinal fasciculus, superior longitudinal fasciculus and many other fibers in AD, compared to normal controls, but no significant results were found between AD and MCI or MCI and NC. None results was found for FA. As a tract-based voxel grouping analysis technique, TBSS partially overcome the problem of each voxel not totally on the same fiber after registration in VBA. But the inconsistent shape of fiber tract across participants may lead to error registration, such as the corpus callosum and so on. Jin et al. reported that Tractspecific analysis (TSA) can reveal detailed local alterations in each tract, which provide a 3D detailed profile that reflects local alterations in a particular tract associated with AD or MCI [bib_ref] 3D tractspecific local and global analysis of white matter integrity in Alzheimer's..., Jin [/bib_ref]. Meanwhile the AFQ showed more specific locations in fibers of MD difference in AD and NC, while it is sensitive to detect some mild alteration between MCI and NC or AD and MCI, which indicate that it has advantages, and could be a complementary method to the classic ones. Neurobiological systematical variation along each tract has been considered as the potential result of different patterns of white matter abnormalities across neuronal fiber tracts. Using AFQ, we found that most of the examined fiber tracts showed widespread white matter abnormalities in AD, while in MCI, only a few of the fiber tracts showed FA and MD alterations, whose findings were similar in the ADNI cohort. Moreover, among these changed tracts, only the right cingulum cingulate and the left arcuate fasciculus displayed obvious disruption of myelin and/or fiber axons in MCI and aggravated deterioration in AD. This finding was supported by FA/MD changes through both the mean, the TBSS and point wise method. We also found the location of white matter abnormalities to be different across 20 examined neuronal fiber tracts; for example, the MCI and AD patients showed similar FA reduction in the middle part of the right cingulum cingulate, whereas the anterior part were reserved. However, the left arcuate fasciculus showed MD elevation located at temporal part of the fibers in MCI and expanded to the temporal and middle part of the fibers in AD.
In the limbic system, the fMRI studies have suggested that hypometabolism or hypo-perfusion initially occur in the posterior cingulate cortex (PCC) [bib_ref] Retrosplenial cortex (BA 29/30) hypometabolism in mild cognitive impairment (prodromal Alzheimer's disease), Nestor [/bib_ref] [bib_ref] Limbic hypometabolism in Alzheimer's disease and mild cognitive impairment, Nestor [/bib_ref]. We found the middle part or the middle component of the right cingulum cingulate showed FA reduction in both MCI and AD patients with AFQ analyses. Previous studies found hypo-metabolism of the middle cingulate gyrus in AD using 18F-FDG PET [bib_ref] Relationships between hippocampal atrophy, white matter disruption, and gray matter hypometabolism in..., Villain [/bib_ref] and showed an Aβ load in the right posterior cingulate using 11 C-PIB EPT, which were partially consistent with our results. Among association fiber tracts, the uncinated fasciculus is associated with episodic memory, the formation and retrieval of memory and cognition [bib_ref] Topography of the uncinate fascicle and adjacent temporal fiber tracts, Ebeling [/bib_ref]. Previous studies have reported that FA values in the uncinated fasciculus are significantly lower and MD values are significantly higher in patients with AD than in healthy controls [bib_ref] Diffusion anisotropy and diffusivity of white matter tracts within the temporal stem..., Taoka [/bib_ref] [bib_ref] Diffusion abnormalities of the uncinate fasciculus in Alzheimer's disease: diffusion tensor tractspecific..., Yasmin [/bib_ref]. In this study, we noted supportive results with TBSS and AFQ, and the ADNI cohort showed same findings with AFQ. Multiple AD-risk studies have reported reduced white matter integrity in the inferior fronto-occipital fasciculus [bib_ref] Altered brain white matter integrity in healthy carriers of the APOE ε4..., Persson [/bib_ref] [bib_ref] Alterations in multiple measures of white matter integrity in normal women at..., Gold [/bib_ref] [bib_ref] White matter diffusion alterations in normal women at risk of Alzheimer's disease, Smith [/bib_ref]. One study revealed that the Aβ42/p-Tau181 ratio was positively correlated with FA in the bilateral inferior fronto-occipital fasciculus, suggesting this tract is involved in AD pathology [bib_ref] White matter changes in preclinical Alzheimer's disease: a magnetic resonance imagingdiffusion tensor..., Molinuevo [/bib_ref]. An MD increase more than an FA reduction in the inferior fronto-occipital fasciculus indicates that the deterioration from the MCI to AD might be primarily caused by the myelin degradation progress. We also found that even in AD, FA reduction and MD elevate was not significant in terms of mean diffusion measures in the genu and splenium of the corpus callosum, whereas using AFQ we found the left part of splenium of corpus callosum and the right part of genu of corpus callosum showed significant FA reduction. The ADNI cohort also showed similar pointwise changes of FA reduction in the corpus callosum. A previous study showed that Aβ deposition was correlated with reduced FA in the splenium of the corpus callosum but not in the entire corpus callosum [bib_ref] Associations between white matter hyperintensities and beta amyloid on integrity of projection,..., Chao [/bib_ref]. Interestingly, we also found that there were no differences among AD, MCI or NC subjects in terms of the mean FA and MD, but there were some abnormal locations in the tract profiles using AFQ.
The above abnormality of white matter bundles indicated the pathological process of AD. The pathology of neurofibrillary tangles shows a significantly correlation with cognitive impairment in AD, which develops first in the trans entorhinal cortex and then may spread to the entorhinal cortex in stages I and II, adjacent to the hippocampus, which is part of the limbic system in stages III and IV, followed by the medial temporal isocortex ("isocortical", stages V/VI), and finally into the brain [bib_ref] Hippocampal plasticity during the progression of Alzheimer's disease, Mufson [/bib_ref]. We found that in MCI, FA reduction occurs primarily in the right cingulum cingulate, bilateral anterior thalamic radiation and uncinated fasciculus, and the genu and splenium of the corpus callosum. At the AD stage, these pathological changes occur in additional fibers, including the bilateral corticospinal tract, left cingulum cingulate, and left cingulum hippocampus, which is consistent with previous reports of the Aβ progression pathway.
Although we found more abnormalities of myelin or/and fiber axons in the 20 examined fiber tracts by pointwise tract profiles among the groups than mean diffusion measures, and prove AFQ is an important supplement of VBA and TBSS, there are a few limitations in this study. First, given that the AD patients are at a late disease stage, it is possible that gray matter atrophy in these patients may have a technical influence on the tractographic results, and therefore such atrophy may contribute to the observed abnormality of the tract profile. Second, DTI measurement could be associated with the prominent white matter hyper-intense lesions found in these patients. The current data are limited in their ability to distinguish these two issues. Third, because of A, difference in anterior part of the fiber; P, difference in posterior part of the fiber; M, difference in middle part of the fiber; T, difference in the temporal lobe part of the fiber; F, difference in the frontal lobe part of the fiber. FDR correction was used in comparison in mean FA with a p value < .05. FWE correction was used in point wised location changes with a p value < .05.
[formula] ATR_L - - - A - - ATR_R - - - A - - CST_L - - - - - - CST_R - - - - - - CCing_L - - - - - - CCing_R - - - M - - CHippo_L - - - A - M CHippo_R - - - - - - CC Splenium - - - R - - CC Genu R - - - - - IFOF_L - - - M - - IFOF_R - - - M - - ILF_L - - A/M/P - M/P ILF_R - - - M/P - - SLF_L - - - M/P - - SLF_R - - - M/P - - UF_L F - F F/M/T - M UF_R - - F - - - AF_L - - - M/F - - AF_R - - - - - - Note: AD, [/formula]
the threshold setting in the fiber tracking, some fibers like bilateral cingulum hippocampus tracts are adjacent to the gray matter and the FA in this part were low, this may lead to fail track some fibers. Forth, the AD diagnosis was not based on biomarkers but only on the neuropsychological scales, which was subjected by our research conditions and might cause imprecise diagnosis.
In conclusion, AFQ can automatically reconstruct white matter tracts in human brains and detect at equivalent locations along these fasciculi, which can be a complementary method for VBA and TBSS technique. We used this method in AD, MCI patients and NC subjects, and examined two diffusion parameters (FA and MD) at 100 locations along 20 white matter fiber tracts. Our results indicated that AFQ can detect changes in specific location along fibers, and the pattern of the disrupted location was different among the fibers, but in most, the disruption started from a certain region in MCI before, expanding to include entire fibers in AD, whose findings were validated by the ADNI cohort. These results may enrich the study on neurodevelopmental alterations and the progression of AD, which can provide new insights into the determination of white matter in the progression from MCI to AD with AFQ. . The percentage of the significant FA reduction region of VBA, TBSS and AFQ, respectively, among AD, MCI, and NC groups. 0% means there were no differences of FA in the comparison between AD vs NC, MCI vs NC and AD vs MCI with TBSS. The y-axis is the percentage of significant voxels/points over the total number of evaluated voxels/points in VBA, TBSS and AFQ. . The percentage of the significant MD elevation region of VBA, TBSS and AFQ respectively, among AD, MCI, and NC groups. The y-axis is the percentage of significant voxels/points over the total number of evaluated voxels/points in VBA, TBSS and AFQ.
contributions are facilitated by the Foundation for the National Institutes of Health (www.fnih.org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer's Therapeutic Research Institute at the University of Southern California. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California.
## Table 8
Summary of change in the location of points with significant difference in pointwise comparison of tract profiles across 20 fiber tracts among the groups between our cohort and the ADNI cohort.
[formula] - - - - - A/M A - - - - ATR_R - - - - - - - A - - - - CST_L - - - - - - S/M - - - - - CST_R - - - - - - S - - - - - CCing_L - - - - - - - - - - - - CCing_R P/M - M - - - P M - - - - CHippo_L - - - - - - P A - - - M CHippo_R - - - - - - - - - - - - CC Splenium L - - - - - - R - - - - CC Genu R R - - - - - - - - - - IFOF_L - - - - - - P/M M - - P - IFOF_R - - - - - - P/M M - - - - ILF_L P - - - - A/M A/M/P - - - M/P ILF_R - - - - M - A M/P - - - - SLF_L - - - - - - A/P M/P - - - - SLF_R - - - - - - - M/P - - - - UF_L F F - - - F M/F F/M/T - - - M UF_R - - - - - F W - - - M - AF_L - - - - - - T/M M/F T - - - AF_R - - - - - - - - - - - - [/formula]
Note: AD, Alzheimer's disease; MCI, mild cognitive impairment; NC, normal control; ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus; AF, arcuate fasciculus. L, left; R, right. +, difference between the groups; −, no difference between the groups; A, difference in anterior part of the fiber; P, difference in posterior part of the fiber; S, difference in superior part of the fiber; M, difference in middle part of the fiber; T, difference in the temporal lobe part of the fiber; W, difference in the entire fiber. FDR correction was used in comparison in mean FA with a p value < .05. FWE correction was used in point wised location changes with a p value < .05.
[fig] Figure 1: The Voxel-based analysis (VBA) of FA among AD, MCI, and NC groups. [/fig]
[fig] Figure 2: The Voxel-based analysis (VBA) of MD among AD, MCI, and NC groups. A: comparison between AD and MCI. B: comparison between MCI and NC. C:comparison between AD and NC. The yellow cluster in white matter region represent the elevation MD in AD compared to NC with Gaussian Random Field (GRF) correction (p < .05). The MD difference in A and B was showed without correction and there were no differences after GRF correction (p < .05). The statistic was displayed with z-values. [/fig]
[fig] Figure 3: The Tract-based spatial statistics (TBSS) of MD among AD, MCI, and NC groups. A: comparison between AD and MCI. B: comparison between MCI and NC. C:comparison between AD and NC. The FA skeleton is displayed in green, which represent the center of the white matter tracts. Red-yellow color bar indicates significant MD elevate (p < .05 corrected for multiple comparisons using threshold-free cluster enhancement, the statistic value beside the color bar is 1-p). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)X. Zhang, et al. NeuroImage: Clinical 22 (2019) 101723 3.5. Tract profiles of FA and MD among the group from our cohort In general, group comparisons of tract profiles between the NC, MCI, and AD groups using a p < .05 threshold(FWE-corrected)identified many points of significant difference. The position of the 20 fiber tracts and tract profiles of 100 locations along the fiber tracts and the results of a pointwise comparison of FA and MD profiles among AD, MCI, and NC groups are illustrated in Figs. 4 and 5. [/fig]
[fig] Figure 4: The Pointwise Comparison of FA Profiles among AD, MCI, and NC groups from our cohort. The plots of the FA profiles of 20 tracts from AD patients (red), MCI patients (green) and NC subjects (blue) as means (SD) (solid lines for means and shaded areas for SDs). The color bars under the FA profiles indicate the regions of significant difference between AD and NC (red), MCI and NC (green), and between MCI and AD (blue). The X-axis represents the location between the beginning and termination waypoint regions of interest. Abbreviations in context: ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus. AF, arcuate fasciculus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)X. Zhang, et al. NeuroImage: Clinical 22 (2019) 101723 20) of examined fiber tracts and an MD increase in 5%(1/20) of examined fiber tracts, primarily in the right CCing and the left AF. Compared with the MCI patients, the AD patients showed FA reduction in 5% (1/20) of examined fiber tracts and an MD increase in 10%(2/20) examined fiber tracts. [/fig]
[fig] Figure 5: The Pointwise Comparison of MD Profiles among AD, MCI, and NC groups from our cohort. [/fig]
[fig] Figure 6: The Pointwise Comparison of FA Profiles among AD, MCI, and NC groups from the ADNI cohort. The plots of the FA profiles of 20 tracts from AD patients (red), MCI patients (green) and NC subjects (blue) as means (SD) (solid lines for means and shaded areas for SDs). The color bars under the FA profiles indicate the regions of significant difference between AD and NC (red), MCI and NC (green), and between MCI and AD (blue). The X-axis represents the location between the beginning and termination waypoint regions of interest. Abbreviations in context: ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus. AF, arcuate fasciculus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) [/fig]
[fig] Figure 7: The Pointwise Comparison of MD Profiles among AD, MCI, and NC groups from the ADNI cohort. The plots of the MD profiles of 20 tracts from AD patients (red), MCI patients (green) and NC subjects (blue) as means (SD) (solid lines for means and shaded areas for SDs). The color bars under MD profiles indicate the regions of significant difference between AD and NC (red), MCI and NC (green) and between MCI and AD (blue). The X-axis represents the location between the beginning and termination waypoint regions of interest. Abbreviations in context: ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus. AF, arcuate fasciculus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) [/fig]
[table] Table 1: Comparison of demographic variables between the groups from our cohort and the ADNI cohort. [/table]
[table] Table 2: Comparison of mean FA among the groups from our cohort.Note: Data are presented as means ± standard deviations. AD, Alzheimer's disease; MCI, mild cognitive impairment; NC, normal control; ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus. AF, arcuate fasciculus. L, left; R, right. Least significant difference test was used in multiple comparisons. n1: n2: n3 means the successfully traced number of AD, MCI, and NC subjects in each fiber. ⁎ Significant difference with a p value < .05 with ANOVA or Post-hoc LSD multiple comparisons. ⁎⁎ Significant difference with a p value < .05(FDR corrected). [/table]
[table] Table 3: Comparison of mean MD among the groups from our cohort.Note: Unless otherwise specified, data reflect the MD values × 10 −3 mm 2 s −1 presented as means ± standard deviations. AD, Alzheimer's disease; MCI, mild cognitive impairment; NC, normal control; ATR, anterior thalamic; CST, corticospinal tract; CCing, cingulum cingulate; CHippo, cingulum hippocampus; CC, corpus callosum; IFOF, inferior fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus; SLF, superior longitudinal fasciculus; UF, uncinated fasciculus; AF, arcuate fasciculus. L, left; R, right. Least significant difference test was used in multiple comparisons. n1: n2: n3 means the successfully traced number of AD, MCI, and NC subjects in each fiber. ⁎ Significant difference with a p value < .05 with Post-hoc LSD multiple comparisons. ⁎⁎ Significant difference with a p value < .05(FDR corrected). [/table]
[table] Table 4: Number of points with significant difference in pointwise comparison of tract profiles across 20 fiber tracts after permutation based FWE correction from our cohort. [/table]
[table] Table 6: Number of points with significant difference in pointwise comparison of tract profiles across 20 fiber tracts from the ADNI cohort after permutation based FWE correction. [/table]
[table] Table 7: Summary of change in the location of points with significant difference in pointwise comparison of tract profiles across 20 fiber tracts among the groups from the ADNI cohort. [/table]
|
E2 Proteins of High Risk Human Papillomaviruses Down-Modulate STING and IFN-κ Transcription in Keratinocytes
In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-k that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-k and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the transactivation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-k and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-k were further determined in clinical specimens by real time PCR. STING and IFN-k were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-k transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.
# Introduction
Human papillomavirus (HPV) infection is the most common sexually transmitted infection with approximately 50-80% of sexually active adults acquiring one or more HPV types during their lifetime. The risk of infection is depending on numerous factors including host factors such as the number of sexual partners, age of sexual activity and genetic background as well as viral factors such as the specific viral genotype [bib_ref] Epidemiology of genital human papillomavirus infection, Koutsky [/bib_ref] [bib_ref] Cervical HPV infection and neoplasia in a large population-based prospective study: the..., Peto [/bib_ref]. HPV genotypes infecting the ano-genital mucosal epithelium are categorized as 'low-risk' (LR-HPV) or 'high-risk' types (HR-HPV), depending on their association with either benign lesions or lesions that can progress to cancers [bib_ref] Papillomavirus infections-a major cause of human cancers, Zur Hausen [/bib_ref]. Specifically, HR-HPVs are now recognized as the etiological agents of cervical cancer [bib_ref] The causal relation between human papillomavirus and cervical cancer, Bosch [/bib_ref]. HPV life cycle is dependent on the differentiation program that keratinocytes undergo within the infected stratified epithelium. Once HPV infects the epithelium through micro-abrasion, the incubation period for production of mature viral particles is from few weeks to years depending on many factors including the infectious dose. HPV infections are very common and most of them remain asymptomatic and are cleared within 6-12 months through an effective host immune response [bib_ref] Papillomaviruses and cancer: from basic studies to clinical application, Zur Hausen [/bib_ref]. The cell mediated immune responses to viral early proteins, such as E2 and E6 for instance [bib_ref] A prospective study on the natural course of low-grade squamous intraepithelial lesions..., Woo [/bib_ref] , are probably involved in spontaneous regression of cervical lesions that is accompanied or followed by circulating antibodies or sero-conversion to the major capsid protein L1 [bib_ref] Human papillomavirus type 16 E2-and L1-specific serological and T-cell responses in women..., Davidson [/bib_ref]. However, approximately 10-20% of infected women have persisting HR-HPV infection and progress to high grade cervical intra-epithelial neoplasia, CIN2/3 and then invasive cancer [bib_ref] Human papillomavirus testing in the prevention of cervical cancer, Schiffman [/bib_ref]. Thus, lack of effective immune responses is a potent factor to carcinogenic progression. The innate immune system has evolved as the first line of host defense against a variety of pathogens. Nucleic acids of pathogens are potent inducers of cellular innate immune defenses after infection [bib_ref] Pattern recognition receptors and control of adaptive immunity, Palm [/bib_ref] [bib_ref] Innate immunity to virus infection, Takeuchi [/bib_ref] [bib_ref] TLRs and innate immunity, Beutler [/bib_ref]. The initiation of innate immune responses relies on the recognition of pathogen components by germline encoded pattern-recognition receptors (PRRs), including the membranebound Toll-like receptors family (TLRs) [bib_ref] Toll-like receptor function and signaling, Kaisho [/bib_ref] , the retinoic acidinducible gene I-like receptors family (RLRs) [bib_ref] Viral evasion and subversion of patternrecognition receptor signaling, Bowie [/bib_ref] , the nucleotide oligomerization domain-like receptors family (NLRs), and cytosolic DNA sensors [bib_ref] Pattern recognition receptors and the innate immune response to viral infection, Thompson [/bib_ref]. Upon recognition, they initiate signal transduction pathways leading to the induction of type I IFN and pro-inflammatory cytokines, which are required for innate immune responses. In this regards, several previous reports indicated that HPV early proteins play major roles in immune evasion of virally induced lesions. For example, E5, E6, and E7 proteins were shown to inhibit delivery of major histocompatibility (MHC) class I molecules, as well as antigen processing and presentation [bib_ref] E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class..., Ashrafi [/bib_ref] [bib_ref] HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not..., Bauer [/bib_ref] [bib_ref] Antigen processing defects in cervical carcinomas limit the presentation of a CTL..., Evans [/bib_ref]. E6 and E7 also disrupt the expression and functions of type I IFN [bib_ref] Interferon response depends on viral transcription in human papillomavirus-containing lesions, Arany [/bib_ref] [bib_ref] Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein...., Park [/bib_ref] via interferon response factors (IRF) type -1 [bib_ref] Abrogation of IRF-1 response by high-risk HPV E7 protein in vivo, Um [/bib_ref] and type-3 [bib_ref] Identification of a member of the interferon regulatory factor family that binds..., Au [/bib_ref]. E6 can also bind tyrosine kinase (Tyk) 2 and inhibit its binding to the cytoplasmic portion of the IFN-a receptor 1 therefore inhibiting phosphorylation of Tyk2, as well as signal transducers and activators of transcription STAT-1 and STAT-2, causing the defect in Janus kinase (JAK) and STAT activation, which interferes with IFN-a mediated signaling [bib_ref] The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and..., Li [/bib_ref]. Human keratinocytes are the main target of HPV infection in vivo [bib_ref] Papillomaviruses and cancer: from basic studies to clinical application, Zur Hausen [/bib_ref]. The E2 protein, which is expressed at the early stage of the HPV life cycle, is a 42-kDa mostly nuclear protein containing three functional domains that are relatively conserved among all papillomaviruses [bib_ref] Crystal structure of the E2 DNA-binding domain from human papillomavirus type 16:..., Hegde [/bib_ref] , consisting of an amino-terminal transactivation domain (TAD), a proline-rich hinge region and a carboxy-terminal dimerization DNA binding domain (DBD) [bib_ref] Evolutionary and biophysical relationships among the papillomavirus E2 proteins, Blakaj [/bib_ref]. HPV E2 plays essential roles in the viral life cycle by activating viral DNA replication in concert with the E1 viral helicase [bib_ref] Papillomavirus DNA replicationfrom initiation to genomic instability, Kadaja [/bib_ref] and regulating viral genome maintenance by its association with the cellular bromo-domain 4 (Brd4) protein [bib_ref] Variations in the association of papillomavirus E2 proteins with mitotic chromosomes, Oliveira [/bib_ref]. HPV8 E2 protein down-regulates b4-integrin (ITGB4) expression in normal human keratinocytes by displacement of the activator protein 1 (AP-1) cellular transcriptional factor (Jun-B/Fra-1) from the ITGB4 promoter [bib_ref] Human papillomavirus type 8 E2 protein unravels JunB/Fra-1 as an activator of..., Oldak [/bib_ref] , thus mediating transcriptional activation of SF2/ASF [bib_ref] RNA splicing factors regulated by HPV16 during cervical tumour progression, Mole [/bib_ref]. These previous data thus indicate that HPV16 E2 can modulate transcription of both viral and cellular genes. In addition, HPV E2 proteins can interact with many cellular proteins involved in transcriptional regulation including SMCX, EP400, BRD4, TopBP1, hSNF5 [fig_ref] Table 1: Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein [/fig_ref] , p/CAP, CBP, PARP (poly(ADP-ribose) polymerase 1), p300, Sp1, AMF-1/ Gps2, hNAP-1, BRM, and Mdm2 (reviewed in [bib_ref] Tumor suppressor or oncogene? A critical role of the human papillomavirus (HPV)..., Bellanger [/bib_ref]. Since HPV E2 protein is involved in a large variety of cellular functions, we decided to investigate its putative role in the context of immune evasion. Genome-wide expression profiling of undifferentiated human primary keratinocytes (HPK) transduced with HPV16E2 expression vectors was investigated for innate immune associated genes. The results showed that stimulator of interferon genes (STING) and IFN-k were specifically down-modulated in human keratinocytes transiently expressing HPV16E2. STING silencing revealed a link between STING and IFN-k in these cells and IFNk silencing lead to transcriptional inhibition of downstream interferon stimulated target genes (ISGs). Abrogation of IFN-k expression may represent an early and central key event in the progression of HPV-infected lesions and ultimately in the development of cervical cancer.
# Materials and methods
## Clinical specimens
Fresh cervical tissue biopsies were obtained from 148 patients recruited at the Gynecology Department, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. The Ethics Committee of Khon Kaen University has approved this study (no. HE541377) and all subjects gave written informed consents for participation. HPV infection status of the cervical tissue biopsies was determined by PCR and HPV reverse line blot hybridization.
## Cell culture
Human primary keratinocytes were used in this study including human primary neonatal foreskin keratinocytes (HPK) (Lonza, Basel, Switzerland), human primary adult foreskin keratinocytes single donor NUH49 and TB3 (HPEKas.05, CELLnTEC, Bern, Switzerland). The keratinocytes were cultured in supplemented CnT-57 medium (CELLnTEC, Bern, Switzerland). All cells used were tested and found free of mycoplasma.
## Transduction
Recombinant adenoviruses containing GFP, GFP-HPV16 E2, GFP-HPV18 E2, and 2 truncated forms of HPV18 E2, GFP-HPV18 E2TAD (containing the amino-terminal transactivation domain of HPV18 E2), and GFP-HPV18 E2DBD (containing the DNA binding domain of HPV18 E2) were used as previously described [bib_ref] Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18..., Demeret [/bib_ref] [bib_ref] Nucleo-cytoplasmic shuttling of high risk human papillomavirus E2 proteins induces apoptosis, Blachon [/bib_ref]. Keratinocytes were allowed to attach on plate surface for 24 h and were then transduced with recombinant adenoviruses at a multiplicity of infection (m.o.i.) of 50 [bib_ref] Loss of HPV16 E2 protein expression without disruption of the E2 ORF..., Xue [/bib_ref]. These conditions of infection allowed ,90% positively transduced cells expressing GFP that were collected by scraping for RNA extraction after 48 h.
## Gene silencing in hpk
Gene silencing of STING and IFN-k was performed using siRNA prepared against STING, IFN-k, and a siControl (Dharmacon Thermo Fisher Scientific, New Hampshire, USA). The siRNA were transfected to HPK cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA). Briefly, 80 nmol of siRNA was added to 500 ml Opti-MEM (Gibco Life Technologies, Invitrogen, Carlsbad, CA, USA) in 6-well plates. Lipofectamine RNAiMax was added, gently mixed and incubated at room temperature for 15 min. 2610 4 cells of HPK were re-suspended in 2.5 ml of supplemented CnT-57 medium and were gently placed to the well containing the siRNA mixture. The cells were incubated for 48 h and then harvested for RNA extraction.
Total RNA Isolation and Quantitative RT-PCR DNA and RNA were extracted from fresh cervical tissue biopsies by AllPrep DNA/RNA Mini Kit (QIAGEN, Singapore). The extracted RNA was reverse transcribed using reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random hexamer (Promega, WI, USA). PCR reactions were performed with gene-specific primers for STING, IFN-k, and house-keeping gene GAPDH (Sigma-Aldrich, MO, USA).
Total RNA from HPK, TB3, and NUH49, that were transduced with recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD, were isolated using RNeasy mini kit (QIAGEN, Singapore) followed by RNeasy mini protocol. Total RNA (0.2 mg) was reverse transcribed using reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random hexamer (Promega, WI, USA). Duplicate PCR reactions were performed with genespecific primers for STING, IFN-k, IFIT1, IFIT3, OAS1, OAS2, OAS3, MX1, MX2, PKR, GFP, and GAPDH (Sigma-Aldrich, MO, USA). Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Invitrogen, Carlsbad, CA, USA). Threshold cycle numbers (CT) were determined with 7900HT Fast Real-time PCR System (Applied Biosystems, Invitrogen, Carlsbad, CA, USA) and the relative quantities of mRNA per sample were calculated using the DDCT method using GAPDH as the calibrator gene. The relative levels of mRNA were determined by setting the mRNA expression level of the lowest expression control to 1.
## Crna synthesis and microarray hybridization
Triplicate of GFP and GFP-HPV16 E2 transduced HPK cells were harvested after 48 h of transduction. Total RNA were isolated and analyzed on an RNA 6000 Nano Lab-on-a-Chip in the 2100 Bioanalyzer (Agilent Technology, ON, CA), showing RIN score above 9.4. 200 ng of RNA were labeled using Illumina TotalPrep-96 RNA Amplification kit (Ambion Life Technologies, Invitrogen, Carlsbad, CA, USA) as per amplification protocol. 750 ng of cRNA generated from amplification and labeling were hybridized into one Human HT-12 v4.0 BeadChip. The BeadChip was incubated at 58uC, with rotation speed 5 for 18 h for hybridization. The BeadChip was washed and stained as per the Illumina protocol and scanned on the iScan (Illumina, CA, USA). The data files were quantified in GenomeStudio Version 2011.1 (Illumina, CA, USA). All samples passed Illumina's sample dependent and independent QC metrics.
## Analysis of differential gene transcription
Gene expression data were imported into Partek Genomics Suite 6.5 (Partek, St Louis, Mo). Raw data were preprocessed, in steps of background correction, normalization, and summarization using robust multi-array average analyses, and the expression data were transformed to log2. Principal-component analysis (PCA) was performed to identify outliers of sample group. Differential expression analysis for the samples was performed using one way analysis of variance (ANOVA). Gene lists were created using a cut-off of P#0.05, 1.5-fold change. Hierarchical clustering was performed using the Gene Expression module. Gene Ontology (GO), an established gene database, was used to define biological categories and functions. Network analysis was performed using core analysis of Ingenuity Pathways Analysis (IPA) to construct the network from microarray gene list. Signaling pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database to define the top of signaling pathways that were altered by HPV16 E2. The microarray data was deposited at Gene Expression Omnibus with accession no GSE54008.
# Statistical analysis
All real-time PCR data were expressed as mean 6 SD. Statistical analyses were performed with SPSS using ANOVA. The groups in each experiment were three or more. Following ANOVA, a post hoc test was performed using the Least Significant Difference test (LSD) and Tukey's Honestly Significant difference test (Tukey's HSD). If P#0.05, results are considered statistically significant.
# Results
## Hpv gfp-e2 fusion proteins expression and function from recombinant adenoviruses -transduced cells
The recombinant adenoviruses containing GFP-HPV E2s were used as described in previous studies [bib_ref] Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18..., Demeret [/bib_ref] [bib_ref] Nucleo-cytoplasmic shuttling of high risk human papillomavirus E2 proteins induces apoptosis, Blachon [/bib_ref] [bib_ref] Loss of HPV16 E2 protein expression without disruption of the E2 ORF..., Xue [/bib_ref] to transduced several cell types. Since HPV target cells in vivo are basal epithelium, undifferentiated human keratinocytes (HPK) were used to study genes modification by HPV16 E2 in vitro. Recombinant adenoviruses containing GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD were transduced to HPK at various m.o.i. (200, 100, 50, and 25, respectively, data not shown). The m.o.i. 50 was defined as the suitable m.o.i. that gave rise to ,100% GFP positive cells and quantifiable E2 mRNA expression in human keratinocytes. Intensity of the GFP signals under a fluorescence microscope were comparable in cells transduced with the various recombinant adenoviruses although GFP-HPV16 E2 exhibited a weaker GFP signal than other GFP fusion proteins [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref]. Partial cytoplasmic localization of the GFP fusion proteins was found in the cells transduced with GFP-HPV16E2, GFP-HPV18E2, and GFP-HPV18E2TAD whereas the GFP-HPV18E2DBD was mostly found in nucleus. Western blot analysis revealed a specific low expression of GFP-HPV16 E2 compared with other fusion proteins [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref] whereas the mRNA levels were comparable in all transduced HPK [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref].
Overexpression of wild type HPV E2 proteins can induce growth arrest of HeLa cells by repressing E6/E7 transcription via repression of the endogenous integrated HPV18 E6/E7 promoter [bib_ref] Suppression of cellular proliferation by the papillomavirus E2 protein, Dowhanick [/bib_ref] [bib_ref] Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis, Desaintes [/bib_ref]. Several papillomaviruses such as the carcinogenic HPV31 express, in addition to full length E2, an E8E2C fusion protein, which is required for repression of replication and transcription [bib_ref] The E8E2C protein, a negative regulator of viral transcription and replication, is..., Stubenrauch [/bib_ref] [bib_ref] The E8 repression domain can replace the E2 transactivation domain for growth..., Stubenrauch [/bib_ref]. Thus, the GFP-tagged DBD domain of HPV E2 protein alone exhibits a repressor function on HPV E6/E7 gene promoter. We evaluated the transcriptional repression activity of the GFPtagged HR-HPV E2 by transducing the various isoforms into HeLa cells and determining the HPV18 E6/E7 mRNA levels by real-time PCR. These experiments indicated that both full-length GFP-HPV16E2 and HPV18E2 showed comparable repressive activity on endogenous HPV18 E6/E7 gene expression in HeLa cells. Analysis of the deleted proteins revealed that HPV18E2DBD is also a potent repressor of HPV18 E6/E7 gene expression but not the TAD domain [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref]. These results thus show that the GFP-HPV E2 fusion proteins exhibit similar activities as reported earlier for the untagged HPV E2s.
## Genome-wide profiling of hpv16 e2-transduced hpk
To identify cellular gene expression profile modified by HPV16 E2, the expression profiles of GFP-HPV16 E2 HPK transduced cells were compared with those of GFP HPK transduced cells as a negative control. Data analysis indicated that HPV16 E2 upregulated 178 genes and down-regulated 601 genes. Gene Ontology analysis further revealed that 21 up-regulated genes and 71 down-regulated genes are involved in immune responses. Hierarchical clustering was constructed using the 92 immune associated genes modulated [fig_ref] Figure 2: Genome-wide profiling in HPK transduced by HPV16 E2 [/fig_ref]. The complete list of genes (fold-change 61.5 compare to GFP control) is shown in supplement Table S1. These data demonstrate that HPV16 E2 is a potent modulator of keratinocyte gene expression, including immune associated genes. Altered cellular signaling pathways in HPK expressing HPV16 E2 based on KEGG database are shown in [fig_ref] Table 1: Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein [/fig_ref]. The top 10 canonical pathways include metabolic pathway, cytokine-cytokine receptor interaction, pathway in cancer, viral carcinogenesis, protein processing in endoplasmic reticulum, PI3K-AKT signaling pathway, MAPK signaling pathway, leukocyte trans endothelial migration, chemokine signaling, and focal adhesion. The top 10 canonical pathways of up-regulated and down-regulated genes are shown in [fig_ref] Table 2: Top 10 modulated pathways in HPK expressing HPV16 E2 protein [/fig_ref].
To identify the transcriptional regulation of cellular genes involved in the control of basal transcription by HPV16 E2, the HPV16 E2 regulated genes were analyzed based on Animal Transcription Factor Database (AnimalTFDB) in 4 main functions, chromatin remodeling factors, transcription factors, transcription co-factors, and transcription elongation factors [fig_ref] Figure 2: Genome-wide profiling in HPK transduced by HPV16 E2 [/fig_ref]. The transcription networks modulated by E2 are depicted as upregulated genes and down-regulated genes [fig_ref] Table 3: Transcription network-associated genes modulated by HPV16E2 in HPK [/fig_ref].
The first immune network analysis was constructed using 90 signature genes from the literature and high-throughput screening in Core Analysis of Ingenuity Pathways Analysis (IPA). It was merged from 5 networks based on main functions of immune responses including immune cell trafficking, inflammatory response, cell-mediated immune response, and humoral immune response. This analysis demonstrated the connectivity of genes in MAPK, NF-kB pathways, as well as the link of MHC class I molecule, complement pathway, cytokines, and chemokines (supplement [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref]. The second network was merged from 72 down-modulated genes based on cell-mediated immune response and cell-to-cell signaling and interaction (supplement [fig_ref] Figure 2: Genome-wide profiling in HPK transduced by HPV16 E2 [/fig_ref]. STAT4 stands as a central factor that links several genes such as type I IFN and ISGs, playing roles in the immune response to viral infections. Notably, IFN-k, a member of the type I IFN family, was down-regulated by HPV16 E2 as well as TMEM173 (STING) which has previously been shown to have a link with type I IFN although not specifically with IFN-k. We therefore decided to determine whether these two genes could be linked in human keratinocytes for the immune response to viral infection.
## Sting and ifn-k were down-modulated by full-length e2 and its amino-terminal domain
To verify the modulation of STING and IFN-k transcription in human keratinocytes, recombinant adenoviruses containing GFP, GFP-HPV16E2, GFP-HPV18E2 were transduced into 3 different primary human keratinocytes cells HPK, TB3, and NUH49. Realtime PCR analyses demonstrated that E2 proteins of HPV16 and 18 potently down-modulated STING mRNA levels in HPK and NUH49 keratinocytes [fig_ref] Figure 3: STING and IFN-k transcripts were down-regulated by HPV E2s [/fig_ref]. IFN-k was also downregulated by HPV16 and HPV18 E2s in HPK, TB3, and NUH49 when compared to adenovirus GFP control transduced keratinocytes [fig_ref] Figure 3: STING and IFN-k transcripts were down-regulated by HPV E2s [/fig_ref].
To evaluate which domain of HPV E2 is responsible for STING and IFN-k regulation, recombinant adenoviruses containing GFP, GFP-HPV18 E2TAD, GFP-HPV18 E2DBD were transduced to HPK, TB3, and NUH49. STING transcripts levels were down- in HPK and NUH49 expressing HPV18 E2TAD when compared to HPV18 E2DBD expressing cells [fig_ref] Figure 3: STING and IFN-k transcripts were down-regulated by HPV E2s [/fig_ref]. IFN-k transcripts levels were also down-modulated by HPV18 E2TAD in HPK, TB3, and NUH49 [fig_ref] Figure 3: STING and IFN-k transcripts were down-regulated by HPV E2s [/fig_ref] , indicating that the amino-terminal domain of E2 (TAD) plays an essential
## Ifn-k is regulated by sting in human keratinocytes
STING can activate both NF-kB and IRF3 transcription pathways to induce expression of members of the type I IFN (IFNa and IFN-b) and thereby exert a potent antiviral effect when expressed [bib_ref] STING is an endoplasmic reticulum adaptor that facilitates innate immune signaling, Ishikawa [/bib_ref]. However, the association of STING and IFN-k in keratinocytes is not known. To determine whether a functional link exists between these two genes, gene silencing experiments were performed in human keratinocytes to knockdown STING or IFN-k transcription upon siRNA tranfection reaching a ,70% efficiency [fig_ref] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I [/fig_ref]. IFN-k transcription was down-modulated to around 20% by silencing of STING (P = 0.06) whereas silencing of IFN-k did not affect STING transcriptional level [fig_ref] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I [/fig_ref] , suggesting that STING might act as an upstream effector of IFN-k gene expression in human keratinocytes. Silencing of IFN-k reduced ISGs transcription such as IFIT1, IFIT3, OAS1, and OAS2 transcription levels as expected [fig_ref] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I [/fig_ref].
## Hpv18 e2tad abrogated sting and ifn-k induction and suppressed isgs transcription
To further evaluate the role of the E2 amino-terminal domain (TAD), HPK cells were transduced by recombinant adenoviruses GFP, and GFP-HPV18 E2TAD for 48 hrs after which transduced keratinocytes were stimulated with poly I:C (10 mg/ml) for an additional 3, 6, and 12 hrs. As measured by RT-PCR, STING and IFN-k transcription levels were significantly reduced in HPV18 E2TAD infected HPK cells at each time point of poly I:C activation compared to GFP-control cells [fig_ref] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I [/fig_ref] , respectively). HPV18 E2TAD also down-modulated several ISG gene expression including, IFIT3, IFIT1, MX1, MX2, OAS1, OAS2, OAS3, whereas in contrast PKR was up-regulated [fig_ref] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I [/fig_ref]. These data clearly demonstrated that the E2TAD alone could efficiently down modulate the stimulated IFN pathway in transduced human keratinocytes in vitro and that E2TADmediated effect was even more potent than siRNA knock-down of IFN-k.
## Sting and ifn-k were down-modulated in hpv positive clinical specimens
To determine the effect of HPV infection on STING and IFN-k expression in vivo in patients lesions, 148 clinical specimens were collected and their HPV infectious status and grade were examined by histology, HPV detection, and HPV genotyping. Based on histological analyses, clinical samples were graded into cervicitis or no intra-epithelial lesion (inflammation) and low grade squamous intra-epithelial lesion (LSIL). Samples of both stages were examined for HPV DNA and HPV E2 transcription. HPV E2 transcriptional levels were significantly increased in HPV positive normal and LSIL when compared to HPV negative normal cases [fig_ref] Figure 5: STING and IFN-k down-regulation in HPV positive clinical specimens [/fig_ref]. STING and IFN-k mRNAs expression levels were also examined. STING transcriptional level was significantly lower in HPV positive LSIL cases compared with HPV negative or inflammation group [fig_ref] Figure 5: STING and IFN-k down-regulation in HPV positive clinical specimens [/fig_ref]. IFN-k transcription level was also significantly down-regulated in normal and HPV positive LSIL cases compared to the HPV negative inflammation group [fig_ref] Figure 5: STING and IFN-k down-regulation in HPV positive clinical specimens [/fig_ref].
# Discussion
Our study revealed an unexpectedly large modulation of cellular genes by HPV16 E2 in human keratinocytes in the absence of any other viral gene expression. Several cell signaling pathways were modulated [fig_ref] Table 1: Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein [/fig_ref] and 2) including immune and non-immune pathways, which showed some overlap with the gene profiling of E2 expression in a HPV negative cervical cancer cell line, C33A [bib_ref] HPV16 E2 could act as down-regulator in cellular genes implicated in apoptosis,..., Ramirez-Salazar [/bib_ref]. In the present report, the transcriptional networks were analyzed based on AnimalTFDB and the gene profiling data indicated that HPV16 E2 can modulate the cellular transcriptional apparatus, including many transcription factors and co-factors [fig_ref] Table 3: Transcription network-associated genes modulated by HPV16E2 in HPK [/fig_ref]. In addition, several histones were up-regulated while the repressor histone deacetylase HDAC6 was down-modulated. These transcriptional modulations might affect downstream genes and lead to epigenetic modification of the host cell genome. The cytosolic DNA recognition leads to activation of TANKbinding kinase 1 (TBK1) and IRF-3 and production of type I IFNs. TBK1 associates with DDX3, a DEAD box upstream RNA helicase, which regulates IFN-b transcription via IRF-3. TBK1 interacts with the exocyst protein Sec5 in a complex that includes the recently identified endoplasmic reticulum adaptor STING [bib_ref] STING is an endoplasmic reticulum adaptor that facilitates innate immune signaling, Ishikawa [/bib_ref] [bib_ref] The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation, Zhong [/bib_ref]. STING interacts with TBK1 and IRF-3 leading to the phosphorylation of IRF-3 and production of type I IFN [bib_ref] STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway, Tanaka [/bib_ref]. IFNk is a member of type I IFN family that is expressed in human keratinocytes and also can be detected in dendritic cells [bib_ref] Interferonkappa, a novel type I interferon expressed in human keratinocytes, Lafleur [/bib_ref]. IFNk is reduced in HPV-infected keratinocytes [bib_ref] IFNkappa, a novel type I IFN, is undetectable in HPV-positive human cervical..., Decarlo [/bib_ref] via de novo methylation on a CpG island near the transcriptional start site induced by HPV16 E6 [bib_ref] Epigenetic silencing of interferon-kappa in human papillomavirus type 16-positive cells, Rincon-Orozco [/bib_ref] [bib_ref] High-risk human papillomaviruses repress constitutive kappa interferon transcription via E6 to prevent..., Reiser [/bib_ref]. The reduction of IFN-k results in the decrease of several ISGs and leads to impaired host cell antiviral responses. STING contributes to stimulating IFN-b as well as IFN-a, but the correlation between STING and IFN-k was not demonstrated. The present report shows that the E2TAD can reduce the poly I:C induced activation of several ISGs. The well characterized IFN-induced pathways are the anti-viral pathways, the PKR, the OAS, and the MX pathways [bib_ref] Transcriptional responses to polypeptide ligands: the JAK-STAT pathway, Schindler [/bib_ref] [bib_ref] Type I interferons, De Maeyer [/bib_ref] [bib_ref] How cells respond to interferons, Stark [/bib_ref]. IFN-k activates the ISRE pathway and signal transduction through the type I IFN receptor [bib_ref] Interferonkappa, a novel type I interferon expressed in human keratinocytes, Lafleur [/bib_ref] which leads to induction of ISGs. Interestingly, our results indicate that HPV16 E2 mediated downregulation of STING in HPK might lead to concomitant reduction in IFN-k expression. The reduction of IFN-k by HPV18 E2 TAD leads to the down modulation of the ISGs genes tested in this report, namely IFIT3, IFIT1, MX1, MX2, OAS1, OAS2, and OAS3. Transcriptional expression of the selected ISGs in polyI:C stimulated HPK cells was more efficiently down modulated by E2 than in IFN-k knockdown cells. This might be explained by a better efficiency of polyI:C activation together with E2TAD expression. The E2 TAD has been shown to interact with numerous cellular regulatory proteins (reviewed in [bib_ref] The papillomavirus E2 proteins, Mcbride [/bib_ref] , as well as the full length E2 protein [bib_ref] Tumor suppressor or oncogene? A critical role of the human papillomavirus (HPV)..., Bellanger [/bib_ref]. E2TAD also interacts with the E1 helicase that activates viral DNA replication [bib_ref] Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational..., Yasugi [/bib_ref] [bib_ref] The X-ray structure of the papillomavirus helicase in complex with its molecular..., Abbate [/bib_ref] and with Brd4 involved in episomal segregation during mitosis [bib_ref] Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral..., You [/bib_ref]. Other E2 functions, such as stability of the protein [bib_ref] Stability of the human papillomavirus type 18 E2 protein is regulated by..., Bellanger [/bib_ref] as well as induction of apoptosis, involve this domain [bib_ref] Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18..., Demeret [/bib_ref]. Induction of apoptosis is caused by accumulation of E2 in the cytoplasm and involves caspase 8 activation [bib_ref] Nucleo-cytoplasmic shuttling of high risk human papillomavirus E2 proteins induces apoptosis, Blachon [/bib_ref]. An high-through put yeast two hybrid analysis of several HPV E2 proteins revealed the interaction of HPV E2s with numerous cellular proteins that can alter the cellular functions including transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking [bib_ref] Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new..., Muller [/bib_ref] [bib_ref] The HPV E2-host protein-protein interactions: A complex hijacking of the cellular network, Muller [/bib_ref]. Accordingly, the suppression of STING and IFN-k gene transcription in human keratinocytes by E2TAD might be mediated by protein interaction of HPV18E2-TAD with several specific cellular proteins. These interactions might be direct or indirect either in the nucleus or cytoplasm of transduced cells that ultimately would lead to transcriptional repression. Moreover, our microarray data contain several cellular transcription factors which are regulated by HPV16E2, that could be regulators of STING and IFN-k gene expression.
IFN-k was strongly down-regulated in normal and LSIL HPV positive cases compared to HPV negative control tissues. This phenomenon might be induced by several HPV early proteins including the viral oncoproteins E6 and E7, but in the early stage of HPV infection, E2 is highly expressed and might therefore also play a major role to abrogate IFN-k transcription [bib_ref] Tumor suppressor or oncogene? A critical role of the human papillomavirus (HPV)..., Bellanger [/bib_ref] [bib_ref] Epigenetic silencing of interferon-kappa in human papillomavirus type 16-positive cells, Rincon-Orozco [/bib_ref] [bib_ref] High-risk human papillomaviruses repress constitutive kappa interferon transcription via E6 to prevent..., Reiser [/bib_ref]. The mechanisms of the suppression of IFN-k production by HPV16 E2 should be clarified in future work. However this is the first report focusing on a putative role of HPV E2 in mechanisms of immune evasion. This novel function of HPV E2 in regulating immune response associated genes thereby implicates this viral protein in the persistence of viral infection ultimately leading to cervical cancer. [fig_ref] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes [/fig_ref] The immune response-associated network. The network was merged based on main functions of immune responses, immune cell trafficking, inflammatory response, cellmediated immune response, and humoral immune response, which involved 90 gene products. This network demonstrated the connectivity of genes in MAPK, NF-kB pathways, as well as the link between MHC class I molecule, complement pathway, cytokines, and chemokines. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Green, down-regulated genes; red, up-regulated genes; gray, not differentially expressed; solid line, direct interaction; dashed line, indirect interaction; yellow line, merged interaction. (TIF) [fig_ref] Figure 2: Genome-wide profiling in HPK transduced by HPV16 E2 [/fig_ref] The cell-mediated immune responses-associated network. The network was merged from 72 E2 mediated down-regulated cellular genes based on cell-mediated immune response and cell-to-cell signaling and interaction. This network showed a connection between IFN-k and STING (TMEM173) with type I IFN being central. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Green, down-regulated genes; gray, not differentially expressed; solid line, direct interaction; dashed line, indirect interaction; yellow line, merged interaction.
## Supporting information
[fig] Figure 1: HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes. (A) GFP signals of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD under fluorescence microscope on live cells (10X). (B) GFP protein expression was detected by western blotting analysis. GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD showed the expected protein sizes of ,32 kD, 62 kD, 64 kD, 50 kD, and 45 kD, respectively and are indicated by asterisks. The b-actin protein was used as loading control. (C) The mRNA expression levels of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD are shown as CT values (mean6SD). (D) The suppressive effect of HPV E2 on HPV18 E6/E7 transcription in HeLa cells is shown as relative transcripts levels compared to cells not expressing E2 using real-time PCR. (*P#0.05). doi:10.1371/journal.pone. [/fig]
[fig] Figure 2: Genome-wide profiling in HPK transduced by HPV16 E2. Microarray was performed 48 h after HPV16 E2 transduction. Differential expression analysis was performed using one way ANOVA. Gene lists were created using a cut-off of P,0.05, 1.5-fold change. (A) Gene Ontology (GO) was used to define biological categories and functions. (B) Hierarchical clustering was constructed from immune associated genes as defined by GO. Cluster 1 and 2 were up-regulated cluster genes and cluster 3 and 4 were down-regulated cluster genes. (C) Signature genes altered in HPK expressing HPV16 E2, immune associated genes were analyzed by GO. Chromatin remodeling factors, transcription factors, and transcription cofactors were analysed by Animal Transcription Factor Database (AnimalTFDB), red numbers represent up-regulated genes; Green numbers represent down-regulated genes. doi:10.1371/journal.pone.0091473.g002 [/fig]
[fig] Figure 3: STING and IFN-k transcripts were down-regulated by HPV E2s. (A) STING transcripts level analyzed by RT-PCR was significantly down-regulated by HPV16 E2, HPV18 E2 and the HPV18 E2 TAD in HPK and B) IFN-k transcription level was significantly down-regulated by HPV16 E2, HPV18 E2 and HPV18 TAD in HPK. C) STING transcripts level was modulated by E2 in NUH49 as shown in (A). D) IFN-k transcripts level was modulated by E2 in ITB3 and NUH49 as in (B). (A-D) HPV18 E2TAD significantly down-regulated STING and IFN-k genes transcription when compared to HPV18 E2DBD. (*P#0.05, **P#0.01, ***P#0.001). doi:10.1371/journal.pone.0091473.g003 [/fig]
[fig] Figure 4: Transcriptional reduction of IFN-k in STING-silenced HPK and poly I:C stimulated HPK. (A) STING and IFN-k transcription level in HPK cells after transfection with siSTING and siIFNK for 48 h. IFN-k transcription level in STING-knockdown HPK was reduced by ,20% (P = 0.06). STING transcription level in IFN-k-knockdown HPK was not changed. (B) ISGs transcription levels were down-modulated in IFN-k-knockdown HPK. (C) TAD of HPV18 E2 abrogated STING transcription in Poly I:C induced HPK cells. Cells transduced with GFP as control and GFP-E218TAD were stimulated with poly I:C (10 mg/ml) for 3, 6, and 12 h. STING transcription level was measured at each time point. (D) IFN-k transcription level was abrogated by HPV18 E2TAD when examined at each time point of IFN-k induction by poly I:C (E) ISGs were determined after stimulation of HPK expressing HPV18 E2TAD with poly I:C (10 mg/ml) for 3 h. (*P#0.05, **P#0.01, ***P#0.001). doi:10.1371/journal.pone.0091473.g004 [/fig]
[fig] Figure 5: STING and IFN-k down-regulation in HPV positive clinical specimens. (A) HPV E2s transcription level was significantly increased in HPV positive normal and LSIL when compared to HPV negative normal cases (P,0.05). (B) STING transcription level was significantly decreased in LSIL with HPV positive and HPV E2 positive cases (P,0.05). Normal cases with HPV positive also showed low STING transcription level when compared to normal HPV negative cases (P, 0.05). (C) IFN-k transcription level was significantly down-regulated in normal HPV positive cases and HPV positive LSIL cases when compared to normal HPV negative cases (P,0.05). (*P#0.05, **P#0.01, ***P# 0.001). doi:10.1371/journal.pone.0091473.g005 (TIF) [/fig]
[table] Table 1: Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein. [/table]
[table] Table 2: Top 10 modulated pathways in HPK expressing HPV16 E2 protein. [/table]
[table] Table 3: Transcription network-associated genes modulated by HPV16E2 in HPK. [/table]
[table] Table S1: Immune responses associated genes modulated by HPV16 E2 in HPK. Gene Ontology analysis indicated that 21 up-regulated genes and 71 down-regulated genes by HPV16 E2 are involved in immune responses. (DOC) [/table]
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Use of the Pediatric Symptom Checklist for the detection of psychosocial problems in preventive child healthcare
Background: Early detection and treatment of psychosocial problems by preventive child healthcare may lead to considerable health benefits, and a short questionnaire could support this aim. The aim of this study was to assess whether the Dutch version of the US Pediatric Symptom checklist (PSC) is valid and suitable for the early detection of psychosocial problems among children.Methods:We included 687 children (response 84.3%) aged 7-12 undergoing routine health assessments in nine Preventive Child Health Services across the Netherlands. Child health professionals interviewed and examined children and parents. Before the interview, parents completed an authorised Dutch translation of the PSC and the Child Behavior Checklist (CBCL). The CBCL and data on the child's current treatment status were used as criteria for the validity of the PSC.Results:The consistency of the Dutch PSC was good (Cronbach alpha 0.89). The area under the ROC curve using the CBCL as a criterion was 0.94 (95% confidence interval 0.92 to 0.96). At the US cut-off (28 and above), the prevalence rate of an increased score and sensitivity were lower than in the USA. At a lower cut-off (22 and above), sensitivity and specificity were similar to that of the US version (71.7% and 93.0% respectively). Information on the PSC also helped in the identification of children with elevated CBCL Total Problems Scores, above solely clinical judgment.Conclusion:The PSC is also useful for the early detection of psychosocial problems in preventive child healthcare outside the USA, especially with an adjusted cut-off.
# Background
Early detection and treatment of psychosocial problems may lead to considerable health benefits. Psychosocial problems have a high prevalence rate and lead to high costs of disease. They also cause substantial restrictions in daily functioning in later life and are the major cause of long-term work disability in young adults. Only a minority of children with psychological or psychosocial problems are under treatment [bib_ref] Factors associated with child mental health service use in the community, Verhulst [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems by preventive child health..., Brugman [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems among toddlers in Dutch..., Reijneveld [/bib_ref]. If untreated, problems are likely to persist in later life and can lead to serious limitations in daily functioning [bib_ref] Factors associated with child mental health service use in the community, Verhulst [/bib_ref] [bib_ref] Childhood predictors differentiate lifecourse persistent and adolescence-limited antisocial pathways among males and..., Moffitt [/bib_ref]. Research has shown that early detection and treatment improves these children's prognosis substantially, but a complete analysis of its cost effectiveness has yet to be carried out.
The community child health service is an ideal setting for the early detection of psychosocial problems among children as routine health examinations are provided through it for the entire population, as a standardised part of preventive child healthcare (PCH). In the Netherlands, municipalities are obliged by law to guarantee proper access to this type of care, free of charge.
However, the predictive value of early detection of psychosocial problems by PCH is still too low [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems by preventive child health..., Brugman [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems among toddlers in Dutch..., Reijneveld [/bib_ref]. For instance, Brugman et al. show that even though Dutch PCH identifies psychosocial problems in 25% of all children of school age, they miss 43% of the children with a clinical score on the Child Behavior Checklist (CBCL) [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems by preventive child health..., Brugman [/bib_ref]. Similarly, Murphy et al. reported that paediatricians had identified psychosocial problems in less than half of the children with elevated scores on the Pediatric Symptom Checklist (PSC) or the Child Global Assessment Scale [bib_ref] Screening for psychosocial dysfunction in inner-city children: further validation of the Pediatric..., Murphy [/bib_ref].
The PSC is a 35-item parent-completed questionnaire that supports the identification of psychosocial problems by paediatricians [bib_ref] Screening for psychosocial dysfunction in inner-city children: further validation of the Pediatric..., Murphy [/bib_ref] [bib_ref] Screening for psychosocial disorders in pediatric practice, Jellinek [/bib_ref] [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref] [bib_ref] Screening for psychosocial problems in 4-5-year-olds during routine EPSDT examinations: validity and..., Pagano [/bib_ref] [bib_ref] Use of the Pediatric Symptom Checklist in a low-income, Mexican American population, Jutte [/bib_ref] [bib_ref] A mental health intervention for schoolchildren exposed to violence: a randomized controlled..., Stein [/bib_ref]. It takes less than 5 minutes to complete and score, and reflects the parent's impression of his or her child's psychosocial functioning. Its validity has been demonstrated in various paediatric settings in the USA, nationally [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref] in inner-city children [bib_ref] Screening for psychosocial dysfunction in inner-city children: further validation of the Pediatric..., Murphy [/bib_ref] , in Hispanic children [bib_ref] Screening for psychosocial problems in 4-5-year-olds during routine EPSDT examinations: validity and..., Pagano [/bib_ref] [bib_ref] Use of the Pediatric Symptom Checklist in a low-income, Mexican American population, Jutte [/bib_ref] and in children of substance-abusing parents. Moreover, the PSC has recently been used as an outcome measure in the assessment of interventions to reduce the impact of trauma [bib_ref] A mental health intervention for schoolchildren exposed to violence: a randomized controlled..., Stein [/bib_ref]. Given its good validity and applicability in US community child health services, the PSC is a likely candidate for use in other countries with similar systems of preventive child healthcare, such as the Netherlands.
The aim of this study was to assess the test properties of the Dutch version of the PSC and determine whether it would be suitable for and contribute to the early detection of psychosocial problems in children aged 7-12 by PCH.
# Methods
This study is based on a community sample of children for whom PSC and CBCL data are available, and data on the identification and management of psychosocial problems by CHPs.
## Population
The sample was obtained using a two-stage selection procedure. In the first stage, a national sample from 9 of the 41 Dutch Preventive Child Health Services was taken. In the second stage, each Service provided a sample of children aged 7-12 who were invited for routine well-child examinations. We aimed at a sample size of 700 respondents for evaluation, as earlier studies [bib_ref] Detecting psychosocial problems among 5-6 years old children in Preventive Child Health..., Vogels [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Early detection of psychosocial problems in adolescents: how useful is..., Reijneveld [/bib_ref] demonstrated that short questionnaires used in PCH settings allow for an area under the ROC curves (AUC) of about 0.90 with a clinical CBCL Total Problems score as criterion. A sample of 700 suffices to estimate this AUC with a 95% confidence interval of +/-0.02.
Of the total sample of 815 eligible children, 687 participated and 674 provided complete data on both questionnaires (84.3% and 82.7% of the original sample, respectively). Both groups were representative of the total sample regarding age and gender, but non-response was higher for children of immigrant/minority origin (27.4% vs. 12.2%). Analyses were restricted to children with complete data for both questionnaires to make interpretation easier.
## Data collection
The data were collected according to a standardised procedure during routine well-child examinations, from September 2004 to July 2005. The study was performed in compliance with the Helsinki Declaration. The design of the study was approved by the local TNO Medical-Ethical Committee and includes verbal informed consent by parents.
The PSC [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref] and the CBCL were mailed to children, along with the standard invitation for the preventive health assessment. Before attending the assessments, parents completed the questionnaires, placed them in sealed envelopes and gave them to the CHPs, who in turn passed them on to the researchers without opening them (in contrast with routine use, where the CHP would partially base the interview on the information from the PSC). The CHP interviewed each child and its parents regarding mental health and background, and examined each child. After each assessment, the CHP answered the following question: 'Does the child have a psychosocial problem, at this moment?' (yes, no) and scored its severity (mild, moderate or severe) and the type of problems identified using a pre-coded list. Children who only had risk indicators for the development of psychosocial problems, such as having parents with psychiatric problems or other family problems, had to be coded as having no problems.
The PSC was translated following the procedure proposed by Guillemin et al. [bib_ref] Cross-cultural adaptation of health-related quality of life measures: literature review and proposed..., Guillemin [/bib_ref]. Firstly, the original US English version of the questionnaire was translated into Dutch by three certified translators working independently of each other. Secondly, three further certified translators each translated one Dutch translation back into US English. The resulting US English versions were compared to the originals and all discrepancies were discussed by three researchers (SAR, MRC and AGCV) who spoke both Dutch and English. Discrepancies were also discussed with the developers of the PSC, Dr J.M. Murphy and Dr M.S. Jellinek, especially where items raised questions as to their intended meaning. The PSC consists of 35 items that are rated as never, sometimes or often present (0, 1 and 2, respectively). Item scores are summed; we dichotomised at 0-27 vs. 28-70, following the US cut-off [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref].
The CBCL was used to assess parents' reports of the behavioural and emotional problems of their children over the preceding six months. Its (good) reliability and validity has been established . We used only the 120 problem items from the CBCL and computed scores for two broad-band groups of syndromes designated as Internalising and Externalising, and a Total Problems score. Children were also allocated to a normal range or a clinical range, using the 90th percentile of the Dutch normative sample as the cut-off.
# Analysis
In the analysis we assessed the psychometric properties of the PSC and its added value in identifying psychosocial problems. Regarding psychometric properties, we first computed its internal consistency and examined the fit between the scale structure and the observed data using confirmatory factor analysis (CFA) with structural equation modelling. Next, we assessed the validity of the PSC using dichotomised CBCL scores (Total Problems score and Internalising/Externalising scales) and referral by the CHP due to psychosocial problems as criteria. Finally, we assessed whether mean PSC scores differed with the children's background.
Regarding the added value of the PSC in identifying psychosocial problems, we assessed the odds of identification of mental health problems (i.e. a clinical CBCL Total Problems score) using an elevated score on the PSC. This was repeated with adjustment for social and demographic risk indicators known to the CHP that might have helped in the identification of psychosocial problems [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems by preventive child health..., Brugman [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Identification and management of psychosocial problems among toddlers in Dutch..., Reijneveld [/bib_ref]. Regarding social and demographic risk indicators, we retained children with missing data in the logistic regression models by creating separate dummies for the missing category of each variable.
All analyses were done with SPSS 12.0 for Windows, except the CFA, which was done with Amos 5. All analyses were repeated for boys and girls separately.
Results for these subgroups are provided only if they differed in a statistically significant way (p < 0.05).
# Results
## Demographics
The average age of the children in the study was 9.7 years (standard deviation 1.4 years) and there were slightly more girls than boys. Further demographic information is presented in [fig_ref] Table 1: Demographic characteristics of the participating children, and mean Pediatric Symptom Checklist scores... [/fig_ref].
## Scores on psc and cbcl
Mean scores on the PSC are slightly higher for boys than for girls, which also holds for the CBCL [fig_ref] Table 2a: Scores on the PSC and CBCL Total Problems, Internalising and Externalising scales,... [/fig_ref]. The internal consistency of the PSC was very good (Cronbach's alpha 0.89), though the CFA revealed that the items could not be fully represented by a single factor (Chi-square = 2715 at 560 df; p < 0.001; Goodness-of-Fit Index (GFI) = 0.75; Parsimony corrected GFI = 0.66). [fig_ref] Table 2b: Numbers and percentages of children with elevated scores on the PSC and... [/fig_ref] shows the prevalence rates of elevated scores on the same questionnaires using their established cut-offs [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref]. Of all the children, 4.5% had elevated scores on the PSC and 8.9% had elevated scores on the CBCL. The latter closely resembles its distribution in the Dutch normative sample. In US populations, the prevalence of elevated PSC scores ranges from 12-14%. This corresponds to a cut-off of 0-21 vs. 22+ among Dutch children, when compared in [fig_ref] Table 2b: Numbers and percentages of children with elevated scores on the PSC and... [/fig_ref]. To enable comparisons with US data on the PSC, all further analyses are presented for this cut-off too.
## Validity
Subsequently, the degree to which the score on the PSC is truly elevated in the case of psychosocial problems as measured by these four criteria (i.e. sensitivity) and the degree to which it is 'normal' in the case of the absence of these problems (i.e. specificity) were assessed. For the recommended cut-off of the PSC at 28 and above, scores were 0.33 and 0.98 respectively, using a clinical CBCL Total Problems score as the criterion, and 0.19 and 0.97 respectively, using being under treatment for mental health problems as the criterion. shows the Receiver Operating Characteristic (ROC) curve for all possible cut-off points. The curve is close to the upper-left corner of the figure, particularly when the CBCL is used as the criterion, indicating a high validity of the PSC if this gold standard is used. Curves for CBCL Internalising and Externalising Problems are largely similar but slightly more off the upper-left corner (i.e. less favourable; not shown). The same holds for problems detected by the CHP when compared with the curve for 'under treatment' (not shown). [fig_ref] Table 3: Receiver Operating Characteristic [/fig_ref] shows the resulting areas under the ROC curves (AUC) and positive and negative predictive values for both cut-offs. Results regarding AUCs did not differ by gender (not shown).
## Differences in scores by background characteristics
Mean PSC scores were higher for boys, for children from minority backgrounds, single-parent families and unemployed families [fig_ref] Table 1: Demographic characteristics of the participating children, and mean Pediatric Symptom Checklist scores... [/fig_ref].
## Added value
Finally, we examined the degree to which information from the PSC contributed to the diagnosis of psychosocial problems as measured by the CBCL over and above the clinical opinion of the child health physician without knowledge of the PSC. This yielded an odds ratio of 21.3 (95% confidence interval 8.7 to 52.2), with the only predictive background characteristic being family composition. Using the alternative PSC cut-off of 22+ yielded slightly higher odds ratios.
## Parent opinion of the psc
A large majority of parents completed the PSC fully (91.1%) and no parent missed more than 3 items. However, 20% of parents made critical remarks about the PSC, mainly concerning lack of fit between questions and answer categories (7%) and unclear questions (5%).
# Discussion and conclusion
This study assessed the psychometric qualities of the Dutch version of the PSC and whether it is suitable for and contributes to the early detection of psychosocial problems among Dutch children aged 7-12 by PCH. Results reveal a good internal consistency and validity using the CBCL as gold standard. However, lower cut-offs have to be used for Dutch children than for children from the USA because of the Dutch children's on-average lower scores.
# Limitations
Methodological factors are unlikely to have affected these results. In general, the response rate was high (84%). Moreover, we used the CBCL as a criterion, which has been proven to be a valid measure for psychosocial problems. Because of complexity and high costs, structured clinical interviews such as the Diagnostic Interview Schedule for Children were not used as criteria. Doing so may have provided additional information but differences with questionnaire-based information have been shown to be small [bib_ref] Adequacy of interviews vs checklists for classifying childhood psychiatric disorder based on..., Boyle [/bib_ref]. . We found the internal consistency of the PSC similar to that found by Jellinek et al. [bib_ref] Brief psychosocial screening in outpatient pediatric practice, Jellinek [/bib_ref] but the results of our confirmatory factor analyses cast some doubt as to whether it measures a single latent, as did a previous study of Gardner et al. [bib_ref] Multidimensional adaptive testing for mental health problems in primary care, Gardner [/bib_ref].
## Fit with previous research on psc and on other questionnaires used in pch
We found much lower mean scores on the PSC than have been found for comparable US samples. Mean scores on other symptom checklists such as the CBCL are also lower for Dutch children than for children from the USA [bib_ref] Comparisons of problems reported by parents of children in 12 cultures: total..., Crijnen [/bib_ref]. Therefore, the Dutch children's lower mean PSC scores probably reflect real differences between these countries in the levels of symptoms reported by parents. This also implies that the cut-off for an elevated score on the PSC should be set lower for Dutch children than for children from the US. At this lower cut-off, the sensitivity and specificity of the Dutch version is similar to that of the US version. Moreover, the test characteristics of the PSC are comparable with or slightly better than those of most questionnaires currently used in Dutch PCH [bib_ref] Detecting psychosocial problems among 5-6 years old children in Preventive Child Health..., Vogels [/bib_ref] [bib_ref] Verloove-Vanhorick SP: Early detection of psychosocial problems in adolescents: how useful is..., Reijneveld [/bib_ref].
Finally, we found higher mean PSC scores for boys and for children from single-parent families, similar to those found by Jellinek et al. for children in the USA [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref]. We did not find differences in terms of parental education level, in contrast to the findings of Jellinek et al. [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref] , but we did find elevated scores among children with unemployed parents, an indicator of familial socioeconomic status that was not studied by Jellinek et al. [bib_ref] Use of the Pediatric Symptom Checklist to screen for psychosocial problems in..., Jellinek [/bib_ref].
## Implications
The results of our study imply that the PSC is useful for the early detection of psychosocial problems by PCH, especially if an adjusted cut-off is used. The PSC mostly detects behavioural and emotional problems, which are common in this age group. However, questions on more extreme behaviours such as the abuse of alcohol and drugs are not asked. Screening using the PSC is best carried out as a first step in a two-step process on the way to referral. A relatively low-cut-off can then be used to avoid missing too many cases. In a second step, cases flagged by the PSC should then be assessed by a CHP before making a final decision about referral. Parental responses show that some questions may require revision. In any event, the PSC is a useful aid for the early detection of psychosocial problems that could be considered for use in other countries as well.
## Competing interests
The author(s) declare that they have no competing interests.
# Authors' contributions
SAR had the original idea for the project and wrote the study protocol. AGCV coordinated the study. All authors discussed the protocol and formulated the final design. MRC supervised the data collection. AGCV, FH, and SAR did the statistical analyses, which were discussed by all authors. SAR wrote the final manuscript, which was discussed, edited and revised by all authors. All authors read and approved the final manuscript.
## Appendix us version of the pediatric symptom checklist (dutch version available on request from the authors)
## Pediatric symptom checklist (psc)
Emotional and physical health go together in children.
Because parents are often the first to notice a problem with their child's behavior, emotions or learning, you may help your child get the best care possible by answering these questions. Please indicate which statement best describes your child.
Please mark under the heading that best describes your child:
[formula] NEVER [/formula]
[table] Table 2a: Scores on the PSC and CBCL Total Problems, Internalising and Externalising scales, for all children and by gender (mean, standard deviation, range). [/table]
[table] Table 1: Demographic characteristics of the participating children, and mean Pediatric Symptom Checklist scores for selected sociodemographic groups (n = 674)*. [/table]
[table] Table 2b: Numbers and percentages of children with elevated scores on the PSC and CBCL Total Problems, Internalising and Externalising scales, and of children currently under treatment for psychosocial problems, for all children and by gender. [/table]
[table] Table 3: Receiver Operating Characteristic (ROC) curve for all possi-ble cut-off points of the Dutch version of the PSC, using a clinical CBCL score and Currently Under Treatment or Psy-chosocial Problems as criteria Figure 1 Receiver Operating Characteristic (ROC) curve for all possible cut-off points of the Dutch version of the PSC, using a clinical CBCL score and Currently Under Treatment or Psychosocial Problems as criteria.10. Is afraid of new situations........................................... 10 _______ _______ _______ 11. Feels sad,unhappy................................................... 11 _______ _______ _______ 12. Is irritable, angry..................................................... 12 _______ _______ _______ 13. Feels hopeless......................................................... 13 _______ _______ _______ 14. Has trouble concentrating......................................... 14 _______ _______ _______ 15. Less interested in friends........................................... 15 _______ _______ _______ 16. Fights with other children.......................................... 16 _______ _______ _______ 17. Absent from school........................................... 17 _______ _______ _______ 18. School grades dropping............................................ 18 _______ _______ _______ 19. Is down on him or herself........................................... 19 _______ _______ _______ 20. Visits the doctor with doctor finding nothing wrong..... 20 _______ _______ _______ 21. Has trouble sleeping................................................. 21 _______ _______ _______ 22. Worries a lot............................................................ 22 _______ _______ _______ 23. Wants to be with you more than before........................ 23 _______ _______ _______ 24. Feels he or she is bad................................................. 24 _______ _______ _______ 25. Takes unnecessary risks............................................. 25 _______ _______ _______ 26. Gets hurt frequently.................................................. 26 _______ _______ _______ 27. Seems to be having less fun....................................... 27 _______ _______ _______ 28. Acts younger than children his or her age..................... 28 _______ _______ _______ 29. Does not listen to rules............................................. 29 _______ _______ _______ 30. Does not show feelings............................................. 30 _______ _______ _______ 31. Does not understand other people's feelings................. 31 _______ _______ _______ 32. Teases others.......................................................... 32 _______ _______ _______ 33. Blames others for his or her troubles........................... 33 _______ _______ _______ 34. Takes things that do not belong to him or her.............. 34 _______ _______ _______ 35. Refuses to share...................................................... 35 _______ _______ _______ Total score ______________ Does your child have any emotional or behavioral problems for which she/he needs help? () N () YAre there any services that you would like your child to receive for these problems? () N () Y [/table]
|
Identifying an efficient, thermally robust inorganic phosphor host via machine learning
## Supplementary
Electron density (Gilman valence per V per atom) 150
Electron density (outer shell per V per atom)
## Supplementary
[fig] Figure 1 Supplementary, Figure 2 Supplementary, Figure 3 Supplementary, Figure 5: Powder X-ray diffraction (XRD) patterns of the NaBa1−xEuxB9O15 phosphor. XRD patterns of the synthesized NaBa1-xEuxB9O15 (x = 0.005, 0.01, 0.02, 0.03, 0.04, 0.05). All diffraction peaks match well with the calculated pattern of NaBaB9O15 crystal structure. Excitation and emission spectra of NaBa1−xEuxB9O15 phosphor. Normalized (a) excitation (λem = 416 nm) and (b) emission (λex = 315 nm) spectra for NaBa1-xEuxB9O15 (x = 0.005, 0.01, 0.02, 0.03, 0.04, 0.05) at room temperature. Luminescence decay curve of NaBa0.97Eu0.03B9O15. The fit (red line) is obtained following Equation 5 in the main text of the manuscript to obtain the decay time (τ).SupplementaryFigure 4: Temperature-dependent emission spectra of NaBa0.97Eu0.03B9O15. The data are collected under 340 nm excitation in the temperature range 80−500 K with a temperature interval of 20 K. Temperature-dependent powder X-ray diffraction patterns of NaBa0.97Eu0.03B9O15 and refined unit cell volume as a function of temperature. (a) The patterns were measured in the temperature range 300−500 K. All diffraction peaks match well with the calculated pattern of NaBaB9O15 crystal. (b) Refined unit cell volume (black circles) obtained with Le Bail method as a function of temperature, which is fit by a second order function (dashed line). [/fig]
[table] Table 1: Descriptor set for predicting the Debye temperature (ΘD,SVR). [/table]
|
Medical Data Acquisition and Internet of Things Technology-Based Cerebral Stroke Disease Prevention and Rehabilitation Nursing Mobile Medical Management System
This research was aimed at exploring the application value of a mobile medical management system based on Internet of Things technology and medical data collection in stroke disease prevention and rehabilitation nursing. In this study, on the basis of radio frequency identification (RFID) technology, the signals collected by the sensor were filtered by the optimized median filtering algorithm, and a rehabilitation nursing evaluation model was established based on the backpropagation (BP) neural network. The performance of the medical management system was verified in 32 rehabilitation patients with hemiplegia after stroke and 6 healthy medical staff in the rehabilitation medical center of the hospital. The results showed that the mean square error (MSE) and peak signal-to-noise ratio (PSNR) of the median filtering algorithm after optimization were significantly higher than those before optimization (P < 0:05). When the number of neurons was 23, the prediction accuracy of the test set reached a maximum of 89.83%. Using traingda as the training function, the model had the lowest training time and root mean squared error (RMSE) value of 2.5 s and 0.29, respectively, which were significantly lower than the traingd and traingdm functions (P < 0:01). The error percentage and RMSE of the model reached a minimum of 7.56% and 0.25, respectively, when the transfer functions of both the hidden and input layers were tansig. The prediction accuracy in stages III~VI was 90.63%. It indicated that the mobile medical management system established based on Internet of Things technology and medical data collection has certain application value for the prevention and rehabilitation nursing of stroke patients, which provides a new idea for the diagnosis, treatment, and rehabilitation of stroke patients.
# Introduction
Cerebral stroke is a common acute cerebrovascular disease, and its clinical manifestations are local neurological deficits [bib_ref] Cerebrovascular disease: primary and secondary stroke prevention, Caprio [/bib_ref]. Cerebral stroke is the second largest cause of death and the first cause of disability worldwide, with high morbidity, high disability rate, high mortality, and high recurrence rate [bib_ref] Causes of acute stroke: a patterned approach, Knight-Greenfield [/bib_ref]. In recent years, with the intensification of population aging in China, the incidence of cerebral stroke patients increased significantly. According to statistics, there are about 2.46 million new cases of cerebral stroke each year in China, of which about 75% of the patients cannot live independently due to disability, and more than 40% are severely disabled [bib_ref] Sémantique, épidémiologie et sémiologie des accidents vasculaires cérébraux [semantics, epidemiology and semiology..., Boursin [/bib_ref]. Due to the characteristics of cerebral stroke disease, its treatment activities have strong real-time performance and are often accompanied by complications such as dysfunction after rehabilitation [bib_ref] Perinatal stroke, Dunbar [/bib_ref]. The current research results show that early rehabilitation nursing can promote the cerebral cortex reorganization of patients with cerebral nerve injury and help to restore the body [bib_ref] Stroke in the young: a global update, Hathidara [/bib_ref]. Due to the influence of medical conditions and hospitalization expenses, patients cannot be treated in hospitals for a long time, and the number and quality of nursing staff in community rehabilitation centers cannot meet the needs of nursing for cerebral stroke patients [bib_ref] Advances and challenges in stroke rehabilitation, Stinear [/bib_ref].
In recent years, with the continuous development of computer technology and Internet of Things technology, mobile medical and health service systems are gradually applied to the prevention, diagnosis, treatment, and nursing of diseases and have become a new type of medical and health care model [bib_ref] The scope and impact of mobile health clinics in the United States:..., Yu [/bib_ref]. The Internet of Things is an intelligent network system that realizes information exchange and communication through network connection of information sensing devices such as radio frequency identification (RFID) technology, infrared sensing, and global positioning system [bib_ref] Mobile health technologies in cardiopulmonary disease, Mac Kinnon [/bib_ref]. The medical mobile APP based on Internet of Things technology realized the detection and management of patients' body temperature, blood pressure, blood glucose, and heart rate. It can communicate with doctors in real time through remote medical system and improve the accuracy of disease diagnosis and treatment and the quality of medical service [bib_ref] Mobile health application and e-health literacy: opportunities and concerns for cancer patients..., Kim [/bib_ref] [bib_ref] Using informatics and mobile health to improve medication safety monitoring in kidney..., Taber [/bib_ref]. In addition, the rise of big data also provides favorable conditions for the rapid development of medical informatization. Medical data ensure the continuity of individual medical treatment, and at the same time, the health status of patients can be analyzed and predicted based on the data of patients' health information [bib_ref] Mobile apps for medication management: review and analysis, Tabi [/bib_ref]. While the medical and health service platform based on Internet of Things technology provides convenience for patient diagnosis and treatment, it also has the disadvantages of lack of sharing of health information, lack of continuous monitoring and management of health physiological parameters, and inability to meet multilevel health needs. The prevention and rehabilitation tracking of cerebral stroke patients can timely evaluate the rehabilitation of patients and prevent the recurrence of the disease. At present, most medical institutions focus on the diagnosis and treatment of cerebral stroke patients [bib_ref] Development and clinical evaluation of a web-based upper limb home rehabilitation system..., Chae [/bib_ref] , ignoring the prevention and rehabilitation tracking of cerebral stroke patients.
In summary, there are few studies on the mobile management system for disease prediction and rehabilitation nursing tracking of cerebral stroke patients. Based on the mobile medical monitoring system and combined with RFID technology, this study studies the tracking and management of the prevention and rehabilitation process of cerebral stroke patients, so as to establish a mobile medical management system for cerebral stroke disease prevention and rehabilitation nursing, and improve the timeliness and efficiency of cerebral stroke disease diagnosis.
# Materials and methods
## Design of mobile medical system based on internet of
Things. The remote mobile medical monitoring system for cerebral stroke patients based on Internet of Things is mainly composed of three layers: physiological parameter acquisition node (data access layer), business logic layer, and intelligent mobile Android terminal layer. The data access layer mainly identifies and transmits the basic clinical data of patients through PDA equipment according to the RFID tag carried by patients, including the hospital information system (HIS) such as patients' physiological characteristics and signs. The business logic layer mainly processes and analyzes the data through mobile hospital information system (MobHIS) and network platform. The intelligent mobile Android terminal layer receives, processes, and displays patient disease information. The framework of the mobile medical system based on Internet of Things in this study is illustrated in .
In this study, the wireless intrusion detection system (WIDS) and wireless network controller (WNC) are mainly used as the medical dedicated wireless network system. WIDS realizes that multiple networks cover a target area at the same time through multifrequency combination and can realize the linear expansion of the wireless network capacity [bib_ref] Advanced machinelearning methods for brain-computer interfacing, Lv [/bib_ref]. WNC can ensure the security and stability of the wireless network system [bib_ref] Comparison of EEG measurement of upper limb movement in motor imagery training..., Suwannarat [/bib_ref].
## Structure design of cerebral stroke rehabilitation
Mobile Monitoring System. In this study, a mobile monitoring system model of human-computer interaction is established by combining remote communication technology, computer intelligence technology, and rehabilitation nursing methods. Cerebral stroke prevention and rehabilitation mobile monitoring system is mainly composed of two modules: patient clinical data acquisition and remote cerebral stroke rehabilitation nursing. The clinical data acquisition mainly obtains the patient's identification (ID) and clinical information by scanning RFID wristband tags and acceleration sensors; transmits the data to the rehabilitation nursing database through the Internet; preprocesses and identifies the data through the rehabilitation nursing system, such as denoising and normalization; and finally obtains the rehabilitation nursing results. The doctor shall timely feedback the recovery status of patients according to the results of the rehabilitation nursing system. The structure diagram of the cerebral stroke rehabilitation nursing mobile monitoring system is shown in [fig_ref] Figure 2: Structure diagram of cerebral stroke rehabilitation nursing mobile monitoring system [/fig_ref].
## Data acquisition and processing of cerebral stroke
Rehabilitation Mobile Monitoring System. The data related to rehabilitation training are collected by binding threedimensional acceleration sensors to the patient's forearm and upper arm. The collected data are transmitted to the wireless data receiver through the built-in Zigbee wireless transmission module and uploaded to the client software system. It is assumed that the accelerations of the acceleration sensor in three directions are A x , A y , A z , and the calculation method of sensor roll angle α and pitch angle β is as follows.
[formula] α = arctan A x A z , β = arctan A y A z :ð1Þ [/formula]
Signals are often disturbed by random noise or Gaussian noise in the process of generation and transmission. The commonly used signal filtering methods include linear filtering and nonlinear filtering [bib_ref] Interventions to improve hospital patient satisfaction with healthcare providers and systems: a..., Davidson [/bib_ref]. Linear filtering has better denoising effect on Gaussian white noise but worse on [bib_ref] Cardiothoracic surgical volume within the military health system: fiscal years, Johnson [/bib_ref]. The median filtering method in nonlinear filtering has good suppression ability for a narrow pulse signal, but its filtering effect on Gaussian white noise needs to be further optimized [bib_ref] Rendszer az egészségügyi weboldalak hitelesítésére [an objective scoring system to evaluate the..., Horváth [/bib_ref]. For the median filter with filter window side length n, it can be expressed as follows.
[formula] y i = med f i−n , f i−n+1 ,⋯,f i+n ð Þ :ð2Þ [/formula]
med represents the extraction median function and f i−n , f i−n+1 , ⋯, f i+n represents the result sequence of onedimensional odd window. The two-dimensional median filter slides in the image in order, and its calculation method is as follows.
[formula] y ij = med x ij È É :ð3Þ [/formula]
x ij is the pixel value of the input area for the window, and y ij represents the intermediate value after sorting the pixel values in the window area. The median filter input signals are Gaussian distributed, and the approximate noise variance can be expressed as follows.
[formula] χ 2 med = 1 4mg 2 n ′ = χ 2 i n + π/2 − 1 × π 2 :ð4Þ [/formula]
n ′ is the noise mean, χ 2 i represents the input noise variance, n represents the filter window length, and g 2 ðn ′ Þ represents the noise density function.
The output noise distribution of the median filter has a certain correlation with the input noise model and the probability density distribution [bib_ref] An adaptive median filter based on sampling rate for R-peak detection and..., Bae [/bib_ref]. The frequency response calculation method of median filtering system can be expressed as follows.
[formula] A = C F :ð5Þ [/formula]
A represents the frequency response of median filter system, and C and F represent the spectrum of input signal and output signal.
For the check noise processing in the data, pixelmedianfilerðN, i, j, AÞ function is introduced to optimize it. It is assumed that the pixel position before filtering is ði, jÞ; the row vector of the filtered pixel position template matrix can be expressed as follows:
[formula] pixelset = adipixel N, i, j, A ð Þ :ð6Þ [/formula]
Due to the limited motor function of cerebral stroke rehabilitation patients, most patients cannot accurately complete the prescribed rehabilitation nursing actions. The obtained data need to further extract the physical characteristics of the signal. The physical characteristics of the signal mainly include the root mean square (RMS), variance, and energy characteristics. The calculation method is as follows.
[formula] RMS = 1 N 〠 N i=1 x 2 i ! 1/2 , Var = 1 N − 1 〠 N i=1 x i − δ ð Þ 2 , En = 〠 N i=1 B i j j:ð7Þ [/formula]
RMS is the root mean square, Var is the variance, En is the energy characteristic, N is the sequence length, δ is the mean, and B i is the signal Fourier transform amplitude.
Mean square error (MSE), root mean square error (RMSE), and peak signal-to-noise ratio (PSNR) were used to evaluate the filtered data. The MSE, RMSE, and PSNR were calculated as follows. ði, jÞ is the pixel position, m is the number of measurements, X is the measured value, Y is the true value, L is the peak signal, and MSEðX, YÞ is the MSE of the image.
[formula] MSE = 1 m 〠 m i=1 〠 m j=1 X i, j ð Þ− Y i, j ð Þ ½ 2 , RMSE = ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 m 〠 m i=1 〠 m j=1 X i, j ð Þ− Y i, j ð Þ ½ 2 v u u t , PSNR X, Y ð Þ= 10 log 10 L 2 MSE X, Y ð Þ :ð8Þ [/formula]
## Establishment of cerebral stroke rehabilitation nursing
Evaluation Model. The back propagation (BP) neural network learns a certain number of sample pairs. After the hidden layer and output layer are calculated, the predicted values of each neuron output in the output layer are calculated. Through the back propagation error, the error between the network, output, and expected output is gradually reduced [bib_ref] Three-dimensional mandibular motion trajectorytracking system based on BP neural network, Tian [/bib_ref]. For the sample pair ðX, YÞ, the calculation method of the network weight matrix W 1 between the input layer and the hidden layer neurons and the network weight matrix W 2 between the hidden layer and the output layer neurons are as follows.
[formula] W 1 = w 1 11 w 1 12 ⋯ w 1 1m w 1 21 w 1 22 ⋯ w 1 2m ⋮ ⋯ ⋮ w 1 l1 w 1 l2 ⋯ w 1 [/formula]
The threshold σ 1 of the hidden layer neuron and the threshold σ 2 of the output layer neuron can be expressed as follows.
[formula] σ 1 = σ 1 1 , σ 1 2 ,⋯,σ 1 l  à , σ 2 = σ 2 1 , σ 2 2 ,⋯,σ 2 n  à :ð10Þ [/formula]
The output O j of the hidden layer neuron and the output Z k of the output layer neuron are expressed as follows.
[formula] O j = f 〠 m i=1 w l ij x i − σ i l ! = f net j À Á , j = 1, 2, ⋯, l, Z k = g w 2 kj O j − σ 2 k = g net k ð Þ, k = 1, 2, ⋯, n:ð11Þ [/formula]
f ðÞ represents the transfer function of the hidden layer, and gðÞ is the transfer function of the output layer.
The error between network output and expected output of the BP neural network can be expressed as follows.
[formula] E = 1 2 〠 n k=1 y k − z k ð Þ= 1 2 〠 n k=1 y k − g 〠 l j=1 w 2 kj O j − σ 2 k ! " # 2 :ð12Þ [/formula]
The number of hidden layer neurons has a significant impact on the performance of the BP neural network. The number of hidden layer neurons can be calculated by empirical equation.
[formula] n = a + b 2 + c:ð13Þ [/formula]
a and b are the number of neurons in the input layer and the output layer, respectively, and c is the constants within 10.
## Database design and test environment of cerebral
Stroke Rehabilitation Mobile Monitoring System. The database and its application system are the core and foundation of the database design of the mobile monitoring system. The design of the database needs to meet the principles of integrity, small number, small number of fields, and high efficiency [bib_ref] Health service delivery and workforce in northern Australia: a scoping review, Edelman [/bib_ref]. Storage and access methods are the main physical structure of the database [bib_ref] What health system challenges should responsible innovation in health address? Insights from..., Lehoux [/bib_ref]. The physical structure of the database needs to meet the minimum storage space and improve the effective access efficiency of the database [bib_ref] Obesity prevention and the role of hospital and community-based health services: a..., Pearce [/bib_ref]. The physical object of this database involves the patient entity and rehabilitation data entity, and the main physical structure includes the patient information The specific information in the database is shown in Tables 1-4.
The test system running the platform environment is as follows: computer operating system Windows 10 flagship version 64-bit operating system, server-side scripting language PHP 5.3.5, relational database management system MySQL 5.5.8, and web server software Apache 2.2.17. The development environment is as follows: project development
## Verification of cerebral stroke rehabilitation mobile
Monitoring System. The rehabilitation of cerebral stroke patients was evaluated by the Brunnstrom staging method. According to the results of Wang et al. [bib_ref] Brunnstrom stage automatic evaluation for stroke patients by using multi-channel sEMG, Wang [/bib_ref] , the motor function recovery of patients was divided into 6 grades: stage I: no muscle contraction, stage II: combined reaction occurred, stage III: collaborative movement was launched at will, stage IV: separation movement occurred, phase V: relatively independent comovement occurred, and stage VI: near normal or basically normal. 32 hemiplegic rehabilitation patients after cerebral stroke and 6 healthy medical staff from the hospital from December 2019 to March 2021 were selected as the research objects. The motion quality was evaluated according to the acceleration physical characteristics collected in the process of the rehabilitation nursing exercise. Most of the cerebral stroke patients were in Brunnstrom stages II-V, and the 6 medical staff were in the stage VI rehabilitation group. Patients with severe cognitive or communication disorders were excluded. 75% of the data were randomly selected from Brunnstrom cerebral stroke patients at different stages as the training set and the remaining 25% as the test set.
The rehabilitation nursing method refers to the method of Ikbali et al. [bib_ref] Virtual reality in upper extremity rehabilitation of stroke patients: a randomized controlled..., Afsar [/bib_ref] to carry out the standard movement training of "patient's hand touching shoulder," and it is modified accordingly. The patient's upper limb rehabilitation training is mainly completed according to the motion guidance map. At the beginning of the training, the user can choose the guidance map freely according to preferences and the upper limb use habits. The action of each cycle takes 10 seconds as the sampling cycle. Computational and Mathematical Methods in Medicine expressed by mean ± standard deviation ( x ± s), and t-test was used. The counting data was expressed by percentage (%). P < 0:05 indicated that the difference was statistically significant.
# Results and analysis
## Comparison of indicators before and after filter
Optimization. The signal processing results before and after the optimization of the median filtering algorithm are compared and analyzed. With the increase of noise intensity, the mean square error (MSE) of the signal showed a significant upward trend. When the noise intensity was 5%, 10%, 20%, 40%, and 60%, the MSE values of the optimized median filtering algorithm were 54:17 ± 4:52, 103:52 ± 8:63 , 215:42 ± 17:95, 1302:17 ± 108:51, and 4865:22 ± 455:26, respectively, and the MSE values of the median filtering algorithm before optimization were 2:17 ± 0:34, 15:41 ± 1:48, 21:52 ± 1:99, 52:42 ± 4:87, and 116:92 ± 8:63, respectively. At different noise intensities, the MSE values of the median filtering algorithm after optimization were significantly inferior than those before optimization, and there was a highly significant difference between the two (P < 0:001) . The peak signal-to-noise ratio (PSNR) value of the optimized median filtering algorithm was significantly higher than that before optimization . It suggested that the median filtering algorithm has better denoising and smoothing performance for the image after optimization, which may be due to the introduction of pixelmedianfilerðN, i, j, AÞ function in the optimization algorithm, which can filter all noise points at one time, but also well preserve the image details and enhance the noise reduction effect of the median filtering algorithm.
## Signal
Filtering Processing Result Analysis. The signals recorded by the upper arm sensor during the rehabilitation nursing training of patients were analyzed. Before filtering, the signals in different directions contained obvious linear noise and calibration noise. The optimized median filter was used to filter the signals. The interference waveforms in the signal curves of the upper arm sensors in different directions of X, Y, and Z axes were significantly reduced .
## Determination of neuron number in cerebral stroke
Rehabilitation Nursing Evaluation Model. The root mean square error (RMSE) values of the model under different numbers of neurons in the hidden layer were analyzed [fig_ref] Figure 6: RMSE value distribution of the model under different numbers of neurons [/fig_ref]. With the increase of the number of neurons, the RMSE value of the model showed a trend of increasing and then decreasing. When the number of neurons was greater than 23, the RMSE value of the model tended to be stable and the change was small. Under different numbers of neurons, the prediction accuracy of the training set and the test set was different. When the number of neurons was 23, the prediction accuracy of the test set reached the maximum value of 89.83%, the prediction accuracy of the test set decreased rapidly, and the prediction accuracy of the training set showed a stable state . Therefore, the number of neurons in the hidden layer was selected as 23 in this study.
## Function selection of cerebral stroke rehabilitation
Nursing Evaluation Model. At present, the training functions for the BP neural network algorithm mainly include the traingd function of the gradient descent algorithm, traingdm function of the momentum backpropagation gradient descent, and traingda function of the dynamic adaptive learning rate [bib_ref] Verification and comparison of three prediction models of ischemic stroke in young..., Chen [/bib_ref]. In this study, the training time and RMSE values under three different training functions are compared . Traingda was used as the training function, the training time and RMSE value of the model were the lowest, which were 2.5 s and 0.29, respectively. The training time and RMSE value of traingda were significantly lower than those of traingd and traingdm functions (P < 0:01). The training steps of the traingda training function were significantly different from those of traingd and traingdm functions (P < 0:001), so traingda was selected as the training function.
The transfer function has a significant influence on the prediction accuracy of the neural network. At present, the commonly used node transfer functions are logsig, tansig, and purelin functions [bib_ref] Medical image fusion method by deep learning, Li [/bib_ref]. The error percentage and RMSE values under different transfer functions in the hidden layer and the input layer are compared [fig_ref] Figure 9: Performance comparison of transfer function between hidden layer and output layer [/fig_ref]. When the transfer in the hidden layer and the input layer is tansig, the error percentage and RMSE values of the model are the minimum, which are 7.56% and 0.25, respectively.
## Result analysis of cerebral stroke rehabilitation mobile
Monitoring System. The Brunnstrom staging results of the subjects included in the study were compared with the prediction results of the mobile monitoring system [fig_ref] Figure 10: Distribution of predicted staging and clinical staging [/fig_ref]. In 32 samples, the prediction results of stages I and II were completely consistent with the clinical staging results. There were 3 samples (9.37%) with difference between normal prediction results and clinical stage results in stages III-VI, and the prediction accuracy was 90.63%. With the increase of the stage grade, the error rate of prediction results increases. It is analyzed that the reason may be caused by the unstable rehabilitation status of the research object and the lack of test data.
# Conclusion
In this study, a mobile medical management system for stroke prevention and rehabilitation care was established based on Internet of Things technology and medical data collection, and the results revealed that the system was feasible. However, there are still some shortcomings: the number of cases is small and the amount of collected data is insufficient, so the number of cases will be increased in the future work to further evaluate the rehabilitation nursing model and management system. In conclusion, the rehabilitation nursing mobile medical management system established based on Internet of Things technology and medical data collection has certain application value for the prevention and rehabilitation nursing of stroke patients, which provides a new idea for the diagnosis, treatment, and rehabilitation of stroke patients.
## Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
## Conflicts of interest
The authors declare no conflicts of interest.
[fig] Figure 2: Structure diagram of cerebral stroke rehabilitation nursing mobile monitoring system. [/fig]
[fig] 2. 7, Figure 3 0, Figure 4 0, Figure 5: Statistical Methods. The test data were processed by SPSS19.0 statistical software. The measurement data were Comparison of MSE values of median filtering algorithm under different noise intensities. ( * * represents a significant difference compared to preoptimization, P < Comparison of PSNR values of median filtering algorithm under different noise intensities. ( * * represents that there is significant difference compared to preoptimization, P < Motion signal curves in different directions: (a) upper arm sensor X-axis; (b) upper arm sensor Y-axis; (c) upper arm sensor Z -axis. [/fig]
[fig] Figure 6: RMSE value distribution of the model under different numbers of neurons. [/fig]
[fig] Figure 7, Figure 8: Prediction accuracy of test set and training set under different numbers of neurons. Indicator comparison of different training functions: (a) training time and RMSE value; (b) training steps. ( * * indicates a significant difference compared with traingda, P < 0:01. * * * indicates a highly significant difference compared with traingda, P < 0:001.). [/fig]
[fig] Figure 9: Performance comparison of transfer function between hidden layer and output layer. [/fig]
[fig] Figure 10: Distribution of predicted staging and clinical staging. [/fig]
[table] Table 1: Basic information of patients. [/table]
[table] Table 2: Patient rehabilitation data. [/table]
[table] Table 3: Patient rehabilitation exercise prescription. [/table]
[table] Table 4: Patient rehabilitation data statistics. [/table]
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Neonatal Neutrophils Stimulated by Group B Streptococcus Induce a Pro-Inflammatory T Helper Cell Bias
Background-Group B Streptococcus (GBS) infection causes inflammatory co-morbidities innewborns. While the mechanisms remain unclear, evidence suggests that impaired innate-adaptive immune interactions may be contributory. We hypothesized that GBS-stimulated neonatal neutrophils provide a milieu that may drive pro-inflammatory T helper cell programming.Methods-Neutrophils were stimulated with Type III GBS (COH1); supernatants or intact neutrophils were co-cultured with CD4 + T cells or Treg cells. Resulting intracellular cytokines and nuclear transcription factors were determined by multicolor flow cytometry.Results-GBS-stimulated neutrophils released soluble mediators that induced greater IL-17responses in neonatal vs. adult CD4 + T cells in the absence of added polarizing cytokines. GBSstimulated neonatal neutrophils also induced robust expression of the canonical nuclear transcription factors for Th1 (Tbet) and Th17 (IL-17) cells in CD4 + T cells. Following GBS Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
# Introduction
Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive bacterium that frequently colonizes the intestines and lower genital tract. GBS is particularly pathogenic in immunocompromised individuals [bib_ref] Group B streptococcal infections in elderly adults, Edwards [/bib_ref] , and GBS infections are a leading cause of fetal and infant death in the United States and around the world [bib_ref] Epidemiology of invasive group B streptococcal disease in the United States, Phares [/bib_ref]. Imbalanced fetal and neonatal immune responses facilitate the inflammatory morbid complications and mortality associated with GBS infection [bib_ref] SIRS and group-B streptococcal sepsis in newborns: pathogenesis and perspectives in adjunctive..., Henneke [/bib_ref]. The exact mechanism(s) by which GBS induces neonatal inflammatory responses remain(s) enigmatic, however, neutrophils appear to be a key cellular component in this process [bib_ref] Neutrophils directly recognize Group B streptococci and contribute to interleukin-1beta production during..., Mohammadi [/bib_ref]. The findings of neutrophil infiltration in the fetal placenta and in preterm neonatal lungs were also particularly striking in association with fatal intrauterine GBS infection [bib_ref] The histologic fetoplacental inflammatory response in fatal perinatal group B-streptococcus infection, De Paepe [/bib_ref].
Neutrophils play a sentinel role in modulating immune responses to invading pathogens (reviewed in (7)). Although neonatal neutrophils exhibit intrinsic functional impairments that increase host susceptibility to infections (reviewed in [bib_ref] Age-appropriate functions and dysfunctions of the neonatal neutrophil, Lawrence [/bib_ref] , paradoxically they can also exhibit exaggerated inflammatory responses to microbial stimulation [bib_ref] Toll-like receptor 1/2 stimulation induces elevated interleukin-8 secretion in polymorphonuclear leukocytes isolated..., Thornton [/bib_ref]. Neutrophils collaborate with the adaptive immune Th17 cells to amplify inflammatory processes that may be protective against infections or that can foster the pathogenesis of inflammatory disorders [bib_ref] Evidence for a cross-talk between human neutrophils and Th17 cells, Pelletier [/bib_ref]. Although the contributions of neutrophil-Th17 cell interactions to neonatal inflammation have not been described, evidence in humans and mice indicates a neonatal capacity for robust Th17 responses [bib_ref] Developmental regulation of Th17-cell capacity in human neonates, Black [/bib_ref] , particularly under conditions also associated with exacerbated neutrophil responses [bib_ref] Augmented Th17-type immune responses in preterm neonates exposed to histologic chorioamnionitis, Rito [/bib_ref] [bib_ref] Maternal inflammation modulates infant immune response patterns to viral lung challenge in..., Gleditsch [/bib_ref]. Thus, neutrophil-Th17 interactions likely contribute to inflammatory morbidities in young infants, including those associated with GBS infections.
Studies have shown the induction of Th1 or Th17-type immune responses during GBS infection and colonization [bib_ref] Group B Streptococcus induces a robust IFN-gamma response by CD4(+) T cells..., Clarke [/bib_ref] [bib_ref] Characterization of host immunity during persistent vaginal colonization by Group B Streptococcus, Patras [/bib_ref]. Inflammatory monocytes can promote Th17 cell propagation by releasing the polarizing cytokines, IL-1β and IL-6 [bib_ref] TNFalpha promotes Th17 cell differentiation through IL-6 and IL-1beta produced by monocytes..., Zheng [/bib_ref] , which are also produced by neutrophils (reviewed in [bib_ref] Neutrophil-derived cytokines: facts beyond expression, Tecchio [/bib_ref]. However, whether neonatal neutrophils have a direct role in the generation of Th1 and Th17 cells, particularly in the context of infection, has not been reported. The present studies were designed to test our hypothesis that GBS can stimulate neonatal neutrophils to drive inflammatory adaptive immune responses.
# Materials and methods
# Ethics statement
This study was performed under an approved protocol and in strict accordance with the guidelines and policies of the Institutional Review Board for Human Studies of Saint Louis University.
## Blood samples
Human umbilical cord blood (CB) samples from healthy full term newborns were obtained with parental consent immediately after delivery for all neutrophil studies. For other studies, anonymous CB samples (less than 12 hours post-delivery) were obtained from the SSM Health St. Louis Cord Blood Bank. Consented healthy adult volunteers were used as controls in some studies.
## Neutrophil and cd4 + t cell isolation
Mononuclear cells and neutrophils were isolated by density centrifugation from CB or adult peripheral blood using Ficoll-Paque PLUS gradient (GE Healthcare). CD4 + T cells or naïve CD4 + T cells were isolated by negative selection (EasySep™ Human CD4 + T cell enrichment kit or EasySep™ Human Naïve CD4 + T Cell Isolation Kit, StemCell Technologies, Vancouver, Canada) according to the manufacturer's instructions. Tregs and T effector cells (Teff) were isolated from purified CD4 + cells by positive selection (EasySep™ Human CD25 Positive Selection Kit, StemCell Technologies). Neutrophil purity, determined by H&E stain (Dif-Quik, Sigma, St. Louis), was > 95%. Neutrophil viability, determined by trypan blue staining, was >94% following isolation procedures, while 60-70% viability was observed following a 1 h incubation with heat-killed (HK) GBS.
## Group b streptococcus
COH1, a virulent serotype III GBS strain from a neonate with sepsis (a generous gift of Dr. Morven Edwards), was grown to log phase in Todd-Hewitt broth (Difco, Detroit, MI), washed, and re-suspended in phosphate-buffered saline (PBS) [bib_ref] Nonopsonic binding of type III Group B Streptococci to human neutrophils induces..., Albanyan [/bib_ref]. To obtain HK-GBS for studies, bacteria diluted in PBS were incubated at 56°C for 30 min; bacterial death was confirmed by re-plating HK-GBS samples. Aliquots of the HK-GBS stock solution of these GBS isolates were stored at -70°C.
## Gbs stimulation studies
Isolated neutrophils (2.5 × 10 6 cells/mL) suspended in RPMI 1640, 10% FBS were cocultured at 37° C, 5% CO 2 for 1 h in media alone or in the presence of heat-killed GBS (hereafter referred to as 'GBS') at a ratio of 10 bacteria per neutrophil [bib_ref] Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage..., Carlin [/bib_ref]. Harvested cellfree culture supernatants (unstimulated neutrophil-conditioned medium, NCM; or GBSstimulated neutrophil-conditioned medium, NCM-GBS) were stored at -80° C until bioassay. For cell-cell (neutrophil-CD4 + T cell) contact experiments, neutrophils cultured in media alone (PMN) or in the presence of GBS (PMN-GBS) were pelleted then re-suspended in complete T cell medium (2 mM glutamine, 50 μM β-mercaptoethanol, 10% heatinactivated human AB type serum, 100 U penicillin/100 μg streptomycin/mL) before addition to CD4 + T cell cultures. For studies to compare the effects of neutrophil supernatants and intact neutrophils on CD4 + target cells, GBS-stimulated or control CB neutrophils, or the resulting CB neutrophil supernatants, were added to autologous CB CD4 + T cell cultures in same-day experiments, as described below. To assess a direct effect of GBS, bacteria (2.5 × 10 7 /mL) suspended in media were cultured for 1 h; cell-free bacterial supernatants were stored at -80° C until bioassay.
## Cd4 + t cell co-cultures
Purified CD4 + T cells (2 × 10 6 cells) were suspended in media and cultured either in media alone, or in media containing 50% (vol/vol%) of NCM or 50% NCM-GBS. For cell-cell contact studies, neutrophils suspended in media were co-cultured with CD4 + T cells at a 2:1 ratio. All CD4 + T cells were cultured on 24 well plates coated with anti-CD3 Ab (2 μg/mL) and in the presence of IL-2 (50 U/mL) at 37° C, 5% CO 2 for 3-6 d [bib_ref] Human tumor-infiltrating Th17 cells have the capacity to differentiate into IFN-gamma+ and..., Ye [/bib_ref]. For 6 d cultures, culture media was refreshed on d 3 with starting media containing IL-2.
## Antibodies
The following fluorochrome-labeled Ab and their respective isotype IgG controls (purchased from Becton-Dickinson, NJ, unless otherwise indicated) were used for surface or intracellular staining of cells: CD4-V500 (clone RPA-T4), CD25-BV605 (2A3), IFNγ-FITC (B27), IL-17A-AF700 (N49-653), FoxP3-APC (eBiosciences, clone PCH101), Tbet-V450 (O4-46), GATA-3-PECy7 (L50-823), and RORγt-PE (Q21-559).
## Flow cytometry and t helper (th) cell subset analyses
To identify Th-specific intracellular cytokines and nuclear transcription factors, at the end of culture CD4 + T cells were stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin for 1 h prior to the addition of 5 μg/ml of Brefeldin A (Golgi-Stop®, BD), then incubated for an additional 4 hours at 37°C. Stimulated cells stained for surface antigens (CD4, CD25) were then fixed and permeabilized (anti-human FoxP3 staining set, eBioscience) as per the manufacturer's instructions. Permeabilized cells were stained for intracellular antigens using fluorochrome-labeled mAbs or mAb type-specific, fluorochrome-labeled IgG controls. Samples were acquired within 24 hours of staining using a 16-color BD LSRII Flow Cytometer. Acquired samples were analyzed using the FlowJo 7.2.2 software (Tree Star, Ashland, OR). Within the gated CD4 + T cell population, Th1 cells were identified by their expression of Tbet and/or IFNγ, and Th17 cells were identified by their expression of RORγt and/or IL-17A. Tregs were identified as CD4 + CD25 + Foxp3 + or CD4 + CD25 + populations; the CD4 + CD25populations represented T effector cells (Teff).
# Statistical analysis
Experimental data were compared using one-way ANOVA (multiple comparisons) or the paired Student ttest as appropriate using Prism v6.03 (GraphPad Software, Inc., La Jolla, CA). A P value <0.05 was considered to be significant.
# Results
## Soluble mediators released by gbs-stimulated neutrophils induce higher frequencies of il-17 + cd4 cells in neonatal vs. adult cultures
To determine if GBS stimulation of neonatal and adult neutrophils might induce differential responses in target CD4 + T cells, neonatal or adult naïve CD4 + T cells [fig_ref] Figure 1: GBS-stimulated neonatal neutrophils release soluble mediators that enhance IFNγ and IL-17 expression... [/fig_ref] , b) were incubated with supernatants of autologous GBS-stimulated neutrophils, and the resulting T helper (Th) phenotypes identified by intracellular staining and flow cytometric analysis. For these and all subsequent studies, a culture period of 6 d was chosen in order to maximize Th17 responses in cultured CD4 + T cells, based on preliminary data and our previous work [bib_ref] Human tumor-infiltrating Th17 cells have the capacity to differentiate into IFN-gamma+ and..., Ye [/bib_ref]. As shown [fig_ref] Figure 1: GBS-stimulated neonatal neutrophils release soluble mediators that enhance IFNγ and IL-17 expression... [/fig_ref] , supernatants of GBS-stimulated neutrophils induced the expression of the Th1 cytokine, IFNγ, in both neonatal and adult co-cultures. Although there was a trend towards higher IFNγ + CD4 + T cell frequencies in neonatal cultures, this difference did not reach significance (P = 0.08). In contrast, supernatants of GBS-stimulated neutrophils enhanced the frequencies of CD4 + T cells that expressed the Th17 cytokine, IL-17A (hereafter referred to as IL
## Gbs-stimulated neonatal neutrophils release factors that induce th1-, th17-, and tregspecific markers in neonatal cd4 + t cells
To assess the effects of neutrophil-derived soluble mediators on the expression of Th1 and Th17-related nuclear transcriptions factors, in the next series of studies neonatal CD4+ T cells were co-cultured with supernatants of unstimulated or GBS-exposed neonatal neutrophils, or in media only. Co-culture with supernatants of GBS-stimulated but not unstimulated neutrophils induced expression of the canonical Th1 nuclear transcription factor, Tbet [fig_ref] Figure 2: Soluble mediators released by neonatal neutrophils induce Tbet and RORγt expression in... [/fig_ref]. Neither unstimulated nor GBS-stimulated neutrophil supernatants induced significant expression of GATA-3, the Th2 nuclear transcription factor, although the latter showed a positive trend (P = 0.07) [fig_ref] Figure 2: Soluble mediators released by neonatal neutrophils induce Tbet and RORγt expression in... [/fig_ref]. In contrast, supernatants from both unstimulated and GBS-exposed neutrophils robustly induced CD4 + T cell expression of the respective master nuclear transcription factors for Th17 and Treg cells, RORγt and FoxP3 [fig_ref] Figure 2: Soluble mediators released by neonatal neutrophils induce Tbet and RORγt expression in... [/fig_ref]. In both cases, supernatants of GBS-stimulated neutrophils had the greatest effects relative to those of unstimulated neutrophils.
## Gbs-stimulated neonatal neutrophils promote the generation of neonatal treg cell populations that co-express th1-and th17-type cytokines
Based on the observed induction of FoxP3 expression in CD4 + T cells by supernatants of neonatal GBS-stimulated neutrophils, we next sought to determine if GBS-stimulated neonatal neutrophils might also influence the generation of neonatal CD4 + CD25 + FoxP3 + Tregs. As shown [fig_ref] Figure 3: GBS-stimulated neonatal neutrophils promote Treg generation via soluble mediators and cell-cell contact... [/fig_ref] , neonatal CD4 + T cell cultures containing supernatants of unstimulated or GBS-exposed neutrophils increased the mean frequencies of CD4 + CD25 + Foxp3 + cell populations, with the greatest effect observed for GBS-stimulated neutrophils. In subsequent studies, neonatal CD4 + T cells were cultured either with supernatants of GBS-stimulated neonatal neutrophils or the intact GBS-stimulated neutrophils.
As shown [fig_ref] Figure 3: GBS-stimulated neonatal neutrophils promote Treg generation via soluble mediators and cell-cell contact... [/fig_ref] , both supernatants and intact neutrophils induced Treg populations in autologous CD4 + T cell populations. However, GBS-stimulated neutrophil supernatants induced a moderately but significantly greater response in CD4 + T cells than that observed following their contact with GBS-stimulated neutrophils.
We recently reported elevated frequencies of Tregs with Th17-type characteristics in preterm neonates exposed to histologic chorioamnionitis, a neutrophil-driven inflammation of the placenta [bib_ref] Characterization of host immunity during persistent vaginal colonization by Group B Streptococcus, Patras [/bib_ref]. Thus, we next sought to determine if GBS-stimulated neonatal neutrophils release factors that promote the generation of Treg cells with an inflammatory phenotype in vitro. In parallel studies, culture supernatants from both unstimulated and GBS-stimulated neonatal neutrophils increased the frequencies of CD4 + CD25 + FoxP3 + Tregs that coexpressed IFNγ [fig_ref] Figure 4: GBS-stimulated neonatal neutrophils release soluble mediators that promote the generation of IFNγ... [/fig_ref] , c) or IL-17 [fig_ref] Figure 4: GBS-stimulated neonatal neutrophils release soluble mediators that promote the generation of IFNγ... [/fig_ref]. As was observed in related studies, soluble mediators derived from GBS-stimulated neutrophils had the more striking effect on the generation of Tregs with inflammatory cytokine expression compared with those of unstimulated neutrophils.
## Gbs-stimulated neonatal neutrophils and derived mediators enhance treg cell coexpression of tbet and rorγt
We next sought to determine whether in addition to the de novo generation of Tregs with inflammatory properties, GBS-stimulated neutrophils might also promote phenotypic alterations of existing Treg populations. To achieve this we co-cultured purified neonatal CD4 + CD25 + Tregs with supernatants of autologous GBS-stimulated neutrophils or the resulting intact GBS-stimulated neutrophils, or with supernatants of GBS bacteria . As shown, both GBS-stimulated neutrophil supernatants and intact GBS-stimulated neutrophils promoted marked enhancements in Treg co-expression of Tbet and RORγt . In contrast, co-incubation of neonatal CD4 + T cells with GBS supernatants alone did not significantly increase Treg co-expression of Tbet (P=0.06) or RORγt (P = 0.12) over that in media only.
## Gbs-stimulated neonatal neutrophils release mediators that induce ifnγ or il-17 expression in neonatal treg and teff populations
To determine if neonatal Treg cells might be particularly susceptible to neutrophil-mediated induction of Th1 and Th17-type responses, we compared the effects of supernatants of unstimulated or GBS-stimulated neonatal neutrophils on the intracellular expression of IFNγ and IL-17 in neonatal naïve Teff (CD4 + CD25 -) and Treg (CD4 + CD25 + ) populations [fig_ref] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in... [/fig_ref]. As shown, co-culture with GBS-stimulated, but not unstimulated, neutrophil supernatants induced co-expression of both IFNγ [fig_ref] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in... [/fig_ref] and IL-17 [fig_ref] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in... [/fig_ref] in Teff and Treg cell populations. A higher mean frequency of IFNγ + cells was observed in Treg vs.
Teff populations [fig_ref] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in... [/fig_ref]. In contrast, frequencies of IL-17 + Treg cells were not significantly different when compared to IL-17 + Teffs (P=0.11) [fig_ref] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in... [/fig_ref].
# Discussion
The present studies were designed to assess the potential effects of GBS-stimulated neonatal neutrophils on the generation of inflammatory CD4 + T cell populations. We now report that GBS stimulation of neonatal neutrophils induces robust Th1-and Th17-type responses in neonatal CD4 + T cell and Treg populations through mechanisms that can involve cell-cell contact and soluble mediators.
We observed that GBS-stimulated neonatal neutrophils, and to a lesser degree unstimulated neutrophils, biased the CD4 differentiation program towards the generation of Th1 and Th17-type cells. The inductive effects of GBS-stimulated neutrophils on expression levels of IL-17 were most striking in neonatal CD4 + T cell cultures relative to those of adults. These findings are consistent with studies showing that GBS more strongly promotes inflammatory responses in neonatal monocytes relative to those of adults [bib_ref] Interleukin-6 production by human neonatal monocytes stimulated by type III group B..., Vallejo [/bib_ref] [bib_ref] Cytokine expression of cord and adult blood mononuclear cells in response to..., Berner [/bib_ref]. The higher IL-17 expression levels we observed in neonatal CD4 + T cells were not entirely surprising given the intrinsic Th17 bias previously reported in neonates [bib_ref] Developmental regulation of Th17-cell capacity in human neonates, Black [/bib_ref]. However, the capacity of GBSstimulated neonatal neutrophils to promote Th17-type responses in target CD4 + T cells, without the assistance of exogenous Th17-propagating cytokines, was a novel finding. We also determined inductive effects of GBS-stimulated neonatal neutrophils on IFNγ expression, findings supportive of potent neonatal Th1-type immune responses to microbial stimulation [bib_ref] Direct TLR-2 Costimulation unmasks the proinflammatory potential of neonatal CD4+ T cells, Sinnott [/bib_ref]. In contrast, GBS-stimulated neonatal neutrophils induced a nominal effect on the expression of the Th2 transcription factor, GATA-3. Although neonates characteristically exhibit predominantly Th2-type immunity (reviewed in (25)), Th2 responses may be suppressed under conditions that promote Th17 cells [bib_ref] IL-4 abrogates T(H)17 cell-mediated inflammation by selective silencing of IL-23 in antigen-presenting..., Guenova [/bib_ref]. We also did not find an independent effect of GBS bacteria on the induction of Th1 or Th17 responses. Our combined in vitro observations suggest that GBS promotes pro-inflammatory responses in neonates in part through actions mediated by targeted neutrophils.
Neonatal neutrophil-derived soluble mediators were also found to promote the propagation of Tregs in CD4 + T cell populations and to modulate the proportions of pro-inflammatory Tregs that co-expressed IFNγ + or IL-17 + , independent of a requirement for cell contact. These observations are consistent with a recent study showing that signaling via TLR2 promotes the generation of Th17-type Tregs in the absence of antigen-presenting cells [bib_ref] TLR2 stimulation drives human naive and effector regulatory T cells into a..., Nyirenda [/bib_ref] , which has relevance to our present findings given that GBS-mediated inflammatory signaling is driven in part by TLR2 activation [bib_ref] Induction and termination of inflammatory signaling in group B streptococcal sepsis, Wennekamp [/bib_ref]. We also determined that GBS-stimulated neonatal neutrophils directly enhanced the in vitro generation of Treg cell populations via physical contact with CD4 + T cells, similar to findings of an inductive effect of neutrophil-CD4 cell contact on Th1 and Th17 differentiation in studies of adult mice and humans [bib_ref] Mouse neutrophils are professional antigenpresenting cells programmed to instruct Th1 and Th17..., Abi Abdallah [/bib_ref] [bib_ref] Neutrophils and T cells: bidirectional effects and functional interferences, Thewissen [/bib_ref].
Our present studies have identified a unique role of neonatal neutrophils in the induction of Th cell populations with protective and inflammatory potential in the context of GBS infection. While it is well established that Th1-type responses are critical in protecting the neonatal host against infections, the protective role of Th17 cells in neonates is less well defined [bib_ref] Protecting the newborn and young infant from infectious diseases: Lessons from immune..., Kollmann [/bib_ref]. The present studies provide additional evidence of the neonatal capacity to increase potentially protective Th1 responses to infectious stimuli, particularly through TLR2 signaling mechanisms [bib_ref] Direct TLR-2 Costimulation unmasks the proinflammatory potential of neonatal CD4+ T cells, Sinnott [/bib_ref] [bib_ref] Induction and termination of inflammatory signaling in group B streptococcal sepsis, Wennekamp [/bib_ref]. Our findings of enhanced generation of IL-17producing immune cells in the context of a GBS stimulus also suggest their contribution to the neonatal host response to infection [bib_ref] Microbial-induced Th17: superhero or supervillain?, Mcgeachy [/bib_ref]. However, although IL-17 is critical to protective immunity, recent studies in neonates also suggest its role in potentiating sepsis-related inflammatory morbidity in neonates through interactions with IL-18 and in mediating the pathogenesis of necrotizing enterocolitis [bib_ref] Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18, Wynn [/bib_ref] [bib_ref] Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis, Egan [/bib_ref]. Furthermore, crosstalk between neutrophils and Th17 cells can amplify injurious inflammatory responses [bib_ref] Evidence for a cross-talk between human neutrophils and Th17 cells, Pelletier [/bib_ref] , although the specific contribution of this mechanism to neonatal inflammation is unclear. In addition, our observation that GBS-stimulated neutrophils enhanced frequencies of RORγt + Tregs suggests a potentially pathogenic phenotype, since RORγt expression coincides with loss of Treg suppressive function and facilitates inflammatory Treg-Th17 conversion [bib_ref] Expression of RORgammat marks a pathogenic regulatory T cell subset in human..., Blatner [/bib_ref]. Pertinently, the TLR2 signaling associated with GBS infection (28) could potentially drive the generation of Th17-type Tregs with diminished suppressive capacity [bib_ref] TLR2 stimulation drives human naive and effector regulatory T cells into a..., Nyirenda [/bib_ref]. Whether the Tregs induced by GBS-stimulated neutrophils exhibit primarily anti-inflammatory or pathogenic function in neonates is an important question that merits further investigation.
GBS infection and other related inflammatory disorders are associated with abundant infiltrative tissue neutrophils (5, 6) that could potentially co-localize with Th17 cells [bib_ref] Evidence for a cross-talk between human neutrophils and Th17 cells, Pelletier [/bib_ref]. These observations and our present studies point to neutrophils as an important environmental source of Th1-and Th17-polarizing cytokines. While the specific neutrophilderived mediators contributing to Th1 and Th17 responses were not examined in the present studies, existing evidence provides some important clues. During microbial infection, neutrophils release IL-12 and IFNγ, cytokines that promote the propagation of Th1 cells [bib_ref] Neutrophil-derived cytokines: facts beyond expression, Tecchio [/bib_ref]. In a model of GBS-induced peritonitis, neutrophils were also found to be the primary producers of IL-1β (5), a key Th17-propagating cytokine [bib_ref] Interleukins 1beta and 6 but not transforming growth factor-beta are essential for..., Acosta-Rodriguez [/bib_ref]. Neonatal neutrophils robustly released IL-1β under inflammatory conditions [bib_ref] Elevated interleukin-1 expression in human neonatal neutrophils, Contrino [/bib_ref] and following exposure to PGN, the major exoskeleton component of gram positive bacteria [bib_ref] Expression profile of cord blood neutrophils and dysregulation of HSPA1A and OLR1..., Fong [/bib_ref]. Thus, neutrophil-derived IL-1β may contribute to a cytokine milieu that supports the generation of Th17 cells and inflammatory IL-17 + Tregs [bib_ref] Lipopolysaccharide-induced chorioamnionitis promotes IL-1-dependent inflammatory FOXP3+ CD4+ T cells in the fetal..., Rueda [/bib_ref]. We also observed reduced survival in neutrophils coincubated with GBS, which may be relevant to our data given that apoptotic signaling in neutrophils involves IL-1β release and Th17-type responses [bib_ref] Expression of Th17-related genes in PHA/IL-2-activated human T cells by Fas signaling..., Su [/bib_ref]. In contrast, neutrophilmediated induction of Th17 cells does not appear to involve IL-6 (29), another key Th17propagating cytokine [bib_ref] Interleukins 1beta and 6 but not transforming growth factor-beta are essential for..., Acosta-Rodriguez [/bib_ref]. Studies are currently underway to characterize the neutrophilderived mediator(s) involved in neonatal CD4 + T cell differentiation and to more fully define the underlying mechanisms.
The key observation made in the present paper, i.e. that GBS can stimulate neonatal neutrophils to promote Th1 and Th17 type responses, adds to existing knowledge of a role for GBS in the induction of neonatal inflammatory injury, such as meningitis [bib_ref] Group B streptococcal beta-hemolysin/cytolysin activates neutrophil signaling pathways in brain endothelium and..., Doran [/bib_ref]. Conversely, GBS has also been shown to suppress neutrophil responses through molecular mimicry mediated by Siglec-9 (20), although whether this or related mechanisms are important in neonates is unclear. We reported diminished expression of Siglec-9 and SHP-1 as well as enhanced basal signaling via PI3K/Akt in neonatal neutrophils [bib_ref] Siglec-9 and SHP-1 are differentially expressed in neonatal and adult neutrophils, Rashmi [/bib_ref]. These findings suggest a possible curtailment of GBS-mediated suppression in neonatal relative to adult neutrophils, which would be consistent with the greater GBS-mediated induction of inflammatory responses in neonatal immune cells observed in the present study and reported by others [bib_ref] Interleukin-6 production by human neonatal monocytes stimulated by type III group B..., Vallejo [/bib_ref] [bib_ref] Cytokine expression of cord and adult blood mononuclear cells in response to..., Berner [/bib_ref] [bib_ref] Direct TLR-2 Costimulation unmasks the proinflammatory potential of neonatal CD4+ T cells, Sinnott [/bib_ref]. These data suggest that severe GBS-infection in fetuses and neonates could promote immune perturbations that simultaneously provoke inflammation while suppressing protective responses, possibilities deserving of further investigation.
In summary, our present data suggest a compelling role for neonatal neutrophils in mediating Th cell responses through a combination of innate and adaptive immune mechanisms. While the resulting transformations could potentially facilitate neonatal protective immune responses against GBS [bib_ref] Microbial-induced Th17: superhero or supervillain?, Mcgeachy [/bib_ref] , conversely, exaggerated inflammatory responses might amplify the pathologic mechanisms associated with neonatal infection to promote a vicious cycle of inflammation [bib_ref] SIRS and group-B streptococcal sepsis in newborns: pathogenesis and perspectives in adjunctive..., Henneke [/bib_ref]. Despite the inherent limitations of in vitro studies, our data provide important new information that advances understanding of inflammatory mechanisms in the vulnerable neonatal population and that sets the stage for future investigations.
[fig] Figure 1: GBS-stimulated neonatal neutrophils release soluble mediators that enhance IFNγ and IL-17 expression in neonatal CD4 + T cells a, b. Naïve CD4 + T cells from term neonates (CB) or from healthy adult (AD) donors were co-cultured in anti-CD3 coated plates in complete T cell medium (M, white bars), or in M containing a 50% (vol/vol%) concentration of supernatants of respective CB or AD neutrophils stimulated with heat-killed GBS (NCM-GBS, black bars). For these and all subsequent studies, IL-2 (50 U/mL) was added to cultures on D0 and again on D3 when media was refreshed. After a 6 d incubation, cells were stimulated prior to staining for intracellular cytokine analysis by flow cytometry, as described in Methods. Shown are mean frequencies of CB and AD CD4 + populations that co-expressed either a. IFNγ or b. IL-17 in the presence of M or NCM-GBS. Data represent paired CB and AD studies, n = 5; X ± SEM. *P<0.05, NCM-GBS vs. M;**P<0.05, AD NCM-GBS vs. CB NCM-GBS. c, d. Frequencies of CD4 + T cell populations that expressed c. IL-17; or d. IFNγ when cultured in the presence of M only, NCM, or NCM-GBS. Scatter-plot data represent the means of 10 individual, replicate donor samples; X ± SEM. * P<0.05, NCM vs. M; NCM-GBS vs. NCM; ** P<0.01, NCM-GBS vs. M; NCM-GBS vs. NCM. [/fig]
[fig] Figure 2: Soluble mediators released by neonatal neutrophils induce Tbet and RORγt expression in CD4 + T cells Neonatal CD4 + T cells were cultured in the presence of M alone (white bars), or M containing 50% (vol/vol%) of culture supernatants from unstimulated (NCM, hatched bars) or GBS-stimulated neonatal neutrophils (NCM-GBS, black bars). Shown are the proportions of cultured CD4 + T cells that expressed a. Tbet; b. GATA-3; c. RORγt and d. FoxP3. Data represent the results of replicate studies from CB donors (n = 4); X ± SEM. [/fig]
[fig] Figure 3: GBS-stimulated neonatal neutrophils promote Treg generation via soluble mediators and cell-cell contact a. Neonatal CD4 + T cells were cultured for 6 d in the presence of M alone (white bars) or in M containing (50% vol/vol) of unstimulated (NCM, hatched bars) or GBS-stimulated (NCM-GBS, black bars) neutrophil supernatants. b. Neonatal CD4 + T cells were cultured in M (white bar), in 50% NCM-GBS (black bar), or in the presence of intact GBS-stimulated CB neutrophils (PMN-GBS, 2:1 PMN:CD4 ratio, grey bar). For both groups of studies, the proportions of CD4 + CD25 + Foxp3 + Treg cells in gated CB CD4 + T cells were determined by flow cytometric analysis of 3-5 CB samples; X ± SEM. *P<0.05; ** P<0.01, NCM-GBS vs. M; PMN-GBS vs. . Author manuscript; available in PMC 2018 June 06. [/fig]
[fig] Figure 4: GBS-stimulated neonatal neutrophils release soluble mediators that promote the generation of IFNγ + and IL-17 + Tregs Neonatal CD4+ T cells were incubated with M (white bars), 50% NCM (hatched bars), or 50% NCM-GBS (black bars). The intracellular contents of a., c. IFNγ and b., c. IL-17 were determined in stimulated CD4 + CD25 + Foxp3 + cell populations by flow cytometry. Data are representative of replicate studies of 8 CB donors. X ± SEM. *P<0.05; ** P<0.01, NCM-GBS vs. M; NCM vs. M. c. Dot plots show the proportionate expression of IFNγ + FoxP3 + or IL-17 + FoxP3 + populations in CD4 + T cells following co-culture with CTCM or NCM-GBS in a representative study from one CB donor. [/fig]
[fig] Figure 6: GBS-stimulated neonatal neutrophils release mediators that induce IFNγ and IL-17 expression in neonatal Tregs and Teff Neonatal CD4 + cells were separated into CD25 -(Teff) and CD25 + (Treg) populations by immunomagnetic selection. Cells were cultured in the presence of M, NCM (50%), or NCM-GBS (50%), then stimulated and stained for intracellular cytokine analysis. Data represent the results of 5 separate studies; X ± SEM. * P<0.05, NCM-GBS vs. M; **P=0.02, CD25 + vs. CD25cell populations. . Author manuscript; available in PMC 2018 June 06. [/fig]
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Bizarre parosteal osteochondromatous proliferation in the lingual area of the mandibular body versus osteochondroma at the mandibular condyle
Background: Bizarre parosteal osteochondromatous proliferation (BPOP) is benign and usually occurs in the small tubular bones of the hands and feet, but it is extremely rare in the oral and maxillofacial region. Methods: The present study compares a case of BPOP occurring in the lingual area of the right mandibular body with a representative case of osteochondroma occurring in the left mandibular condyle using immunohistochemical methods.Results: BPOP showed no continuity to the cortical bone of the mandible on X-ray and was histologically composed of immature cartilage and bone tissues, whereas osteochondroma showed overgrowth of hypertrophic chondrocytes accompanied by mature bone with endochondral ossification. Although BPOP showed no features of cellular atypia or malignant transformation, it expressed more osteogenic proteins, including BMP-2, BMP-4, RUNX2, OC, AP, OPG, RANKL, CTGF, and bFGF, than osteochondroma. Furthermore, the perichondral spindle cells and marrow osteoblasts/ fibroblasts of BPOP showed stronger immunoreaction of PCNA, p53, β-catenin, BCL2, pAKT, survivin, 14-3-3, CEA, EMA, pan-K, and S-100 than the tumor cells of osteochondroma. Conclusions: Therefore, it was presumed that similar to embryonal osteochondroid tissue, BPOP might be activated by osteogenic and oncogenic signaling and that this increased signaling may explain the rapid growth and high recurrence of BPOP.
# Background
Bizarre parosteal osteochondromatous proliferation (BPOP) is a rare, benign osteocartilaginous lesion which was first described in 35 lesions from the small bones of the hands and feet by . Many cases involving various other sites, including the zygoma, maxilla, and mandible, have been reported [bib_ref] Bizarre parosteal osteochondromatous proliferation (Nora's Lesion) of the mandible. A rare bony..., Dashti [/bib_ref] [bib_ref] Lesions of juxtacortical origin (surface lesions of bone), Kenan [/bib_ref] [bib_ref] Bizarre parosteal osteochondromatous proliferation of bone (Nora's lesion), Meneses [/bib_ref] [bib_ref] Magnetic resonance imaging characteristics of bizarre parosteal osteochondromatous proliferation of the hand:..., Orui [/bib_ref]. BPOP usually shows key radiological features, including lack of histological features characterized by three distinct components with variable degrees of representation: (1) hypercellular cartilage with calcification and ossification, with the calcified cartilage having a characteristic basophilic tinctorial quality; (2) cancellous bone undergoing maturation; and (3) spindle cell stroma without cytologic atypia [bib_ref] Bizarre Parosteal osteochondromatous proliferation (Nora's lesion) of the hand: a report of..., Barrera-Ochoa [/bib_ref] [bib_ref] Bizarre parosteal osteochondromatous proliferation: Nora's lesion, Chaabane [/bib_ref] [bib_ref] Bizarre parosteal osteochondromatous proliferation (Nora's lesion): a retrospective study of 12 cases,..., Abramovici [/bib_ref] [bib_ref] Bizarre parosteal osteochondromatous proliferation: a locally aggressive benign tumor, Joseph [/bib_ref]. BPOP, also known as Nora's lesion, often behaves like a malignant tumor, clinically and microscopically. Histological features of BPOP include hypercellular spindle cells, a blue tinctorial quality in the osteocartilaginous interfaces, and a scattering of binucleated or bizarre enlarged chondrocytes [bib_ref] A bizarre parosteal osteochondromatous proliferation (Nora's lesion) of metatarsus, a histopathological and..., Doganavsargil [/bib_ref].
Generally, it is presumed that BPOP arises from periosteal tissues through a process of cartilaginous metaplasia. BPOP can be easily confused, both clinically and microscopically, with other benign and malignant lesions of the bone, including osteochondroma, myositis ossificans, florid reactive periostitis, turret exostosis, parosteal osteosarcoma, and chondrosarcoma [bib_ref] Nora's lesion, a distinct radiological entity?, Dhondt [/bib_ref] [bib_ref] Submandibular osseous choristoma, Johann [/bib_ref]. BPOP has been reported to have a high rate of recurrence (about 50 %) but without metastasis [bib_ref] Bizarre parosteal osteochondromatous proliferation: a locally aggressive benign tumor, Joseph [/bib_ref] [bib_ref] Usual clinical presentation of bizarre parosteal osteochondromatous proliferation (BPOP) with unusual histology, Lynch [/bib_ref]. Translocation between chromosomes 1 and 2, t(1;17)(q32;q21), and inversion of chromosome 7, inv(7)(q22q32), have been observed in BPOP [bib_ref] Bizarre parosteal osteochondromatous proliferation: a new cytogenetic subgroup characterized by inversion of..., Broehm [/bib_ref] [bib_ref] Bizarre parosteal osteochondromatous proliferation with a t(1;17) translocation, Endo [/bib_ref].
Osteochondroma is the most common tumor of the skeletal bones, but it is relatively uncommon in the jaws at the condyle or the tip of the coronoid process [bib_ref] Osteochondroma of the mandibular condyle: literature review and report of a case, Ribas Mde [/bib_ref]. This benign cartilage-capped tumor simulating unilateral condylar hyperplasia is usually discovered incidentally on radiographic examination or on palpation of a protruding mass in the affected area [bib_ref] Osteochondroma of the mandibular condyle. Case report and its management, Simon [/bib_ref]. Malocclusion and progressive facial asymmetry are common findings in most cases of condylar osteochondroma [bib_ref] Review of osteochondroma of mandibular condyle and report of a case series, Roychoudhury [/bib_ref]. Computed tomography and magnetic resonance imaging depicted the central part of the exophytic bone lesion as having continuity to the underlying bone marrow, which is considered to be the typical finding of osteochondroma compared to BPOP [bib_ref] Diagnosis of osteochondroma of the mandibular condyle in atypical facial pain: a..., Nasr [/bib_ref] [bib_ref] Diagnosis and prognosis of chondrosarcoma of bone, Rozeman [/bib_ref]. In addition, an inversion of chromosome 7 [inv (7)(q22q32)] has also been observed in osteochondroma [bib_ref] Bizarre parosteal osteochondromatous proliferation with an inversion of chromosome 7, Sakamoto [/bib_ref].
The present study used immunohistochemical (IHC) staining using different antisera to compare a case of BPOP occurring at the lingual area of the right mandibular body to a representative case of osteochondroma occurring in the left mandibular condyle.
# Methods
## A case of bpop and osteochondroma
A 17-year-old female noticed a hard sublingual mass 3 months prior and experienced stiffness during yawning. The oral mucosa then became ulcerated, and a calcified mass was exposed with whitish and partially bluish color. On computed tomography, the mass was ovoid and lobulated with irregular calcification and measured about 30 × 20 mm on the lingual area of the right mandibular body. The calcified mass showed no continuity with the cortical bone of the mandible on X-ray [fig_ref] Figure 1: Radiographic views of this study [/fig_ref] ; therefore, it was enucleated through simple intraoral dissection at Seoul National University Dental Hospital. The removed specimen was submitted to the Department of Oral Pathology, Gangneung-Wonju National University Dental Hospital (GWNUDH) for analysis. It was composed of cartilaginous and osseous tissues on gross observation. The cartilaginous tissue was approximated to the lingual surface of the mandibular body, while the osseous tissue had grown toward the sublingual area [fig_ref] Figure 2: BPOP [/fig_ref]. The lesion was diagnosed as BPOP through pathological examination (OS2014-25).
A 39-year-old female presented with severe malocclusion and facial asymmetry, which had slowly progressed for 4 years. She was referred to GWNUDH with a chief complaint of slight pain on the left temporomandibular joint during mouth opening. Her left mandibular condyle was severely enlarged with cortico-medullary continuity from adjacent bone structures on orthopantomogram [fig_ref] Figure 1: Radiographic views of this study [/fig_ref]. The tumorous condyle was surgically removed by high condylectomy and diagnosed as osteochondroma through pathological examination (S2014-4).
## Immunohistochemical study
The removed specimens of BPOP and osteochondroma were separately fixed in 10 % neutral formalin, decalcified with 5 % nitric acid, embedded with paraffin, and sectioned to 4-μm thickness. The microsections were routinely stained with hematoxylin and eosin (HE) and immunohistochemical (IHC) staining using the following antisera: proliferating cell nuclear antigen (PCNA; Santa Cruz Biotech, USA), bone morphogenetic protein (BMP)-2 (Santa Cruz Biotech), BMP-4 (Santa Cruz Biotech), runt-related transcription factor 2 (RUNX2; Abcam, Cambridge, UK), osteocalcin (OC; DAKO, Denmark), alkaline phosphatase (AP; DAKO), osteoprotegerin (OPG; Santa Cruz Biotech), receptor activator of nuclear factor-kappaB ligand (RANKL; Santa Cruz Biotech), connective tissue growth factor (CTGF; Abcam), basic fibroblast growth factor (bFGF; DAKO), p53 (Santa Cruz Biotech), v-akt murine thymoma viral oncogene homologue 1 (Santa Cruz Biotech), phosphorylated at Thr 308 (pAKT), β-catenin (Santa Cruz Biotech), B cell lymphoma 2 (BCL2; Santa Cruz Biotech), 14-3-3 (Santa Cruz Biotech), survivin (Santa Cruz Biotech), carcinoembryonic antigen (CEA; Santa Cruz Biotech), epithelial membrane antigen (EMA; Abcam), pan-keratins (pan-K; Santa Cruz Biotech), and S-100 (Santa Cruz Biotech, USA). IHC staining was performed using the indirect triple sandwich method [bib_ref] Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Kim [/bib_ref] [bib_ref] Metastatic leiomyosarcoma in the oral cavity: case report with protein expression profiles, Kim [/bib_ref]. Our institutional review board approved the examination of these biopsy specimens by the Department of Oral Pathology, GWNUDH (IRB 2015-07).
# Results
On histological observation, the BPOP specimen disclosed inner cartilaginous tissue and outer osseous tissue [fig_ref] Figure 2: BPOP [/fig_ref]. The cartilaginous tissue was immature and contained many bizarre proliferating chondrocytes [fig_ref] Figure 2: BPOP [/fig_ref] with multifocally distributed perichondral fibrous tissue mainly composed of spindle cells [fig_ref] Figure 1: Radiographic views of this study [/fig_ref]. The osseous tissue showed linearly elongated trabecular bones that stemmed from the cartilaginous tissue [fig_ref] Figure 2: BPOP [/fig_ref]. The endochondral ossification was almost abortive but extensively produced trabecular bones [fig_ref] Figure 2: BPOP [/fig_ref]. There was no atypical cellular change; however, the perichondral fibrous tissue was thickened with proliferating spindle cells.
Osteochondroma, on the other hand, showed exophytic growth of cartilaginous tissue, followed by trabecular bone ossification at the mandibular condyle. The condylar cartilage almost entirely lacked perichondral fibrous tissue but contained a thick layer of aggregated and proliferative hypertrophic chondrocytes [fig_ref] Figure 5: Osteochondroma [/fig_ref]. Endochondral ossification was evident with features of chondroid tissue embedded in ossifying bony tissue [fig_ref] Figure 5: Osteochondroma [/fig_ref].
IHC staining for osteogenetic protein expression in the BPOP specimen was conspicuously positive for both BMP-2 and BMP-4, which are known to indicate osteogenesis and chondrogenesis, respectively, while osteochondroma was much more weakly positive for these proteins. Particularly, the spindle cells of the perichondral fibrous tissue and marrow osteoblasts and fibroblasts of BPOP were strongly positive for OC, AP, and CTGF and consistently positive for OPG, RANKL, and bFGF (Figs. 2D-G and 3), whereas the proliferating chondrocytes and osteoblasts of osteochondroma [fig_ref] Figure 5: Osteochondroma [/fig_ref] were weakly positive for BMP-2, BMP-4, OC, AP, OPG, CTGF, and bFGF and rarely positive for RUNX2 and RANKL [fig_ref] Figure 5: Osteochondroma [/fig_ref].
Regarding the proliferative activity observed by PCNA immunoreaction, the BPOP specimen showed a conspicuous positive reaction in the spinous cells of the perichondral fibrous tissue and in some osteoblasts/fibroblasts of the osseous tissue [fig_ref] Figure 2: BPOP [/fig_ref] , while osteochondroma was rarely positive [fig_ref] Figure 5: Osteochondroma [/fig_ref].
On IHC staining of oncogenic protein expression, the bizarre chondrocytes of BPOP were strongly positive for β-catenin, BCL2, and pAKT and consistently positive for p53 and survivin [fig_ref] Figure 4 A: -J BPOP [/fig_ref] , while some tumor cells of osteochondroma were positive for β-catenin and 14-3-3 and weakly positive for p53, pAKT, BCL2, and survivin [fig_ref] Figure 5: Osteochondroma [/fig_ref].
On the other hand, the biomarkers of chondrosarcoma, such as CEA, EMA, pan-K, and S-100 [bib_ref] Immunoprofile of mesenchymal chondrosarcoma: aberrant desmin and EMA expression, retention of INI1,..., Fanburg-Smith [/bib_ref] [bib_ref] Chondroid chordomas and low-grade chondrosarcomas of the craniospinal axis. An immunohistochemical analysis..., Wojno [/bib_ref] [bib_ref] Clinicopathologic features of osteochondroma with malignant transformation, Zheng [/bib_ref] , showed a slight positive reaction in the spindle cells of perichondral fibrous tissue [fig_ref] Figure 4 A: -J BPOP [/fig_ref] but an almost negative reaction in the tumor cells of osteochondroma [fig_ref] Figure 4 A: -J BPOP [/fig_ref].
# Discussion
On histological observation of the present cases, osteochondroma showed increased endochondral ossification with overgrowth of hypertrophic chondrocytes, while BPOP showed abortive endochondral ossification. The immature cartilaginous tissue of BPOP abruptly produced the trabecular bones that were elongated linearly in a radiating fashion from the core cartilage. While the hypertrophic chondrocytes of osteochondroma produced trabecular bones that were subsequently connected with the original bone marrow, the cartilaginous tissue of BPOP, probably derived from the parosteal mesenchyme, produced trabecular bones in the external direction from the core cartilage, resulting in marrow discontinuity between BPOP and the original bone. However, the trabecular bone growth of BPOP was more active and extensive than that of osteochondroma, and BPOP had multiple cartilaginous tissues which were immature and diffusely scattered, while osteochondroma had a thick cartilage cap on the condylar head. These findings directly indicate that BPOP may have higher recurrence rate than osteochondroma.
In the IHC stains, BPOP showed more intense expression of osteogenic proteins (BMP-2, BMP-4, RUNX2, OC, AP, OPG, RANKL, CTGF, and bFGF) than the ostoeochondroma. BMP-2 and BMP-4, which are markers of osteogenesis and chondrogenesis, respectively, were consistently positive in BPOP but only weakly positive in osteochondroma. Furthermore, the biomarkers of ossification (RUNX2, OC, AP, OPG, and RANKL) and the biomarkers of mesenchymal growth (bFGF and CTGF) were much stronger in BPOP than in osteochondroma. It was thought that BPOP grew more actively than osteochondroma by producing immature cartilage and bone.
The higher expression of osteogenic proteins in BPOP than in osteochondroma was coincident with increased expression of PCNA in the perichondral spinous cells and marrow osteogenic cells of BPOP. Osteochondroma was rarely positive for PCNA. The present study also performed IHC examination for different oncogenic proteins (p53, β-catenin, BCL2, pAKT, and survivin) and the biomarkers of chondrosarcoma (CEA, EMA, pan-K, and S-100) in BPOP and osteochondroma. For the surgical approach for the osteochondroma, not like that for BPOP, several factors, such as complete excision with condylar reconstruction, mandibular contouring, and reconstruction of normal occlusion, must be considered. Virtual surgical simulation for guidance of excision of the mandibular condyle and combined correction of dentofacial deformities can be recommended recently [bib_ref] Treatment of dentofacial deformities secondary to osteochondroma of the mandibular condyle using..., Li [/bib_ref].
The oncogenic protein expression of perichondral spindle cells and marrow osteoblasts/fibroblasts of BPOP was strongly positive for p53, β-catenin, BCL2, pAKT, and survivin, while osteochondroma was weakly positive for βcatenin and 14-3-3 and rarely positive for p53, BCL2, pAKT, and survivin. Furthermore, BPOP was consistently positive for CEA, EMA, pan-K, and S-100, while osteochondroma was rarely positive for these proteins.
Immunohistochemically, BPOP showed the expression of bFGF and vascular endothelial growth factor (VEGF), similar to those occurring in endochondral ossification in the growth plate. Thus, BPOP is considered as a reparative process which is occasionally confused with other benign or malignant conditions [bib_ref] Bizarre parosteal osteochondromatous proliferation (Nora's lesion) of the foot, Horiguchi [/bib_ref]. It was presumed that BPOP was more strongly activated by the oncogenic proteins (p53, β-catenin, BCL2, pAKT, and survivin) than osteochondroma; BPOP might be affected by oncogenic signaling of cellular proliferation and survival. Eventually, BPOP produced CEA, EMA, pan-K, and S-100, implying that BPOP might have higher potential for malignant transformation than osteochondroma. Since BPOP still showed no cellular atypia and expressed only low levels of embryonic chondrocyte proteins (CEA, EMA, pan-K, and S-100), it was also presumed that the growth of BPOP was primitive and immature, similar to the embryonal development of cartilage and bone, i.e., Meckel's cartilage in mandible development, rather than to the progression of malignant transformation.
# Conclusions
The present study used IHC staining to compare a case of BPOP occurring at the lingual area of the right mandibular body with a case of representative osteochondroma occurring at the left mandibular condyle. Although the BPOP specimen showed no features of cellular atypia or malignant transformation, it did express more osteogenic proteins (BMP-2, BMP-4, RUNX2, OC, AP, OPG, RANKL CTGF, and bFGF) than osteochondroma. Furthermore, the perichondral spindle cells and marrow osteoblasts/fibroblasts of BPOP showed stronger immunoreaction of PCNA, p53, βcatenin, BCL2, pAKT, survivin, CEA, EMA, pan-K, and S-100 than the tumor cells of osteochondroma. Therefore, it was presumed that, similar to the embryonal osteochondroid tissues, BPOP might be activated by the osteogenic and oncogenic signalings and that this increased growth signaling may explain the rapid growth and high recurrence of BPOP.
[fig] Figure 1: Radiographic views of this study. A Computed tomography of BPOP, an irregularly calcified mass located on the lingual side of the mandibular body without cortical attachment (arrows). A1 Frontal plane. A2 Horizontal plane. B Panoramic view of osteochondroma, enlarged left condylar head (arrows) [/fig]
[fig] Figure 2: BPOP. A1 Sublingual ulceration with a whitish calcified mass. A2 Removed mass showing partial bluish color (arrows). A3 BPOP specimen was composed of cartilaginous (1) and osseous(2)tissue. A4 Bizarre chondrocytes (arrows) in cartilaginous tissue. B HE stain. B1-B3 (area 1 of A3). B4, B5 (area 2 of A3). B1 and B3 show core cartilage covered with thick perichondral fibrous tissue. B4 and B5 show anastomosing trabecular bone centered from cartilaginous tissue, mimicking endochondral ossification. C-G IHC stains with no background stain. C PCNA. D BMP-2. E BMP-4. F RUNX2. G OC [/fig]
[fig] Figure 3: BPOP. IHC stains with no background stain. A AP. B OPG. C RANKL. D CTGF. E bFGF. (1-3: cartilaginous tissue of Fig. 2A3. 4-5: osseous tissue of Fig. 2A3) [/fig]
[fig] Figure 4 A: -J BPOP. A HE stain, noted bizarre chondrocytes without cellular atypia. B-J IHC stains with no background stain. B p53. C β-catenin. D BCL2. E pAKT. F Survivin. G CEA. H EMA. I pan-K. J S-100. K-N Osteochondroma. K HE stain noted the tumorous growth of chondrocytes. L CEA. M EMA. N S-100. [/fig]
[fig] Figure 5: Osteochondroma. A, B HE stain noted hyperplastic chondrocytes undergoing abortive endochondral ossification. C-R IHC stains with no background stain. C PCNA. D BMP-2. E BMP-4. F RUNX2. G OC. H AP. I OPG. J RANKL. K CTGF. L bFGF. M p53. N pAKT. O β-catenin. P BCL2. Q 14-3-3. R Survivin. [/fig]
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Structural changes in brains of patients with disorders of consciousness treated with deep brain stimulation
Disorders of consciousness (DOC) are one of the major consequences after anoxic or traumatic brain injury. So far, several studies have described the regaining of consciousness in DOC patients using deep brain stimulation (DBS). However, these studies often lack detailed data on the structural and functional cerebral changes after such treatment. The aim of this study was to conduct a volumetric analysis of specific cortical and subcortical structures to determine the impact of DBS after functional recovery of DOC patients. Five DOC patients underwent unilateral DBS electrode implantation into the centromedian parafascicular complex of the thalamic intralaminar nuclei. Consciousness recovery was confirmed using the Rappaport Disability Rating and the Coma/Near Coma scale. Brain MRI volumetric measurements were done prior to the procedure, then approximately a year after, and finally 7 years after the implementation of the electrode. The volumetric analysis included changes in regional cortical volumes and thickness, as well as in subcortical structures. Limbic cortices (parahippocampal and cingulate gyrus) and paralimbic cortices (insula) regions showed a significant volume increase and presented a trend of regional cortical thickness increase 1 and 7 years after DBS. The volumes of related subcortical structures, namely the caudate, the hippocampus as well as the amygdala, were significantly increased 1 and 7 years after DBS, while the putamen and nucleus accumbens presented with volume increase. Volume increase after DBS could be a result of direct DBS effects, or a result of functional recovery. Our findings are in accordance with the results of very few human studies connecting DBS and brain volume increase. Which mechanisms are behind the observed brain changes and whether structural changes are caused by consciousness recovery or DBS in patients with DOC is still a matter of debate.Human consciousness is often described as a complex phenomenon, consisting of two components, wakefulness and awareness 1,2 . While wakefulness is associated with functional brainstem neurons i.e. the reticular system projecting to both thalamic and cortical neurons, awareness is mostly related to the functional integrity of the thalamus, cerebral cortex, and their connections 1-4 .Disorders of consciousness (DOC) occur as a result of interference with the mentioned systems. DOC can be acute and reversible, as a transient stage in a spontaneous recovery due to trauma, or chronic and irreversible, as in patients with unresponsive wakefulness syndrome (UWS) 1-4 . Prolonged DOC is not only long-lasting, serious, and currently untreatable, but also has severe consequences on a patient's quality of life 5-7 . These conditions impose a serious consideration from both medical and ethical perspectives 7-9 . Clinical conditions such as anoxic or traumatic brain lesions can cause diffuse neuronal damage resulting in serious disorders ranging from minimally conscious state (MCS), UWS, coma, locked-in syndrome, and even brain death 1,10 . The boundaries separating the mentioned conditions are unclear, with each one including severities 1,2 . UWS is currently described as wakefulness without consciousness, including a complete lack of reactions. Additionally,OPEN
it is characterized by variable cycles of sleeping and wakefulness, with preserved spontaneous respiration, digestion, and thermoregulation [bib_ref] Chronic disorders of consciousness, Bernat [/bib_ref] [bib_ref] Unresponsive wakefulness syndrome: A new name for the vegetative state or apallic..., Laureys [/bib_ref] [bib_ref] Meningitis of sinusoid origin with the form of coma vigil, Calvet [/bib_ref] [bib_ref] Persistent vegetative state after brain damage. A syndrome in search of a..., Jennett [/bib_ref]. MCS is on the other hand characterized by inconsistent, but visible evidence of consciousness [bib_ref] The minimally conscious state: Definition and diagnostic criteria, Giacino [/bib_ref] with the patient being awake but unaware. A patient in MCS can follow instructions occasionally, give simple gestural or non-verbal yes/no responses, and show a certain level of purposeful movement. The incidence of UWS is roughly 5 to 25 per million, while the prevalence in the adult population in the USA ranges from 40 to 168 per million [bib_ref] Incidence and prevalence of the vegetative and minimally conscious states, Beaumont [/bib_ref]. Additionally, various neurological impairments occur, including seizures, movement disorders, myoclonus, focal motor, and sensorimotor deficits, as well as emotional, behavioral, and cognitive disturbances [bib_ref] Cognitive sequelae of hypoxic-ischemic brain injury: A review, Anderson [/bib_ref] [bib_ref] Neurological sequelae of hypoxic-ischemic brain injury, Lu-Emerson [/bib_ref].
Despite advances in diagnosis, improved classification, use of modern technology, and well-known pharmacological treatments alongside non-invasive brain stimulation, the surgical therapeutic approaches are still not significantly advanced [bib_ref] Therapeutic interventions in patients with prolonged disorders of consciousness, Thibaut [/bib_ref] [bib_ref] Deep-brain stimulation in a persistent vegetative state: Follow-up results and criteria for..., Tsubokawa [/bib_ref]. Several studies used deep brain stimulation (DBS) of certain nuclei, such as centromedian parafascicle (CM-pf) complex and brain stem reticular formation, to regain consciousness in UWS and MCS patients [bib_ref] DBS therapy for the vegetative state and minimally conscious state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep-brain stimulation in a persistent vegetative state: Follow-up results and criteria for..., Tsubokawa [/bib_ref] [bib_ref] Deep cerebral stimulation in patients with post-traumatic vegetative state 25 cases, Cohadon [/bib_ref] [bib_ref] Deep brain stimulation and spinal cord stimulation for vegetative state and minimally..., Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. Research thus often focused on the thalamus as a functionally important thalamocortical link, where stimulation through key thalamocortical and thalamus-forebrain circuitry was shown to be important and caused widespread effect [bib_ref] The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence..., Van Der Werf [/bib_ref]. The use of the CM-pf complex as a target was based on literature overview and clinical recommendation, as well as due to the connectivity and prospective effect on systems involved in consciousness [bib_ref] DBS therapy for the vegetative state and minimally conscious state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep-brain stimulation in a persistent vegetative state: Follow-up results and criteria for..., Tsubokawa [/bib_ref] [bib_ref] Deep cerebral stimulation in patients with post-traumatic vegetative state 25 cases, Cohadon [/bib_ref] [bib_ref] Deep brain stimulation and spinal cord stimulation for vegetative state and minimally..., Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref] [bib_ref] Functional mapping of thalamic nuclei and their integration into cortico-striatalthalamo-cortical loops via..., Metzger [/bib_ref]. Previous studies were done on a small cohort, mainly as case reports, and did not yield a definite answer whether this highly specific and refined method can be therapeutically successful [bib_ref] DBS therapy for the vegetative state and minimally conscious state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep-brain stimulation in a persistent vegetative state: Follow-up results and criteria for..., Tsubokawa [/bib_ref] [bib_ref] Deep cerebral stimulation in patients with post-traumatic vegetative state 25 cases, Cohadon [/bib_ref] [bib_ref] Deep brain stimulation and spinal cord stimulation for vegetative state and minimally..., Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref] [bib_ref] Behavioural and EEG arousal induced by stimulation of unspecific projection systems in..., Hassler [/bib_ref] [bib_ref] Pathology in diffuse projection system preventing brainstem-electrode arousal from traumatic coma, Mclardy [/bib_ref] [bib_ref] Chronic electrical stimulation of the thalamic unspecific activating system in a patient..., Sturm [/bib_ref] [bib_ref] Behavioural improvements with thalamic stimulation after severe traumatic brain injury, Schiff [/bib_ref] [bib_ref] Results of a prospective study (CATS) on the effects of thalamic stimulation..., Magrassi [/bib_ref]. Even though some studies claimed that there was no definitive evidence of the efficacy of this method for treatment of DOC patients, the usefulness of DBS was shown using controlled studies, as well as our previous study [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref].
Virtually all of the studies mentioned are missing an explanation of the detailed structural and functional cerebral changes induced by DBS. Recently, renewed interest in the grading of DOC and different trials of therapeutic approaches have been noted. Previous studies observed short-term volume changes in the human brain structure following DBS, potentially indicating restorative possibilities in patients with neurodegenerative diseases [bib_ref] Deep brain stimulation influences brain structure in Alzheimer's disease, Sankar [/bib_ref]. Modern magnetic resonance imaging (MRI) techniques play an important role in diagnostic classification and evaluation of cerebral damage in order to improve the gradation of different types and levels of DOC 32 . Moreover, MRI has been shown to be a potential tool for morphometric measurements and changes in a restructured, "reorganized" brain in DOC patients [bib_ref] Thalamic volume as a biomarker for disorders of consciousness. I Proceedings Volume, Rubeaux [/bib_ref] [bib_ref] Regional brain volumetry and brain function in severely brain-injured patients, Annen [/bib_ref]. Modern neuroimaging thus uses different methods to objectively evaluate both structural and functional changes as an effect of different treatments.
Previously, it has been documented that DBS changes function in such structures as the striatum, hippocampus, amygdala, etc. It would thus arguably follow that if electrical stimulation changes the brain's structure and its function, stimulation of damaged circuitry could then induce both functional and structural changes with an eventual improvement of function [bib_ref] The location of the conscious center in the brain: The corpus striatum, Dandy [/bib_ref] [bib_ref] Mental phenomena evoked by electrical stimulation of the human hippocampal formation and..., Halgren [/bib_ref] [bib_ref] & Fried, I. Consciousness and neurosurgery, Crick [/bib_ref].
To determine the impact of CM-pf stimulation on a possible brain structural reorganization after functional recovery, we performed a quantitative volumetric MRI analysis in DOC patients who underwent CM-pf DBS.
Our study aimed to reveal if CM-pf DBS could induce an impact on brain structural reorganization in DOC patients and lead to a changed volume of specific cortical and subcortical structures.
# Methods
Patients. This retrospective study included five patients who underwent CM-pf DBS due to DOC after which they achieved functional recovery [fig_ref] Table 1: Demographic and clinical characteristics of consciousness improved patients after DBS [/fig_ref]. Out of the five patients, three were female, average age 16 ± 1.53. The cause of injury in one patient was anoxic due to cardiac arrest, while other two patients experienced traumatic injuries. Two patients were male with an average age of 20 ± 4.24 years. In both of these patients the cause of injury was anoxia due to cardiac arrest. The length between the injury and time to DBS was 2 months in males and 11 ± 5.13 months (range [bib_ref] DBS therapy for the vegetative state and minimally conscious state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for disorders of consciousness: Systematic review of cases and..., Vanhoecke [/bib_ref] [bib_ref] Deep brain stimulation, neuroethics, and the minimally conscious state: Moving beyond proof..., Schiff [/bib_ref] [bib_ref] The right to die in the minimally conscious state, Johnson [/bib_ref] [bib_ref] Unresponsive wakefulness syndrome: A new name for the vegetative state or apallic..., Laureys [/bib_ref] [bib_ref] Meningitis of sinusoid origin with the form of coma vigil, Calvet [/bib_ref] [bib_ref] Persistent vegetative state after brain damage. A syndrome in search of a..., Jennett [/bib_ref] [bib_ref] The minimally conscious state: Definition and diagnostic criteria, Giacino [/bib_ref] in females. The average follow-up duration in female patients was 88 ± 38.17 months (range 30-102), while in male patients it was 84 ± 38.18 months (range 57-111).
In the present study we included patients previously described in Chudy et al. [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. Forty-nine patients were enrolled in the previous study. Fourteen patients (10 UWS and 4 MCS) fulfilled neurophysiologic, clinical, and neuroimaging criteria and underwent DBS implantation. Four patients (3 MCS and 1 UWS) achieved functional recovery, while other patients were further followed by an attending palliative physician [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. In this study we included one additional UWS patient who underwent DBS implantation after fulfilling the aforementioned criteria and achieved functional recovery. Patients selected for DBS were evaluated utilizing the standard Rappaport Disability Rating (RDR) scale and the Coma/Near Coma (C/NC) scale [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref] [bib_ref] The disability rating and coma/near-coma scales in evaluating severe head injury, Rappaport [/bib_ref]. Inconsistently responsive to simple commands patients were classified as MCS patients (C/NC Level 1), while patients who were not able to respond to any command were classified as UWS (C/NC Level 2-4). DOC patients were selected based on three main factors: their neurophysiologic evaluation, 12/24 h electroencephalography (EEG) and neuroimaging (MRI). The neurophysiological criteria we investigated were recordable somatosensory evoked potential (SEP), motor evoked potentials, and brainstem auditory evoked potentials, even with pathological parameters such as prolonged latencies or central conduction time. The entry criterion for SEPs was recordability via stimulation of median nerves, with or without SEPs elicited by tibial nerve stimulation. The second main factor we looked at was EEG. The entry criterion for this method was the presence of periods of desynchronized EEG activity during 12/24 h of monitoring processed EEG [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. Obtained neuroimaging (MRI) showed absence of a structural defect. After passing extensive selection criteria, the patients underwent DBS electrode implantation into the CM-pf complex of the left thalamic intralaminar nuclei, while in patients with posttraumatic lesions, the electrode was placed in the better-preserved hemisphere [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. On the third postoperative day, monopolar stimulation was initiated using the contact eliciting the strongest arousal response with minimal current (25 Hz frequency, 90 µs pulse duration, voltage 2.5-3.5 V). Stimulation was applied for a 30-min period every two hours during the daytime. Stimulation duration was up to 10 months after DBS electrode implantation. The informed parental/caregivers consent was obtained for all patients in accordance with the Declaration of Helsinki and ethical approval was obtained from the Institutional Review Board of the Dubrava University Hospital, School of Medicine, University of Zagreb, Croatia. MRI acquisition. MRI scans were obtained on a 1.5 T MRI scanner (MAGNETOM Aera, Siemens Healthineers, Erlangen) using 24 channeled head coils. Standard clinical sequences were used, as well as high-resolution 3D T1-weighted magnetization-prepared rapid acquisition gradient echo (MPRAGE) sequence with the following scanning parameters: TR = 2300 ms, TE = 3 ms, flip angle = 15°, matrix size = 256 × 256, field of view = 256 mm, voxel size = 1.0 × 1.0 × 1.0 mm. Obtained MRI scans were closely examined showing the absence of any large brain lesion [fig_ref] Figure 1: Initial structural MRI scans of included patients showing an absence of any... [/fig_ref]. MRI was obtained three times; the first one prior to DBS implantation, the second one between 8 and 12 months after surgery (approximately 1 year), and the third time between 1 and 7 years after DBS implementation.
Quantitative volumetric methods. Volumetric analysis was conducted using an automated CIVET processing pipeline (version 2.1.0., http://www.bic.mni.mcgil l.ca/Servi cesSo ftwar e/CIVET ) of different interdependent algorithms, on a web-based portal, CBRAIN (https ://porta l.cbrai n.mcgil l.ca), providing tools for observer-independent corticometric analysis [bib_ref] The CIVET image-processing environment: A fully automated comprehensive pipeline for anatomical neuroimaging..., Ad-Dab'bagh [/bib_ref] [bib_ref] CBRAIN: A web-based, distributed computing platform for collaborative neuroimaging research, Sherif [/bib_ref] [bib_ref] Region-specific reduction in brain volume in young adults with perinatal hypoxic-ischaemic encephalopathy, Bregant [/bib_ref]. During analysis, the pipeline included the following steps: correction of radiofrequency intensity and nonuniformity artifacts corrected using the N3 algorithm [bib_ref] A nonparametric method for automatic correction of intensity nonuniformity in MRI data, Sled [/bib_ref] , linear registration using the 9-parameter affine process of translation, scaling, and rotation, and the 3D T1 volume registration to the ICBM 152 stereotactic space [bib_ref] Automatic 3D intersubject registration of MR volumetric data in standardized talairach space, Collin [/bib_ref] [bib_ref] A probabilistic atlas and reference system for the human brain: International Consortium..., Mazziotta [/bib_ref]. The brain was masked, including scull stripping and tissue classification as grey matter, cerebrospinal fluid, and white matter, using the discrete classifier with advanced neural network methods [bib_ref] Fast robust automated brain extraction, Smith [/bib_ref] [bib_ref] Automatic quantification of MS lesions in 3D MRI brain data sets: Validation..., Zijdenbos [/bib_ref]. Cortical, grey matter and white matter surfaces extraction using the Laplacian map, partial volume classification [bib_ref] Automated 3-D extraction and evaluation of the inner and outer cortical surfaces..., Kim [/bib_ref] [bib_ref] Automated 3-D extraction of inner and outer surfaces of cerebral cortex from..., Macdonald [/bib_ref] and thickness calculation 49 were final steps. The surfaces were smoothed and registered in order to calculate the regional cortical volume and the average regional thickness of the cerebral cortex of automatically parcellated lobes [bib_ref] An unbiased iterative group registration template for cortical surface analysis, Lyttelton [/bib_ref] [bib_ref] Depth potential function for folding pattern representation, registration and analysis, Boucher [/bib_ref]. Quantification of regional cortical thickness averages where lobe borders were determined by sulcal landmarks and detected by the CIVET pipeline and regional cortical volume estimates were made for parietal, occipital, frontal, temporal lobes, parahippocampal gyrus, cingulate gyrus, isthmus of the cingulate gyrus, and insula [fig_ref] Figure 2: Volumetric analysis was conducted using the CIVET pipeline measuring regional cortical volume... [/fig_ref]. Subcortical structure segmentation (putamen, caudate, thalamus, globus pallidus, hippocampus, amygdala, nucleus accumbens) was performed using an automated MRI brain volumetry system, volBrain [bib_ref] volBrain: An online MRI brain volumetry system, Manjón [/bib_ref] [fig_ref] Figure 2: Volumetric analysis was conducted using the CIVET pipeline measuring regional cortical volume... [/fig_ref]. Volumetric analysis was performed at three measuring points, according to MRI scans: prior to DBS, approximately 1 year after, and 7 years after DBS implementation.
Statistical analysis. Data analysis was performed using the MedCalc Statistical Software version 12.5.0 (MedCalc Software, Ostend, Belgium; https ://www.medca lc.org). Data were plotted as individual values, horizontal lines and markers represent mean ± SD. Comparison of the regional cortical volumes and thicknesses of parcellated lobes and volumes of subcortical structures were obtained. Depending on the distribution and www.nature.com/scientificreports/ type of variables, volumetric data in all measuring points were analyzed using one-way ANOVA, with the Student-Newman-Keuls post hoc test, or Kruskal-Wallis test with pairwise comparisons according to Conover. In order to reduce intersubject variability we have performed the regional volumes normalization by head size. Normalization was done using the subject with lowest head size value as a calibrator value to calculate the coefficient (subject value/calibrated value). Regional volumes were multiplied with the calculated coefficient for each subject and further analyzed using previously mentioned statistical test. We did not correct for multiple comparisons in the present small patient sample. The statistical significance was set at p < 0.05.
Ethics approval and consent to participate. This study was carried out in accordance with the recommendations of the Ethical Board of the Dubrava University Hospital and University of Zagreb, School of Medicine with written informed parents/caregivers consent from all subjects in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the University Hospital Dubrava and University of Zagreb, School of Medicine. [fig_ref] Figure 4: Subcortical structures volumetric analysis in three measuring points [/fig_ref]. While right parahippocampal gyrus regional cortical thickness increase significantly (↑16.9%) (F(2,9) = 1.937, p = 0.02), regional cortical thickness increase was observed in both right cingulate gyrus and insula bilaterally, as well as in the left parahippocampal gyrus, although these values were not statistically significant. Regional All five patients included in the study raised to full awareness and regained the ability to communicate and interact. Two male patients (Patient 1 and 2) were graded as MCS using RDR and C/NC scale [fig_ref] Table 1: Demographic and clinical characteristics of consciousness improved patients after DBS [/fig_ref]. Both of them are currently able to live independently to a large extent [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref] , although both of them are experiencing problems with concentration and memory . Out of the three female patients, one was graded as MCS (Patient 3), while two of them were graded as UWS (Patient 4 and 5) using RDR and C/NC scale [fig_ref] Table 1: Demographic and clinical characteristics of consciousness improved patients after DBS [/fig_ref]. All female patients regained consciousness, although they are currently still unable to live independently [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. The main obstacle for further progress is related to motor difficulties, such as spasticity, contractures, inability to move their limbs, to articulate words, etc. For several years, their communication was in a form of non-verbal signs, predominantly as facial gestures and/or limb movement. Today, these patients are able to form simple words .
# Results
# Analysis
# Discussion
Previous studies reported that DBS in DOC is still in its exploratory phase [bib_ref] DBS therapy for the vegetative state and minimally conscious state, Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the treatment of vegetative state, Yamamoto [/bib_ref] [bib_ref] Deep-brain stimulation in a persistent vegetative state: Follow-up results and criteria for..., Tsubokawa [/bib_ref] [bib_ref] Deep cerebral stimulation in patients with post-traumatic vegetative state 25 cases, Cohadon [/bib_ref] [bib_ref] Deep brain stimulation and spinal cord stimulation for vegetative state and minimally..., Yamamoto [/bib_ref] [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref] [bib_ref] Behavioural and EEG arousal induced by stimulation of unspecific projection systems in..., Hassler [/bib_ref] [bib_ref] Pathology in diffuse projection system preventing brainstem-electrode arousal from traumatic coma, Mclardy [/bib_ref] [bib_ref] Chronic electrical stimulation of the thalamic unspecific activating system in a patient..., Sturm [/bib_ref] [bib_ref] Behavioural improvements with thalamic stimulation after severe traumatic brain injury, Schiff [/bib_ref] [bib_ref] Results of a prospective study (CATS) on the effects of thalamic stimulation..., Magrassi [/bib_ref]. Keeping in mind the ethical criteria and seriousness of DOC, our previous results showed limited but encouraging success [bib_ref] Deep brain stimulation for the early treatment of the minimally conscious state..., Chudy [/bib_ref] [bib_ref] Deep brain stimulation for treatment patients in vegetative state and minimally conscious..., Chudy [/bib_ref]. Although brain atrophy was initially observed on MRI prior to DBS, it was difficult and challenging to explain the volume increase of several brain structures and areas after DBS, which was in accordance with previous studies [bib_ref] Deep brain stimulation influences brain structure in Alzheimer's disease, Sankar [/bib_ref]. Two main aspects are going to be discussed in the following paragraphs. Firstly, a brief overview of consciousness, the role of the thalamus in conscious networks, as well as the general functional effects of stimulation. Secondly, we will discuss the structural changes that occurred after functional improvement, as a result of processes of repair, plasticity, and reorganization.
General effects of deep brain stimulation. All major factors and phenomena of consciousness are not fully understood at the moment. Despite numerous different study explanations and well-elaborated hypotheses, the neurobiological mechanism of consciousness is still largely unexplored [bib_ref] & Fried, I. Consciousness and neurosurgery, Crick [/bib_ref] [bib_ref] A framework for consciousness, Crick [/bib_ref] [bib_ref] Reorganization of the connectivity between elementary functions-a model relating conscious states to..., Mogensen [/bib_ref] [bib_ref] Visual perception from the perspective of a representational, non-reductionistic, level-dependent account of..., Overgaard [/bib_ref] [bib_ref] The status and future of consciousness research, Overgaard [/bib_ref]. What is known however is that various components of the central nervous systems participate simultaneously in this intriguing phenomenon [bib_ref] Reorganization of the connectivity between elementary functions-a model relating conscious states to..., Mogensen [/bib_ref] [bib_ref] Visual perception from the perspective of a representational, non-reductionistic, level-dependent account of..., Overgaard [/bib_ref] [bib_ref] The status and future of consciousness research, Overgaard [/bib_ref] [bib_ref] Neurobiology of consciousness: An overview, Delacour [/bib_ref] [bib_ref] Consciousness, accessibility, and the mesh between psychology and neuroscience, Block [/bib_ref] [bib_ref] Are there levels of consciousness?, Bayne [/bib_ref]. Alongside the reticular ascending systems projecting from the upper reticular formation of the brainstem, other structures participate significantly in forming consciousness. These structures include monoaminergic pathways, the locus coeruleus, serotonergic and dopaminergic systems, the forebrain cholinergic system, as well as the nucleus basalis [bib_ref] Brain stem reticular formation and activation of the EEG, Moruzzi [/bib_ref] [bib_ref] The isodendritic core of the brain stem, Ramón-Moliner [/bib_ref] [bib_ref] Neurobiological basis of consciousness, Young [/bib_ref] [bib_ref] Neuroanatomical correlates of brainstem coma, Parvizi [/bib_ref] [bib_ref] Functional neuroanatomy of the noradrenergic locus coeruleus: Its roles in the regulation..., Samuels [/bib_ref]. In addition, a special system of neurons connecting the brainstem to the interstitial neurons may also participate 67 and form important networks for maintaining and regulating cortical functions [bib_ref] Evaluation of interstitial nerve cells in the central nervous system. A correlative..., Das [/bib_ref] [bib_ref] Cytology and time of origin of interstitial neurons in the white matter..., Kostovic [/bib_ref]. These neurons project towards a number of subcortical structures, in which projections from thalamus nuclei are also received [bib_ref] Brain stem reticular formation and activation of the EEG, Moruzzi [/bib_ref] [bib_ref] The isodendritic core of the brain stem, Ramón-Moliner [/bib_ref] [bib_ref] Neurobiological basis of consciousness, Young [/bib_ref] [bib_ref] Neuroanatomical correlates of brainstem coma, Parvizi [/bib_ref] [bib_ref] Functional neuroanatomy of the noradrenergic locus coeruleus: Its roles in the regulation..., Samuels [/bib_ref]. We propose that the main functional system responsible for the observed structural changes is widespread thalamocortical and thalamosubcortical (feedback) circuitry, modulated by several systems: modulatory monoaminergic systems, the reticular ascending system from the upper brainstem, basal forebrain cholinergic systems, etc.
The thalamus is a crucial relay of the cortico-striatal-thalamo-cortical circuits and its role in arousal, attention to salient stimuli, and processing of information has been documented by both animal models as well as functional connectivity studies in humans [bib_ref] The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence..., Van Der Werf [/bib_ref] [bib_ref] Functional mapping of thalamic nuclei and their integration into cortico-striatalthalamo-cortical loops via..., Metzger [/bib_ref] [bib_ref] Thalamic contributions to attention and consciousness, Newman [/bib_ref] [bib_ref] Activation by attention of the human reticular formation and thalamic intralaminar nuclei, Kinomura [/bib_ref] [bib_ref] Participation of the thalamic CM-Pf complex in attentional orienting, Minamimoto [/bib_ref] [bib_ref] Central thalamic contributions to arousal regulation and neurological disorders of consciousness, Schiff [/bib_ref] [bib_ref] The cortico-basal ganglia integrative network: The role of the thalamus, Haber [/bib_ref]. The CM-pf complex as the main part of the intralaminar nuclei [bib_ref] The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence..., Van Der Werf [/bib_ref] provides particularly strong connections to several subcortical structures [bib_ref] The cortico-basal ganglia integrative network: The role of the thalamus, Haber [/bib_ref]. It also provides strong connections to the basal ganglia [bib_ref] Retrograde axonal transport and the demonstration of non-specific projections to the cerebral..., Jones [/bib_ref] , primarily the caudate nucleus [bib_ref] The thalamo-caudate versus thalamo-cortical projections as studied in the cat with fluorescent..., Macchi [/bib_ref] , putamen [bib_ref] The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence..., Van Der Werf [/bib_ref] , nucleus accumbens 77 , and pallidum, all of which was demonstrated using both tracing and tractographic studies [bib_ref] Preferential networks of the mediodorsal nucleus and centromedian-parafascicular complex of the thalamus-a..., Eckert [/bib_ref]. A relatively weaker subcortical connectivity was noticed between CM-pf and the amygdala [bib_ref] Thalamic midline cell populations projecting to the nucleus accumbens, amygdala, and hippocampus..., Su [/bib_ref] , and the hippocampus [bib_ref] Electrophysiological studies of hippocampal connections and excitability, Green [/bib_ref] [bib_ref] The pathways connecting the hippocampal formation, the thalamic reuniens nucleus and the..., Cavdar [/bib_ref]. On the other hand, excitatory projections from the CM-pf complex toward cortical areas have been reported as much weaker and non-specific [bib_ref] The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence..., Van Der Werf [/bib_ref]. The cortical projections seem to be limited to the agranular cortex with the only exception being the anterior . Neurocognitive and behavioral assessment of consciousness improved patients after DBS. CA cardiac arrest, TBI traumatic brain injury, UWS unresponsive wakefulness syndrome, MCS minimally conscious state. *Under intensive speech therapy at the moment, able to pronounce simple words. [bib_ref] Preferential networks of the mediodorsal nucleus and centromedian-parafascicular complex of the thalamus-a..., Eckert [/bib_ref]. Additionally, the CM-pf complex probably interacts with various cortical areas via the corticostriatal pathways, through strong retrograde projections [bib_ref] Mental phenomena evoked by electrical stimulation of the human hippocampal formation and..., Halgren [/bib_ref].
The selection of the thalamus as a stimulation target thus seems justified because of its potent effect in the thalamocortical circuitry. Our preliminary results showed the thalamic volume decrease less than 1% at the second measuring point, while at the third measuring point thalamic volume decrease of almost 25% was observed [fig_ref] Figure 4: Subcortical structures volumetric analysis in three measuring points [/fig_ref]. Since the thalamic volume was slightly decreased at the period when functional recovery was observed in patients, we presume that the constant volume contributed to functional connectivity of the thalamus and previously mentioned structures. Results of positive stimulation effects point to the thalamus and reorganization of functional circuitry as important factors in recovery after DOC. The DBS electrode, positioned deep inside the brain, provides permanent stimulation. This in turn provides new possibilities for integrating function and structure for consciousness recovery. However, despite the evidence that thalamic intralaminar nuclei have an influence on cerebral function and connectivity, it is unlikely that it can explain such major changes in the brain structure volume. In our patients, a larger volume decrease was observed several years after the functional recovery, thus we can only assume it occurred as a further adaptation of the thalamus to the brain reorganization due to the initial brain injury, either trauma or anoxic lesion. Additionally, the thalamus volume could decrease in response to the lack of adequate stimulus. Once the DBS is removed, the stimulus that maintained thalamic volume by providing sufficient stimulation may cease. The variability of structural changes observed after DBS may be partly explained by the great variety of cortical, subcortical, and brainstem functional systems involved. This variability suggests that there is no single functional system affected. Therefore, besides the thalamus and indirectly affected systems, other factors play a possible role and will be discussed below.
Structural effects observed on MRI. The exact underlying mechanisms contributing to volume changes are still unknown. There are several possible explanations about how long functional stimulation could have led to a volume increase. These include synaptogenesis, gliogenesis, axonal remodeling, micro vascularization, neuronal size increase, and extracellular matrix changes. A combination of all these factors is also possible. Of note is that adult neurogenesis has also been described in certain brain regions (i.e. the hippocampus) [bib_ref] Deep brain stimulation influences brain structure in Alzheimer's disease, Sankar [/bib_ref] [bib_ref] Modifiable factors that alter the size of the hippocampus with ageing, Fotuhi [/bib_ref]. It is well known that after various lesions, the brain volume could be changed by different underlying processes of brain tissue repair (apoptosis, microstructural glial reactivity, changes in the extracellular matrix, etc.), plasticity (sprouting, myelination, dendritic plasticity), and reorganization [bib_ref] Reorganization of the injured brain: Implications for studies of the neural substrate..., Mogensen [/bib_ref] [bib_ref] Reorganization and plastic changes of the human brain associated with skill learning..., Chang [/bib_ref] [bib_ref] Experience-dependent neural plasticity in the adult damaged brain, Kerr [/bib_ref] [bib_ref] Neural plasticity and its contribution to functional recovery, Sharma [/bib_ref]. Therefore, we could expect similar events occurring and leading to the increased brain volume in our patients. Additionally, processes of repair and plasticity probably overlap. The reorganization of pathways is thus expected to be primarily functional (since various pathways transduce electrical information when the brain is stimulated), rather than structural, which is possible primarily during development [bib_ref] Experience-dependent neural plasticity in the adult damaged brain, Kerr [/bib_ref] [bib_ref] Neural plasticity and its contribution to functional recovery, Sharma [/bib_ref]. Nevertheless, considering how the most voluminous components of the brain tissue are cell bodies of neurons, glia, dendrites, and myelinated axons [bib_ref] Modular construction of nervous systems: A basic principle of design for invertebrates..., Leise [/bib_ref] [bib_ref] All brains are made of this: A fundamental building block of brain..., Mota [/bib_ref] , it is unlikely that these cells experience a significant increase in their size receiving more excitatory input. Moreover, an increase of dendritic branching postsynaptic spines of super excited neurons is possible. The data on glia are less consistent, but it is generally expected that astroglia follows a metabolic increase of its function and become hypertrophic when stimulated [bib_ref] Neuronal-astrocyte metabolic interactions: Understanding the transition into abnormal astrocytoma metabolism, Turner [/bib_ref]. Volume increase could therefore occur during an increase in synapsis number and size which are more activate during prolonged stimulation of important excitatory glutamatergic neurons 92 . However, synapsis makes only a small proportion of the brain volume.
Several examples of increased brain volume during development in abnormal conditions have been previously reported. In such cases, hyper-connectivity within the cortex was explained by the changes in the extracellular substance, which makes approximately 70% of the developing brain, while being scarce in the normal adult brain [bib_ref] Cortical sulcal maps in autism, Levitt [/bib_ref] [bib_ref] Neuronal density and architecture (gray level index) in the brains of autistic..., Casanova [/bib_ref] [bib_ref] Reduced gyral window and corpus callosum size in autism: Possible macroscopic correlates..., Casanova [/bib_ref].
The aforementioned structural changes (repair, plasticity, and reorganization) are impossible to follow in patients, even if postmortem material is available. Therefore, MRI is so far the only available method providing general information that something is structurally different. Additionally, it is especially useful to combine different MRI techniques such as tractography with easily performed volumetric measurements, a valuable indicator that structural processes are taking place in the brain after stimulation. Only a few volumetric MRI analyses in patients with DOC were performed so-far [bib_ref] Thalamic volume as a biomarker for disorders of consciousness. I Proceedings Volume, Rubeaux [/bib_ref] [bib_ref] Regional brain volumetry and brain function in severely brain-injured patients, Annen [/bib_ref]. Grey matter volume of the parahippocampal gyrus, thalamus, and caudate were previously presented to be the key features differing healthy subjects and patients with DOC. Additionally, white matter volumes of the parahippocampal gyrus, isthmus of cingulate gyrus, and brainstem were previously described as the most affected white matter regions [bib_ref] Regional brain volumetry and brain function in severely brain-injured patients, Annen [/bib_ref]. Analysis of the subcortical structures was previously performed only for the thalamus; slower atrophy of the thalamus was observed in MCS than in UWS patients [bib_ref] Thalamic volume as a biomarker for disorders of consciousness. I Proceedings Volume, Rubeaux [/bib_ref]. These results suggest that patients with better-preserved levels of consciousness are more likely to preserve an increased brain volume over time. Our results indicate that the most informative region is the parahippocampal area. This area is known to be important for the formation of episodic memories as well as permitting rapid processing which in turn enables contextualization of outside events. It is therefore well located to bind associations from other cortical streams, suggesting an important function in consciousness. Our findings are in the accordance with the results of previous preclinical studies of DBS in rodents which observed structural neuroplasticity (hippocampal neurogenesis [bib_ref] Memory rescue and enhanced neurogenesis following electrical stimulation of the anterior thalamus..., Hamani [/bib_ref] [bib_ref] Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory, Stone [/bib_ref] , as well as an increased complexity of apical dendrites and the length of basal dendritic trees of pyramidal neurons of the hippocampus [bib_ref] High frequency stimulation of the infralimbic cortex induces morphological changes in rat..., Bezchlibnyk [/bib_ref]. Furthermore, our findings are in accordance with results showing short-term brain volume increase, namely the hippocampal area, in Alzheimer's dementia patients following DBS [bib_ref] Deep brain stimulation influences brain structure in Alzheimer's disease, Sankar [/bib_ref].
Several limitations of the presented study should be mentioned, such as a small number of patients, their mixed gender, different age and etiology of injury as well as different times for MRI scans from the initial injury and volumetric analysis in included patients. Still, we believe the presented results are valuable and can be a valid starting point for future research.
# Conclusions
Even on a small sample size included in the present study, we tried to emphasize the importance of morphometric MRI quantification which can expand our understanding of the relationship between brain structures and consciousness recovery. Our results are in accordance with the results of very few human studies connecting DBS and brain volume changes. They also support the idea that DBS in the CM-pf complex may have a widespread effect on cerebral function and structure. Structures with altered volumes belong to cortico-thalamo-cortico-basal ganglia circuitry, limbic circuitry and at the same time have strong modulatory monoaminergic brainstem input. The nature of the initial lesion of neural networks, neurons, and glia, is difficult to determine with the current criteria and methodology applied. Which mechanisms are behind the observed brain changes and whether the observed structural changes are caused by consciousness recovery and/or DBS in DOC patients is yet to be elucidated. In future studies, different MRI volumetric analysis of cortical areas or deep brain structures could help to identify cell types and more details contributing to the regional volume change.
## Data availability
The datasets generated for this study are available on request to the corresponding author.
[fig] Figure 1: Initial structural MRI scans of included patients showing an absence of any large brain lesion (patients 1-5, from left to right). Scientific Reports | (2021) 11:4401 | https://doi.org/10.1038/s41598-021-83873-y [/fig]
[fig] Figure 2: Volumetric analysis was conducted using the CIVET pipeline measuring regional cortical volume and thickness for parietal, occipital, frontal, temporal lobes, isthmus of the cingulate gyrus, parahippocampal, and cingulate gyrus and insula (a), and volBrain software, for subcortical structures segmentation (b). Singlesubject volumetric analysis is presented.Scientific Reports | (2021) 11:4401 | https://doi.org/10.1038/s41598-021-83873-y www.nature.com/scientificreports/ cortical thickness is observed to decrease over time in the parietal, frontal, temporal, and occipital lobe, as well as the isthmus of the cingulate gyrus. Statistical analysis of normalized segregated volumes revealed significantly higher volume of the left insula (F(2,9) = 6.804, p = 0.01). Volumes of the right insula (F(2,9) = 1.407, p = 0.29), the left parahippocampal gyrus (F(2,9) = 1.142, p = 0.36), the right parahippocampal gyrus (F(2,9) = 1.694, p = 0.23), the left cingulate gyrus [/fig]
[fig] Figure 3: Regional cortical volumetric analysis in three measuring points (prior to DBS, 1 year after DBS, and 7 years after DBS). The regional cortical volumetric analysis revealed significant volume increase in right parahippocampal gyrus, left cingulate gyrus, and left insula, while the trend of volume increase was presented in left parahippocampal gyrus volume, right cingulate gyrus volume, and right insula volume. Vertical bars, standard deviation. [/fig]
[fig] Figure 4: Subcortical structures volumetric analysis in three measuring points (prior to DBS, 1 year after DBS, and 7 years after DBS). Significant volume increase of caudate, hippocampus, and amygdala were presented, while putamen and accumbens volumes presented a trend of volume increase during three measuring points. Both globus pallidus and thalamus volume decreased over time. Vertical bars, standard deviation. , p = 0.09) and the right cingulate gyrus (F(2,9) = 2.423, p = 0.14), as well as volumetric analysis of frontal, parietal, occipital, and temporal lobe, and isthmus of the cingulate gyrus, did not significantly differ among groups. Subcortical normalized volumes of the amygdala (F(2,9) = 4.451, p = 0.04) were significantly higher. Normalized volumes of the caudate (F(2,9) = 2.952, p = 0.22), hippocampus (F(2,9) = 3.390, p = 0.08), putamen (F(2,9) = 1.015, p = 0.40), accumbens (F(2,9) = 2.436, p = 0.14), globus pallidus (F(2,9) = 0.032, p = 0.96) and thalamus (F(2,9) = 0.747, p = 0.50) did not significantly differ. [/fig]
[table] Table 1: Demographic and clinical characteristics of consciousness improved patients after DBS. CA cardiac arrest, TBI traumatic brain injury, RDR rappaport disability rating scale, C/NC coma/near coma scale, UWS unresponsive wakefulness syndrome, MCS minimally conscious state. [/table]
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Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops
Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.
# Introduction
The introduction of insect-resistant GE crops began in the 1990s, with a number of today's crops incorporating Bt Cry proteins [bib_ref] The food and environmental safety of Bt crops, Koch [/bib_ref] [bib_ref] The role and use of genetically engineered insect-resistant crops in integrated pest..., Naranjo [/bib_ref]. According to the International Service for the Acquisition of Agribiotech Applications (ISAAA) GM Approval database 1 , 85 transformation events involving Bt Cry protein expressed in 10 crops received regulatory approval somewhere in the world by the end of 2019. These have been further incorporated into 206 stacks (that combine two or more GE traits) that have received additional regulatory approvals 1 . These approvals have each been accompanied by an ERA that has typically focused on identifying the target range and specificity of the Bt Cry proteins using a tiered approach to non-target testing that is very similar to the approach used in the assessment of chemical pesticides [bib_ref] A tiered system for assessing the risk of genetically modified plants to..., Garcia-Alonso [/bib_ref] [bib_ref] Assessment of risk of insect-resistant transgenic crops to nontarget arthropods, Romeis [/bib_ref] ; . Tier-1 testing involves the use of surrogate species tested under worst-case exposure conditions in the laboratory to identify potential hazards to them. Specific endpoints, typically including mortality, are measured in tier-1 tests under conditions of exposure to concentrations, usually several fold higher than concentrations expected in the field [bib_ref] Assessment of risk of insect-resistant transgenic crops to nontarget arthropods, Romeis [/bib_ref]. In the absence of relevant negative effects in test species at high exposures, a conclusion that the likelihood of adverse ecological effects under realistic conditions is low or negligible can be supported [bib_ref] Deriving criteria to select arthropod species for laboratory tests to assess the..., Romeis [/bib_ref]. If negative effects are observed under worst-case conditions, then higher tier studies are conducted to establish if the effect is relevant under more realistic conditions (i.e., lower dose; [bib_ref] Guidance on the environmental risk assessment of genetically modified plants, Efsa [/bib_ref] ; .
After more than 20 years of use in the field, there is a substantial history associated with GE plants incorporating Bt Cry proteins and their safety in the environment [bib_ref] Are Bt crops safe?, Mendelsohn [/bib_ref] [bib_ref] Impacts of Bt crops on non-target invertebrates and insecticide use patterns, Naranjo [/bib_ref] [bib_ref] Extrapolating non-target risk of Bt crops from laboratory to field, Duan [/bib_ref] [bib_ref] The cultivation of Bt corn producing Cry1Ac toxins does not adversely affect..., Guo [/bib_ref] [bib_ref] Adaptation of the ToxRTool to assess the reliability of toxicology studies conducted..., Koch [/bib_ref] [bib_ref] Genetically engineered crops help support conservation biological control, Romeis [/bib_ref]. The utility of a tiered approach using mortality as the primary endpoint is supported by experience with Bt Cry proteins and by an understanding of the mode of action, target specificity and exposure levels for these proteins. However, new pest control technologies, including non-Bt Cry proteins and the use of RNAi, have led to an increased interest in sublethal endpoints testing. This interest is due to several factors, including broader interest in sublethal impacts of chemicals, differences in the mode of action, and the length of time required to observe an effect, and concerns about cumulative or additive effects of multiple stressors in the environment.
While the use of sublethal endpoints is potentially informative for the ERA of non-Bt GE plants, there are a number of challenges to implementing this approach. These include the wide variety of potential endpoints from which to choose and difficulties interpreting the relationship between sublethal endpoints in laboratory studies and observable effects in the field. This paper addresses some of these challenges by examining 1 http://www.isaaa.org/gmapprovaldatabase/ sublethal endpoints in the context of insect-resistant GE plants and considering when inclusion of sublethal endpoints may or may not be warranted in the context of problem formulation for a case-specific ERA. This review has been informed by discussions at a workshop convened by the ILSI Research Foundation (Washington, DC, USA, 4-5 March 2019) that asked participants to consider the relevance of sublethal endpoint testing using a case study approach.
## Non-target organism assessment
## Problem formulation
The process of identifying and refining the information that will be informative for case-specific ERA is referred to as problem formulation . The mechanics of problem formulation can be described in a number of ways [bib_ref] Conceptualizing risk assessment methodology for genetically modified organisms, Hill [/bib_ref] [bib_ref] Problem formulation in the environmental risk assessment for genetically modified plants, Wolt [/bib_ref] [bib_ref] Problem formulation in environmental risk assessment for genetically modified crops: A practitioner's..., Gray [/bib_ref] , but the process involves a series of steps that incorporate context for the decision being made, information about the receiving environment and the societal values or protection goals that are identified in relevant laws and regulations. Because these protection goals are often broad, case specific ERA requires the refinement of operational protection goals and the subsequent identification of assessment endpoints, which allow the testing of relevant hypotheses to inform the assessment [bib_ref] Evaluating environmental risks of genetically modified crops: Ecological harm criteria for regulatory..., Sanvido [/bib_ref] [bib_ref] Optimising environmental risk assessments, Devos [/bib_ref]. Data are then collected under laboratory, semi-field or field conditions for measurement endpoints that are related to those assessment endpoints (Garcia-Alonso and [bib_ref] Protection goals in environmental risk assessment: A practical approach, Garcia-Alonso [/bib_ref]. The advantage of problem formulation is that it provides an explicit rationale for how and why a particular measurement endpoint will be informative to an ERA .
For most risk assessments on non-target invertebrates, operational protection goals are reliant on maintaining populations of value or beneficial arthropods that contribute to important ecosystem services . Among the most relevant have been populations of pollinators, parasitoids, and predators as well as charismatic, protected, threatened, or endangered species, for which an exposure assessment indicates they will have a meaningful exposure to the GE plant. Once the particular species of interest are identified, appropriate surrogate, indicator, or focal species are selected for testing [bib_ref] Guidance on the environmental risk assessment of genetically modified plants, Efsa [/bib_ref].
## Nto study design
As with any research or regulatory study, the design of an non-target organism (NTO) study must be appropriate for the intended end use of the data. Well-designed early-tier studies are intended to identify any hazards that require further study. Careful consideration, therefore, must be given to ensuring that tests are reliable and especially that false negative results (i.e., failing to identify a hazard) are avoided since they would lead to the release of hazardous material. False positive results, on the other hand, also should be avoided since they might have consequences, even beyond the triggering of additional studies [bib_ref] When bad science makes good headlines: Bt maize and regulatory bans, Romeis [/bib_ref]. The first step is to identify appropriate surrogate species [bib_ref] Surrogate species selection for assessing potential adverse environmental impacts of genetically engineered..., Carstens [/bib_ref]. These should be chosen based on the representativeness of species FIGURE 1 | This figure details the tiered structure of testing used during the ERA process. This is hypothesis-based testing, where the initial tests are conducted at doses higher than what is encountered in a natural setting to simulate a worst-case scenario exposure. Example risk hypotheses are provided for NTOs to demonstrate how this structure can be used to test sub-lethal effects. This figure was adapted from [bib_ref] Assessment of risk of insect-resistant transgenic crops to nontarget arthropods, Romeis [/bib_ref].
of importance in the expected receiving environment, and ideally represent a functional group of interest. As a practical consideration, it is useful to select a surrogate from readily available laboratory reared strains. This provides uniformity to the test and control groups as well as reproducibility of results obtained from multiple laboratories. A surrogate species may additionally be selected based on its sensitivity to the test substance, taking into account what is known about the spectrum of activity of the insecticidal protein. For example, when the surrogate species and the target pests are more closely related phylogenetically, it is more probable that the surrogate species will be sensitive to the test substance [bib_ref] When bad science makes good headlines: Bt maize and regulatory bans, Romeis [/bib_ref]. NTO tests should be designed to expose the surrogate to the test substance in a way that maximizes the likelihood of detecting an effect and represents a relevant exposure pathway (e.g., dietary exposure). While juveniles are usually expected to be more susceptible in some species, other species may instead or also have susceptible adults, so it is advantageous to test multiple life stages provided there is a reasonable exposure pathway for those life stages [bib_ref] Recommendations for the design of laboratory studies on non-target arthropods for risk..., Romeis [/bib_ref] [bib_ref] Deriving criteria to select arthropod species for laboratory tests to assess the..., Romeis [/bib_ref].
When designing the study protocol, the most expedient approach is to use an already available artificial diet into which both test substances and positive controls can be easily incorporated. This allows the concentrations of the test substance to exceed the expected environmental exposure in order to test levels that exceed worst-case exposures. If an artificial diet is not available, or not suitable for incorporation of the test substance, then GE plant material can be used. However, this may limit the test dose possibilities and reduces the margin of exposure. Furthermore, it might be a challenge to identify the appropriate non-transformed control material.
The use of appropriate controls is a critical component for ensuring that test results are reliable and meaningful [bib_ref] Recommendations for the design of laboratory studies on non-target arthropods for risk..., Romeis [/bib_ref]. When plant material is used as a test substance, near-isogenic lines are preferred as a control in order to eliminate confounding variables in the composition of the materials, unrelated to the GE trait. For NTO studies, negative controls should be designed to mimic the test conditions as closely as possible, and usually use the same test diet with an inert ingredient in place of the test substance. Negative controls also are essential to evaluate if specific a priori assay performance criteria were met (e.g., level of acceptable control mortality), to ensure that any observed effects are not due to nutritional inadequacies, failure of the test arthropods to eat the diet or inappropriate conditions in the experimental design for the health of the test arthropods. Similarly, positive controls are recommended to ensure that the test system is functioning as intended. This includes verifying that the test animal is consuming the test substance. To ensure this, having a positive control that closely mimics the mode of action, or at least requires the same route of exposure as the test substance is desirable. Moreover, recombinant insecticidal proteins produced in microorganisms are often used as the test substance in place of plant derived proteins because of the impracticality of purifying a FIGURE 2 | This figure provides details of the broad steps used in the risk assessment process. All risk assessments start with problem formulation to develop the hypotheses that need to be tested. Once problem formulation is completed risk characterization is conducted with analysis of exposure and testing to determine the potential effects of the exposure. Data from the risk characterization studies are used in the risk evaluation process. This figure was adapted from [bib_ref] Problem formulation in the environmental risk assessment for genetically modified plants, Wolt [/bib_ref]. sufficient quantity of protein from the plants. For this reason, it is important to assess the functional and biochemical equivalence of the test substance with the protein expressed by the GE crop [bib_ref] Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins..., Raybould [/bib_ref]. Another aspect to consider is the biological activity of the test substance in the diet: a parallel bioassay is usually performed using a target species in order to check that the insecticidal protein is stable and has the expected level of biological activity in the diet. In practice, not every test system is able to meet the definition of an ideal NTO study. However, as long as the methods and protocols are reported accurately and the limitations of the study are understood and explained, these studies should still be considered in a weightof-evidence approach. As a minimum standard, tier 1 studies should include a worst-case-exposure scenario, confirmation that the test species is exposed to the biologically active test substance, and the use of a negative experimental control [bib_ref] Quality of laboratory studies assessing effects of Bt-proteins on non-target organisms: minimal..., De Schrijver [/bib_ref].
## Measurement endpoints in nto testing
Historically, the primary and most common measurement endpoint for an early tier laboratory study is mortality. There are a number of reasons for this, including that it is normally unambiguous, easy to measure and has a clear and direct relationship to potential adverse effects on populations of NTOs and ecosystem services they provide. Because it is a common endpoint, there are study designs and methods described for measuring mortality in multiple test systems and for many test species that are validated. Adherence to these standards is often encouraged or required for submitting study results associated with regulatory reviews.
One advantage of using mortality as a study endpoint is that regulatory agencies have developed policies and practices associated with interpreting the results of mortality in NTO studies in regulatory risk assessments. A white paper generated by a panel of experts suggested 50% mortality or a 50% impact on development or weight at the maximum hazard dose in tier 1 studies with insecticidal proteins as a reasonable threshold value for determining if higher tier studies will be informative. The European Food Safety Authority (EFSA) recommends a multiplicative effect of 20% in tier 1 laboratory studies in order to trigger additional studies (EFSA, 2010). One criticism of looking at mortality as a measurement endpoint is that it may not be protective for sublethal effects that might impact populations and the ecological services provided by NTOs. While this is certainly true, it is mitigated by achieving a sufficient margin of exposure, margin of safety or other conservative features of NTO study design.
While it is often remarked that mortality is the only endpoint used in in the regulatory risk assessment of Bt Cry proteins [bib_ref] Science-based risk assessment for nontarget effects of transgenic crops, Andow [/bib_ref] [bib_ref] Genetically modified crops deserve greater ecotoxicological scrutiny, Desneux [/bib_ref] , this is not the case. As shown in [fig_ref] TABLE 1 |: Laboratory studies with beneficial non-target invertebrates [/fig_ref] , although mortality is the most common measurement endpoint, it is not unusual to see one or more sublethal endpoints recorded for a regulatory study (e.g., larval weight and development time can be collected when conducting lethality testing provided the test is of sufficient duration for the test species to reach developmental milestones). Most regulatory studies on insecticidal proteins have also recorded sublethal endpoints, but these data are not always reported in summaries or subsequent representation of the study. Sublethal endpoints that have been measured in studies for Bt Cry proteins include larval and adult weight, developmental timing, fecundity (number of offspring), percent completing adult development and even mobility. What is equally apparent from [fig_ref] TABLE 1 |: Laboratory studies with beneficial non-target invertebrates [/fig_ref] is that while sublethal endpoints have been measured, sometimes it is not immediately obvious how or why particular data on sublethal endpoints are collected, and there is little consistency in how those endpoints are reported in the literature. This is not unexpected as these measurements are selected and coordinated by individual product managers in the absence of specific data requirements. Standardized and validated test protocols used to assess foliar applications of pesticides published by the International Organization for Biological and Integrated Control of Noxious Animals and Weeds (IOBC) include methods for a total of nine beneficial species (two parasitoids, seven predators) and can inform NTO testing for GE plants. With the exception of one beetle species (Aleochara bilineata, Coleoptera: Staphylinidae), all IOBC protocols assess mortality as an endpoint [bib_ref] Guidelines to Evaluate Side-effects of Plant Protection Products to Non-target Arthropods, Candolfi [/bib_ref]. In addition, the tests also considered sublethal endpoints, mainly based on reproduction but also food consumption and behavior of the organism.
The selection of measurement endpoints associated with NTO assessment should be guided by a proper problem formulation that incorporates case-specific information pertinent to the assessment. In this way, measurement endpoints are selected that are clearly linked to an assessment endpoint and associated with the risk hypothesis. Risk hypotheses should not be generic, but rather case-specific and informed by what is known about the insect active routes of exposure and its effects on sensitive or target species. Also, prior to the study, the project managers and risk assessors should know specifically how the measurement endpoints will be interpreted and how they will be used in the decision process. If it cannot be clearly articulated how study results will inform the assessment, then the measurement endpoints for the study may not be well-aligned with protection goals and decision-making priorities. Finally, the measurement endpoints should be designed to incorporate the practical realities and limitations associated with available and appropriate relevant test species.
## Experience with sublethal endpoint testing in non-bt ge crops
The development of novel, non-Bt Cry proteins for insect control and of non-protein based methods such as RNAi has been accompanied by an increased interest in assessing sublethal impacts. Publications related to these new technologies demonstrate some of the ways that sublethal endpoints are used.
## Rnai and mon87411
The potential use of RNAi for pest control has been widely discussed [bib_ref] RNAi: Future in insect management, Burand [/bib_ref] and regulators are considering the use of RNAi to control insect pest in relation to risk assessment [bib_ref] Biotechnological uses of RNAi in plants: Risk assessment considerations, Casacuberta [/bib_ref] [bib_ref] Biosafety research for non-target organism risk assessment of RNAi-based GE plants, Roberts [/bib_ref]. Presently, a single insect protected crop using RNAi as a mechanism has been approved for commercialization. The effect of the RNAi in the target pest, the western corn rootworm (WCR, Diabrotica virgifera virgifera), is triggered by the presence of a dsRNA targeting a housekeeping gene. MON87411 targets the WCR Snf7 gene and its mode of action has been well-characterized [bib_ref] Characterizing the mechanism of action of doublestranded RNA activity against western corn..., Bolognesi [/bib_ref] [bib_ref] Physiological and cellular responses caused by RNAi-mediated suppression of Snf7 orthologue in..., Ramaseshadri [/bib_ref]. Uptake of DvSnf7 RNA generates eventual mortality or severe growth inhibition, which can be observed 5 days after exposure to the test substance. There is also a relationship between exposure duration and eventual growth inhibition at lower doses, but short exposures at high doses are sufficient to induce mortality [bib_ref] Characterizing the mechanism of action of doublestranded RNA activity against western corn..., Bolognesi [/bib_ref]. A description of the published ERA for MON87411 expressing DvSnf7 RNA, includes a description of NTO testing conducted in support of the risk assessment [bib_ref] Ecological risk assessment for DvSnf7 RNA: A plant-incorporated protectant with targeted activity..., Bachman [/bib_ref]. A range of vertebrate tests were conducted, as well as arthropod testing including toxicity assays for seven species of arthropod. In addition to mortality, sublethal endpoints were observed for each species and included measures of time to adulthood, percent adult emergence, adult biomass (weight), and fecundity (number of surviving offspring produced). Tests were conducted with concentrations of the test substance in excess of 10-fold the maximum expected environmental concentration and the duration of the test period was in excess of the time required to see an effect in the target species. The selection of surrogate species took into account the mode of action, considering that coleopterans (beetles) show significantly greater sensitivity to ingested dsRNA than other arthropod orders [bib_ref] Biosafety research for non-target organism risk assessment of RNAi-based GE plants, Roberts [/bib_ref]. Because this order of insect includes the target species, WCR, it provides a good illustration of how phylogenetic relationships and an understanding of the mode of action can facilitate the choice of surrogates. Additionally, bioinformatics was used as a complementary tool to perform a screening and to identify potential surrogate species based upon the presence of sequence matches. Sequence alignment between the genome and DvSnf7 informed the number and type of species tested, focusing on those which were considered most likely to be informative [bib_ref] Ecological risk assessment for DvSnf7 RNA: A plant-incorporated protectant with targeted activity..., Bachman [/bib_ref].
## Non-bt cry insecticidal proteins: ipd072aa
One example of a non-Bt insecticidal protein that has been subject to extensive NTO assessment is IPD072Aa, isolated from Pseudomonas chlororaphis, which has activity against WCR [bib_ref] A selective insecticidal protein from Pseudomonas for controlling corn rootworms, Schellenberger [/bib_ref]. The IPD072Aa protein has been the subject of bioassays to determine the spectrum of activity in order to inform the NTO risk assessment . As the target pest is a coleopteran, bioassays were conducted with 11 species of Coleoptera, representing four families. Additionally, four species of lepidopteran (moths and butterflies) representing four families in this order were tested. Measurement endpoints included mortality as well as weight for all but one species tested, and time to emergence for two species of ladybird beetles. No observed effects were reported for any of the Lepidoptera species, but both mortality and sublethal effects were observed at varying protein concentrations in some of the tested Coleoptera. In line with best practices for NTO study design (see NTO Study Design above) the criteria for selection of species to characterize the spectrum of activity of IPD072Aa was based on several factors such as the phylogenetic relationship between the species and WCR, established laboratory bioassay methodologies, availability of laboratory reared insects, a known suitable diet and reproducibility of the measurement endpoints .
IPD072Aa has a midgut site of action (SOA) where it targets and disrupts midgut epithelial cells causing breakdown of the epithelial lining in WCR through what appears to be a nonpore forming mechanism [bib_ref] A selective insecticidal protein from Pseudomonas for controlling corn rootworms, Schellenberger [/bib_ref]. The ability of IPD072Aa to kill WCR larvae resistant to mCry3A or Cry34Ab1/Cry35Ab1 indicates that its target site differs from those of the Bt Cry proteins [bib_ref] Safety assessment of coleopteran active IPD072Aa protein from Pseudomonas chlororaphis, Carlson [/bib_ref]. This knowledge related to its mode of action and the phylogentic relationship between the candidate surrogate species and WCR will guide or surrogate species for the soil and aquatic environment to support the regulatory risk assessments of plant expressed insecticidal Bt Cry proteins.
## Species
Life-stage exposed Measurement endpoints References This table only represents sublethal endpoint data collected as part of a regulatory study and does not present sublethal endpoint data collected as part of solely academic studies. a Data provided in [bib_ref] Testing the impact on nontarget organisms of insecticidal proteins expressed in transgenic..., Stacey [/bib_ref] are also listed in [bib_ref] Non-target organism risk assessment of MIR604 maize expressing mCry3A for control of..., Raybould [/bib_ref]. [bib_ref] Non-target organism risk assessment of MIR604 maize expressing mCry3A for control of..., Raybould [/bib_ref] summarizes the data that were submitted to the regulatory authority. Interestingly, for all but one of the organisms tested (O. insidiosus; P. cupreus; and A. bilineata) not all sublethal endpoint measured by [bib_ref] Testing the impact on nontarget organisms of insecticidal proteins expressed in transgenic..., Stacey [/bib_ref] are also reported by [bib_ref] Ecological versus ecotoxicological methods for assessing the environmental risks of transgenic crops, Raybould [/bib_ref] selection of the appropriate surrogate species and measurement endpoints in NTO assays.
## Non-cry insecticidal proteins: vip3a
Vip3A is a Bt vegetative insecticidal protein that is active against lepidopteran pests. It has a different mode of action from Cry proteins, and when delivered as a combined treatment it has the potential to delay the evolution of pest resistance to Bt crops [bib_ref] The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A..., Lee [/bib_ref]. A description of the ERA for MIR162, a maize event expressing the Vip3A protein, has been published [bib_ref] Non-target organism effects tests on Vip3A and their application to the ecological..., Raybould [/bib_ref]. The bioassays were conducted using species representing functional groups of foliar arthropods, soil-dwelling invertebrates, pollinators, wild birds and mammals, aquatic invertebrates and farmed or wild fish [bib_ref] Non-target organism effects tests on Vip3A and their application to the ecological..., Raybould [/bib_ref]. In addition to mortality, sublethal endpoints such as fecundity, weight increase, adult emergence, body weight, and length were recorded for some of the species tested. Depending on the species, worst-case, or conservative maximum expected environmental concentrations were used.
# Discussion
## Sublethal endpoints are addressed in the context of existing frameworks for nto assessment
The rationale for conducting NTO studies in support of regulatory risk assessments is not dependent on whether the studies are designed to measure mortality or sublethal endpoints. Before a study is conducted, the problem formulation process should identify a set of informative tests based on the pathways of environmental exposure, and an identification of relevant taxa and functional groups for which risk should be assessed. Once the appropriate tests are identified, consideration can be given to what sort of measurement endpoints will best inform the assessment. When making these decisions, it is important to keep in mind the practical limitation associated with NTO testing and risk assessment.
## Absence of a single definitive test or sublethal endpoint
As with other types of testing, there is no single definitive test that can address every possibility of sublethal effects. As is typical of the regulatory risk assessment process, consideration of whether a test is necessary should be done on a case-bycase basis, focusing on the value of the information for reaching conclusions about overall risk. Although flexibility in testing allows sublethal tests to be tailored to specific needs of a chosen test system, non-uniformity in testing procedures can make it difficult to compare results obtained by independent researchers conducting experiments and then reporting sublethal impacts in the literature. These comparisons may provide helpful context for regulatory considerations. The endpoints that have been previously utilized to measure sublethal effects for all types of insect-active GE plants (i.e., weight, growth and developmental time, fecundity) appear to be useful and sufficient for sublethal testing of non-Bt Cry insect actives, both proteinbased and dsRNA-based.
## Effect of the active on the target can indicate the utility of sublethal endpoints in nto testing
Knowledge regarding the mode of action and time to effect, as it relates to the effect of the insect active on the target insect, is instructive for ascertaining whether sublethal endpoint testing is likely warranted in NTOs. For example, if an insect active targets a cellular process that broadly affects growth (e.g., protein synthesis) it may be reasonably expected that collection of data measuring sublethal endpoints such as weight, development time, and reproduction may be warranted. If those endpoints are affected in the target organism, they may also be affected in NTOs. Additionally, the time that it takes for sublethal effects to manifest in the target organism during lethality testing may also be an indication that sublethal endpoint testing is warranted for NTO assessment. If mortality in the target organism is delayed, but indications of mortality are evident earlier due to the onset of sublethal effects such as delayed growth or development, then it may make sense to look for these effects in NTO assessment as well.
## Sublethal endpoints need to relate to ecologically relevant assessment endpoints
Sublethal endpoints that relate to measurable ecologically relevant endpoints will be much easier to interpret than more complicated endpoints. Typically, measurement endpoints for sublethal effects that include development time, growth/weight and reproduction are used for data collection, and these endpoints are readily quantifiable and relatable to assessment endpoints such as population size. While a variety of additional sublethal endpoints reflecting more ambiguous measurements have been reported in the published literature (i.e., feeding behavior and learning performance; [bib_ref] Does Cry1Ab protein affect learning performances of the honey bee Apis mellifera..., Ramirez-Romero [/bib_ref] , the interpretation of these endpoints with respect to ecological outcomes is challenging.
## Practical considerations for developing new test systems
Any NTO testing is dependent on the existence or the development of a well-characterized and validated test system. Some existing test systems may lend themselves to the collection of sublethal endpoint data, but others may not. Thus, development of new test systems may be required when and if the problem formulation indicates that a particular insect active is likely to cause sublethal effects in relevant NTOs. In either case, before the tests are conducted it is important to assure that the results of the sublethal measurements will be meaningful for risk assessment.
## Addressing knowledge deficits
For RNAi-based insect actives, bioinformatics will become increasingly important in predicting potential adverse effects associated with exposure to dsRNA. However, there are currently significant gaps in bioinformatics data for both pest organisms and for ecologically important taxonomic groups, as well as a lack of information on which taxonomic groups are (in)sensitive to environmental RNAi. Narrowing those information gaps will provide a better understanding of the extent to which different NTOs need to be assessed for a case-specific assessment for a particular environmental RNAi. More sublethal endpoint data are collected than is widely acknowledged. In part, this is, due to the primary status of mortality in testing guidelines and in summary reports or journal publications where sublethal endpoint measurements often are relegated to supplementary material. Finding new means of sharing this information (and improving access to it) is needed in the future to increase the potential usefulness of this information for risk assessments. Similarly, data collected during early characterization of pesticidal proteins may be considered proprietary in nature, presenting a barrier to broad distribution. Whenever possible, however, mechanisms should be encouraged to improve access to this type of information. There are several recent examples where early characterization of pesticidal molecules have been published [bib_ref] Characterization of the spectrum of insecticidal activity of a double-stranded RNA with..., Bachman [/bib_ref] [bib_ref] Characterization of the activity spectrum of MON 88702 and the plant-incorporated protectant..., Bachman [/bib_ref] [bib_ref] Characterization of the spectrum of insecticidal activity for IPD072Aa: A protein derived..., Boeckman [/bib_ref]. However, the research community is encouraged to find new means of storing and disseminating information that often is omitted from peerreviewed publications but has value as a collective resource in further development of NTO risk assessment methodology.
# Conclusions
NTO testing is conducted in support of regulatory decision making, and therefore must be designed for this purpose, rather than simply to conduct scientifically interesting experiments. The testing associated with any particular insect-active GE crop should be informed by a problem formulation process. Problem formulation takes into account what is known about the insect-active protein, the crop, and the expected interactions between NTOs and the associated insect-active crop, as well as the availability of well-developed test systems that facilitate the interpretation of test results in a regulatory context. The problem formulation process remains fundamentally the same whether the measurement endpoint is mortality or sublethal endpoints. When selecting sublethal endpoints for consideration, a risk hypothesis should link the sublethal endpoint to an assessment endpoint and the associated protection goal. Because most NTOs are protected at the population level and NTO communities at the functional level, typically, sublethal endpoints that are related to growth, development, and reproduction and which can be easily extrapolated to population level effects are most informative. While additional sublethal endpoints might be measurable, they should only be considered for regulatory testing if there is a clear relationship to a protection goal and the results are likely to reduce uncertainties associated with the NTO assessment. A review of recent and past measurement of sublethal endpoints collected to inform regulatory studies of plant incorporated insecticidal Bt Cry proteins is summarized in [fig_ref] TABLE 1 |: Laboratory studies with beneficial non-target invertebrates [/fig_ref]. These data suggest sublethal endpoints for current insect resistant GE crops are observed and measured more routinely than is often claimed in the literature.
A long history of standardization exists that can inform the future of NTO testing. While standard methods are not absolutely required for testing possible sublethal impacts, such studies can be informative for risk assessment. However, for these studies to be informative, there should be a clear understanding of what data are being collected and what is the rationale for collecting them. When published in peer reviewed publications, these sublethal endpoints are often published as supplemental data. If sublethal testing is done, and data are reported, these data should be presented more prominently in research reports. This practice would promote a broader understanding and further discussion of the utility of sublethal endpoints and enhance their usefulness to the risk assessment process.
# Disclosure
The information and views are those of the authors as individuals and experts in the field, and do not necessarily represent those of the organizations where they work. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by Greenlight Biosciences, Inc.,
[table] TABLE 1 |: Laboratory studies with beneficial non-target invertebrates [/table]
|
Exploring patients’ experiences of the whiplash injury-recovery process – a meta-synthesis
# Introduction
The process of designing next-generation outcomes and management methods to better meet the needs of patients with whiplash-associated disorders (WAD) must involve a broader approach than is currently used. Because the outcome measures and management methods used for WAD are mostly based on studies at group level using standard measures but not based on an individual's specific needs, it is important to explore the experiences of patients with WAD during the process from injury to recovery.
In the context of WAD, there is no accepted standardized definition for recovery. Sterling et al 1 defined the prognosis for clinical recovery by identifying 3 pathways through a trajectory-modeling analysis in which the prognostic factor was the severity of Dovepress Dovepress 1264 the initial pain-related disability (mild, moderate, or severe) measured at the group level and using standard measures. However, at the individual level, patients themselves most likely define recovery differently depending on their life situation. Thus, patients' thoughts regarding the injury-recovery process could be significant for their symptom management.
Efforts to manage WAD in current practice still struggle to find the most effective ways to solve pain-and disabilityrelated problems during the recovery process. Exercise as a management method has been studied and is recommended extensively to facilitate recovery, as described in a recent systematic review. [bib_ref] General exercise does not improve long-term pain and disability in individuals with..., Griffin [/bib_ref] However, the conclusion of that review was that there was no evidence that general exercise interventions led to long-term improvements. [bib_ref] General exercise does not improve long-term pain and disability in individuals with..., Griffin [/bib_ref] Patient education 3 and advice [bib_ref] The treatment of neck pain-associated disorders and whiplash-associated disorders: a clinical practice..., Bussières [/bib_ref] are often used, either alone or in combination with other methods, to stimulate recovery from WAD. However, the evidence for education is equivocal, and related trials have shown small effects. [bib_ref] The role of exercise and patient education in the noninvasive management of..., Rebbeck [/bib_ref] The difficulties of finding effective treatment and management strategies for WAD might be due to the complexity of the disorder and a great variation of pain-related problems between individuals, which clinicians and researchers have not fully comprehended. To complement current practice, we must understand patients' experiences of WAD and determine what patients believe would support their recovery process.
The situation is similar for the outcomes used in the WAD context. Quantifying patients' perceptions of their WADrelated life situation is not easy. As a basis for determining what to measure and where to direct the management and treatment, 5,6 the biopsychosocial model plays an important role in the WAD context. However, when only the biopsychosocial model is used, there is no guarantee that patients' perceptions of their WAD-related life situation will be addressed and targeted during evaluation and management. A description of patients' perceptions of their neck pain and how they experience the process from injury to recovery [bib_ref] Patient-centredness: a conceptual framework and review of the empirical literature, Mead [/bib_ref] [bib_ref] Writing patient-centered functional goals, Randall [/bib_ref] is important for modernizing the management of WAD. Thus, the aim of this study was to conduct a meta-synthesis to analyze qualitative research findings and thereby understand patients' experiences of WAD and the injury-recovery process.
## Materials and methods design
A qualitative meta-synthesis was applied. Meta-synthesis is defined being as an interpretive integration of existing published qualitative findings. 9
## Data sources
The PubMed, PsychINFO, Scopus, and Web of Science databases were independently searched. Additionally, the reference lists of the included studies were manually screened to complement the database search. The following keywords were used in the indicated combinations: whiplash, whiplash associated disorders, WAD, expectations or beliefs, recovery, qualitative or phenomenography, and focus group or interview. The search was performed in June 2017, and the range of publication dates was not limited.
The inclusion criteria were as follows: qualitative original studies, reported in the English language, the sample comprised participants with WAD, data collected via individual interviews or via a combination of individual interviews and focus groups, and the results relevant to the aim of this meta-synthesis. The titles and abstracts were screened, and the full text of studies that fulfilled the inclusion criteria was evaluated.
## Data extraction and quality assessment
Data were extracted by the first author and cross-checked by the other authors independently. The Critical Assessment Skills Programme (CASP) was used to assess the quality of the included studies.The CASP has been used extensively in previous qualitative reviews. [bib_ref] Physiotherapists may stigmatise or feel unprepared to treat people with low back..., Synnott [/bib_ref] [bib_ref] The association between chronic low back pain and sleep: a systematic review, Kelly [/bib_ref] [bib_ref] Doctors' attitudes and beliefs regarding acute low back pain management: a systematic..., Fullen [/bib_ref] The CASP includes 10 statements (see [fig_ref] Table 1: The quality assessment by Critical Appraisal Skills Program [/fig_ref] with the response alternatives yes/ no/can't tell. All authors independently assessed the quality of the included studies to increase trustworthiness. Disagreements were resolved by consensus discussions among the authors.
## Data synthesis and analysis
We applied the principles for analysis as recommended by Sandelowski and Barroso 9 and Walsh and Downe. 14 First, we tabulated the key findings from each study. Second, the quality of the studies was assessed using the CASP statements.Third, by reading and re-reading the results of the included studies, we attempted to capture all the relevant descriptors from the original text before synthesizing these finding into the results. Fourth, we used reciprocal translation, which aims to translate the included studies' findings one at a time into categories that the next study's findings can confirm or extend while allowing space for divergent and deviant data to refute the identified categories and add new categories. Finally, we synthesized the categories into themes, thus elucidating more refined meanings than each study alone could identify.
## 1265
Patients' experiences of the whiplash injury-recovery process
## Trustworthiness of the meta-synthesis authors
The authors of this meta-synthesis are all senior researchers. All but one (LN) has extensive clinical and research experience in WAD-related health problems. All the authors have experience in qualitative research, but LN has a particularly extensive and varied experience with different qualitative designs and analysis methods. All the authors are well aware of the possible problems that reflexivity and preconceptions [bib_ref] Qualitative research: standards, challenges, and guidelines, Malterud [/bib_ref] could cause in this type of study, and all authors reflected on these issues during the meta-analysis process.
# Results
A total of 57 studies were identified in the first stage, and 4 studies were ultimately eligible for inclusion in this metasynthesis. presents the study identification process. In total, 81 individuals with WAD were interviewed in the studies; see [fig_ref] Table 2: Characteristics and key findings of the included studies • To move or... [/fig_ref] for more details.
The results of the quality assessment of the included studies are presented in [fig_ref] Table 1: The quality assessment by Critical Appraisal Skills Program [/fig_ref].
The synthesis resulted in 29 codes, 6 categories, and 3 themes. The themes were "distancing from normalcy, selfefficacy in controlling the life situation after the injury, and readjustment and acceptance". The themes, categories, and codes are presented in .
## Distancing from normalcy interference and loss
The participants talked about pain and other symptoms interfering with their lives and about the loss of hope, ability, social roles, autonomy, and spontaneity. The participants also noted that it was difficult to continue exercising even though they believed exercise was beneficial. The participants believed that they had lost the physical capacity they had before the injury, [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref] and they felt sad and helpless about all physical, psychological, and social losses that were consequences of WAD. [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref] [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] The participants sought to resume the normalcy, [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] autonomy, and spontaneity of life experienced before the accident. [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref] existential uncertainty WAD changed the participants' self-image, [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] and the participants felt that their previous self-image was difficult to re-establish. [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref] The changes in self-image likely contributed to the participants' existential uncertainty and feelings of being Abbreviation: wAD, whiplash-associated disorders. The synthesized themes and categories from the initial findings in the included studies, shortened to codes
## Codes categories themes
Loss of ability, confidence, slavery, loss of freedom, loss of hope, abandoned by those around, loss of social roles, interference interference and loss Distancing from normalcy Frustration, fear, distress, sadness, worry, negative moods, stigma, chaos, mystery, a riddle needing answers, incongruences, unsure about future, changed self-image existential uncertainty Controllable pain, to have control over daily demands, self-satisfaction, self-confidence, hope, beliefs influence self-confidence, pain controllability is influenced by pain severity stigmatized. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] The participants felt that other individuals without WAD and pain labeled them negatively because there was no visible evidence of their pain. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] The invisibility of pain also contributed to the participants' belief that others thought they were malingering. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] Self-efficacy in controlling the life situation after the injury
## 1267
Patients' experiences of the whiplash injury-recovery process appeared to lead to low confidence and insecurity regarding the ability to manage injury-related problems and the future. [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] In contrast, perceived positive outcomes of treatment contributed to increased perceived control. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] Additionally, support from health care staff and significant others was an important part of being able to control the situation. [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] At the same time, some participants believed that they needed to take responsibility for the situation themselves to increase their ability to control it. [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] The perceived severity, 17 manageability, [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref] and realistic expectations [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] of symptom development also influenced the participants' feelings of control. Severe symptoms were difficult to tolerate and manage and decreased the participants' beliefs and confidence regarding their ability to control the situation. The participants did not always know what worsened pain and other WAD-related symptoms. The participants tried to determine how to control their situation but did not often succeed. Consequently, their confidence in their ability to manage pain and participate in daily activities decreased.
## Knowledge and understanding
Knowledge of how medication, pain, and other symptoms were associated was believed to make the situation more understandable and could lead to the development of better strategies for coping with the situation. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] Additionally, an understanding of the pain was believed to contribute to its elimination. However, when the expectations of recovery were not fulfilled quickly enough, confusion and conflicting thoughts about outcomes arose. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] The participants needed to prioritize different commitments according to how they believed their pain and other symptoms would be aggravated. [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] Knowledge about and experiences of participation in physical activity were perceived to be beneficial, [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] although physical activity was not always easy to prioritize. [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref]
## Readjustment and acceptance
## Recapture life roles
Returning to work was perceived as a challenging outcome. [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] The combination of expectations regarding recovery and daily experiences of fluctuating symptoms decreased the participants' confidence concerning the future [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] and their return to work. [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] Supportive health care staff, colleagues, and employers were of great importance in increasing the participants' confidence in their ability to return to work. Perceived work-related stress and aggravated symptoms decreased their confidence in their ability to return to work, while different adaptations of working conditions and acceptance of their own physical and psychological limitations increased the participants' confidence regarding return to work. [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref]
## Happiness
The participants talked about the importance of being optimistic despite the pain and other symptoms, [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] and their optimism was reinforced by perceived symptom improvements. [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] They believed that an optimistic outlook on life would help improve their situation. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] Striving for normalcy aroused positive emotions such as hope [bib_ref] Whiplash patients' experience of a multimodal rehabilitation programme and its usefulness one..., Rydstad [/bib_ref] and happiness. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] Additionally, emotional improvement was interpreted as getting better even when the pain and other symptoms did not necessarily decrease. [bib_ref] What does 'recovery' mean to people with neck pain? Results of a..., Walton [/bib_ref]
# Discussion
This meta-synthesis of patients' experiences of WAD and the injury-recovery process resulted in 3 themes -1) distancing from normalcy, 2) self-efficacy in controlling the life situation after the injury, and 3) readjustment and acceptance -describing the participants' beliefs regarding pain, their WAD-related life situation and their future expectations and acceptance related to the recovery process. The content of the 3 themes, which improves our understanding of the complexity of WAD, can be summarized as follows (the numbers refer to the respective themes indicated above): (1) Patients' perceived feelings of not being their "usual self " when pain and other symptoms interfered with activities and social contacts were strong. (1) The changed self-image was difficult to cope with and likely led to perceptions of stigmatization.
(2) Struggling with feelings of a loss of control appeared to lead to low confidence and insecurity. (2) Thus, reinforcing participants' self-efficacy in taking control over pain and daily activities emerged as important. (2) A focus on increasing knowledge and understanding the pain and its consequences was believed to lead to better strategies for handling the situation during the recovery process. Furthermore, (3) resuming life roles, including returning to work, was challenging, but being (3) optimistic reinforced symptom improvements and contributed to feelings of happiness. Furthermore, these studies showed an association between disability 20 or engagement in activities [bib_ref] Predictors before and after multimodal rehabilitation for pain acceptance and engagement in..., Söderlund [/bib_ref] and recovery expectations. Those with low expectations were more likely to report high disability 20 and low engagement in activities. [bib_ref] Predictors before and after multimodal rehabilitation for pain acceptance and engagement in..., Söderlund [/bib_ref] In the current synthesis, one of the emerging categories was existential uncertainty, which consisted of codes such as distress, worry, stigma, chaos, unsure about the future, and changed self-image. These cognitions and emotions are likely to contribute to recovery expectations during the recovery process. Holm et al 20 stated that interventions should be targeted to recovery expectations to be beneficial. However, we might need to identify not only recovery expectations but also the components of the recovery expectations concept and then design intervention around the components that are problematic to the patient. [bib_ref] Predictors before and after multimodal rehabilitation for pain acceptance and engagement in..., Söderlund [/bib_ref] [bib_ref] A qualitative study of the meaning of recovery, Beaton [/bib_ref] [bib_ref] Association between self-perceived recovery and the SF-36 after minor musculoskeletal injuries, Ottosson [/bib_ref] Chronic WAD has been shown to be predicted by initial high pain intensity, coping styles, depression, fear of movement, [bib_ref] Course and prognostic factors for neck pain in whiplash-associated disorders (WAD): results..., Carroll [/bib_ref] [bib_ref] Coping and recovery in whiplash-associated disorders: early use of passive coping strategies..., Carroll [/bib_ref] [bib_ref] The relation between the Fear-Avoidance Model and constructs from the Social Cognitive..., Sandborgh [/bib_ref] [bib_ref] Coping as a mediating factor between selfefficacy and disability in Whiplash Associated..., Söderlund [/bib_ref] [bib_ref] Whiplash Associated Disorders -predicting disability from a process-oriented perspective on coping, Söderlund [/bib_ref] [bib_ref] Is the self-efficacy and catastrophizing in pain related disability mediated by control..., Söderlund [/bib_ref] and catastrophizing. [bib_ref] If they can put a man on the moon, they should be..., Bostick [/bib_ref] [bib_ref] Coping as a mediating factor between selfefficacy and disability in Whiplash Associated..., Söderlund [/bib_ref] [bib_ref] Whiplash Associated Disorders -predicting disability from a process-oriented perspective on coping, Söderlund [/bib_ref] [bib_ref] Psychosocial factors related to return to work following rehabilitation of whiplash injuries, Adams [/bib_ref] [bib_ref] Course of recovery for whiplash associated disorders in a compensation setting, Casey [/bib_ref] Several categories indicated by our results mirrored the predictors, which could be important to the recovery process, found in previous quantitative studies.
## Existential uncertainty and recovery expectations in recovery process
The existential uncertainty category consisted of codes such as feelings of fear, sadness, worry, and negative moods, among others. However, the category also contained codes such as stigma, chaos and mystery, which could contribute to catastrophizing; moreover, all of these codes could contribute negatively to the recovery process. Bring et al 32 studied coping patterns in patients with acute WAD. The authors concluded that on days with high physical and psychological distress, the patients reported a high degree of catastrophizing. [bib_ref] Coping patterns and their relation to daily activity, worries, depressed mood, and..., Bring [/bib_ref] Beliefs of being stigmatized, perceiving chaos in daily life, and believing that one's situation is a mystery or a riddle that requires answers demand different methodological actions from those currently being used in the evaluation and treatment of WAD. These beliefs may not have been addressed effectively in the current management of WAD and thus may not effectively support the recovery process.
## Control and self-efficacy in recovery process
The perception of struggling with control was one of the categories in the theme "self-efficacy in controlling the life situation after the injury". This category consisted of codes related to control, controllable pain, and having control over daily demands but also self-satisfaction, self-confidence, hope, and beliefs that influence self-confidence. These results could be interpreted as indication that we must identify various areas where perceived control is an issue when evaluating the life situation of patients with WAD. We must also identify the meanings and the levels of self-satisfaction, self-confidence, and perceived feelings of hope to offer better tailored treatments for supporting the recovery process. The codes in the perceived struggling with control category also suggest that there is a strong need for coping strategies that affect perceived control over pain and activities, as demonstrated previously. [bib_ref] Multidimensional associative factors for improvement in pain, function, and working capacity after..., Angst [/bib_ref] Furthermore, the knowledge and understanding category included codes related to learning to develop strategies for control and self-management and understanding the need for realistic expectations, indicating important factors to consider in the treatment of WAD. This finding is in line with recent guidelines in which educating through advice as part of treatment has been recommended. [bib_ref] The treatment of neck pain-associated disorders and whiplash-associated disorders: a clinical practice..., Bussières [/bib_ref] However, because evidence of education as an effective treatment strategy remains equivocal, and considering the present results regarding desired topics for gaining knowledge (e.g., strategies for control), there is a need to consider what content knowledgeenhancing education should involve for these patients from an individual's point of view.
Poor self-efficacy in activities is a strong predictor of long-term disability. [bib_ref] The relation between the Fear-Avoidance Model and constructs from the Social Cognitive..., Sandborgh [/bib_ref] [bib_ref] Is the self-efficacy and catastrophizing in pain related disability mediated by control..., Söderlund [/bib_ref] [bib_ref] Cognitive behavioural components in physiotherapy management of chronic Whiplash Associated Disorders (WAD)..., Söderlund [/bib_ref] [bib_ref] The mediating role of self-efficacy expectations and fear of movement and (re)injury..., Söderlund [/bib_ref] Functional self-efficacy and catastrophizing had large direct effects on disability, and self-efficacy had the larger effect of these 2 variables. [bib_ref] Is the self-efficacy and catastrophizing in pain related disability mediated by control..., Söderlund [/bib_ref] In 1 study, patients who had high self-efficacy in activities at inclusion reported lower disability at a 3-month follow-up compared with those who reported low initial self-efficacy. [bib_ref] Cognitive behavioural components in physiotherapy management of chronic Whiplash Associated Disorders (WAD)..., Söderlund [/bib_ref] The theme "self-efficacy in controlling the life situation after the injury" supported the importance of self-efficacy and emotions and beliefs in relation to increasing or decreasing self-efficacy, as captured in previous studies. In contrast, the content of the theme "distancing from normalcy" and the related category interference and loss suggest that there are factors that can decrease one's self-efficacy in managing the WAD-related situation. To date, there has been little or no effort to systematically evaluate and manage the beliefs and emotions in the context of WAD. The closest attempts have involved measuring and increasing self-efficacy beliefs and perceived control over pain. Much remains to be accomplished to better support the recovery process.
## Life roles and acceptance in recovery process
The recapture life roles category in the theme "Readjustment and acceptance" further described possible significant factors contributing to patients' beliefs in their capacity to handle the situation, i.e., a desire to get and stay well, resume preinjury daily functioning, have the expected physical capacity, handle commitments that make it difficult to concentrate on
## 1269
Patients' experiences of the whiplash injury-recovery process recovery, and the challenges in retaining a positive outlook regarding recovery.
Approximately 50% of patients with WAD have longterm symptoms after their injury, 1,24 and a large proportion are reported to have poor quality of life. [bib_ref] Five years after the accident, whiplash casualties still have poorer quality of..., Tournier [/bib_ref] Additionally, 13-50% are unable to work or participate fully in their daily activities. [bib_ref] Prognostic factors of whiplash-associated disorders: a systematic review of prospective cohort studies, Scholten-Peeters [/bib_ref] The category recapture life roles describes the importance of resuming work, gaining independence and autonomy, and participating in valued life roles. Patients with WAD are in the middle of their lives, a period when work and participation in life are highly valued. Additionally, these results indicate that better management strategies are needed to support return to work and daily life in general.
The category happiness described existing positive beliefs and emotions, such as optimism, spontaneity, re-establish a satisfactory sense of self, positive emotions, and improvement in mood, that are important to capture in evaluation and support during management. The happiness category is likely related to the concept of acceptance and thus possibly indicates the possible importance of acceptance and commitment therapy, where psychological inflexibility is targeted, as one of the treatment strategies in WAD supporting the recovery process. [bib_ref] Psychological flexibility as a mediator of improvement in Acceptance and Commitment Therapy..., Wicksell [/bib_ref] We need new ideas for designing evaluation and management methods for the recovery process for patients with WAD. [bib_ref] Toward optimal early management after whiplahs injury to lessen the rate of..., Jull [/bib_ref] Our results can be a starting point for the development of more individualized methods that support recovery better than the current methods do. For example, the 3 recovery pathways suggested by Sterling et al 1 could be complemented with the results of the present study and possibly applied in clinical practice at the individual level.
# Strengths and limitations
The strengths of the study lie in its credibility, trustworthiness, and auditability. The credibility of data handling is important in meta-synthesis, [bib_ref] Meta-synthesis method for qualitative research: a literature review, Walsh [/bib_ref] meaning that the data remain true to their source. In our study, the codes are clearly recognizable in the included studies, thus implying high credibility. Trustworthiness is another important methodological issue. In our study, all the authors, who are senior researchers, independently checked each step of the analysis, implying high trustworthiness. Thus, we believe that our analysis and the results are both credible and trustworthy. Furthermore, to enhance auditability, [bib_ref] Meta-synthesis method for qualitative research: a literature review, Walsh [/bib_ref] we have transparently documented each phase of the meta-analysis, from search to synthesis. Another important methodological issue in qualitative research is transferability, i.e., whether the findings can be transferred to new settings. [bib_ref] Generalizability and transferability of metasynthesis research findings, Finfgeld-Connett [/bib_ref] The present findings are in line with those of previous qualitative studies, e.g., those investigating the life situation of people living with another chronic condition. [bib_ref] Living with moderate-severe chronic heart failure as a middle-aged person, Nordgren [/bib_ref] The number of included studies might be considered a limitation affecting the trustworthiness of the results. However, we conducted an intensive search of studies that was not limited in terms of publication year and found only 4 relevant papers. Several repeated searches of databases and reference lists yielded the same 4 studies, which we ultimately included. In qualitative studies, the sample size should not be so large that it aggravates the intensive analysis required. Ethically, it is also important to listen to patients' voices without delay to facilitate improved management of WAD in the future. The participants in the included studies also repeatedly talked about similar topics, thus providing confirmation that enhances the trustworthiness of our results. We believe that our results generally mirror the beliefs of patients with WAD.
It should be noted that in the study by Williamson et al, [bib_ref] If I can get over that, I can get over anything" -understanding..., Williamson [/bib_ref] only the findings for their first aim were included as their second aim lay beyond the focus of this meta-synthesis.
The quality of the studies could be a limitation. Specifically, 1 study failed the quality evaluation. However, as Sandelowski et al 42 wrote: "In general, studies should not be excluded for reasons of quality, because […] there are wide variations in conceptions of the good, and in quality criteria". Thus, we decided to include the low-quality study.
# Conclusion
The results of the present study with its 3 themes (distancing from normalcy, self-efficacy in controlling the life situation after the injury, and readjustment and acceptance) and the large variation in the contents (codes) of the themes provide a more comprehensive understanding of the complexity of WAD; patients' complex, multifaceted experiences of WAD; and the injury-recovery process. The results can guide the development of new ways to evaluate and manage WAD. These results also indicate that there is great demand for an approach based on individuals' specific needs to determine the depth of problems and how to support the recovery process.
[table] Table 1: The quality assessment by Critical Appraisal Skills Program (CASP) criteria; response options are Yes/No/Can't tell submit your manuscript | www.dovepress.com [/table]
[table] Table 2: Characteristics and key findings of the included studies • To move or not to move (movement is best, reasons not to move) • Believing you can do it (it's up to me, needing support, loss of confidence) • Fitting it all in • The way ahead (optimism, unsure about the future, need for realistic expectations) [/table]
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Female breast cancer survival in Qidong, China, 1972–2011: a population-based study
Background: Based on data from the population-based Qidong Cancer Registry, we report a survival analysis for female breast cancer patients diagnosed during 1972-2011 in order to assess the long-term trends for the prognosis of this cancer.Methods:The last follow-up for survival status of the 3,398 registered female breast cancer cases was April, 2012. Cumulative observed survival (OS) and relative survival (RS) rates were calculated using Hakulinen's method performed by the SURV3.01 Software developed at the Finnish Cancer Registry. Results: The one-, three-, five-, ten-, fifteen-, twenty-, thirty-, and forty-year OS rates were 83.61%, 67.53%, 58.75%, 48.56%, 42.57%, 38.30%, 29.19%, 19.35%; and the RS rates were 84.76%, 70.45%, 63.12%, 56.81%, 55.26%, 56.36%, 62.59%, 84.00%, respectively. Five-year RS rates of age groups 15-34, 35-44, 45-54, 55-64, 65-74, and 75+ were 60.17%, 68.27%, 67.79%, 56.03%, 55.50%, and 57.28%; 10-year RS rates were 54.16%, 59.59%, 61.34%, 47.78%, 51.30%, and 59.28%, respectively. There were statistical differences among the age groups (RS: χ 2 = 152.15, P = 0.000). Remarkable improvement could be seen for the 5-year RS rates from 52. 08% in 197208% in to 69.26% in 200308% in -200708% in , and the 10-year RS rates from 43.16% in 197208% in to 60.85% in 199808% in -2002 Conclusions: Survival outcomes from Qidong registered cases with breast cancer have shown gradual progress during the past 40 years. The disparities between survival rates of this area and developed countries are getting narrower, but there is still great need for improving survival in Qidong.
# Background
This study was based on the data of the Qidong Cancer Registry (QCR), a population-based cancer registry, which was established in 1972 and is administered by the Qidong Liver Cancer Institute (QDLCI). The QCR covers Qidong City, Jiangsu Province, China with an area of 1208 km 2 , and a population of 1.12 M at the end of 2011. Geographically, Qidong is located at the mouth of the Yangtze River (Chang Jiang), by the Yellow Sea (Huang Hai). The QCR is one of the longest standing cancer registries in China. In 1974, the QDLCI established an all-death cause registration system (as an official Vital Statistics source) in the same coverage area for disease monitoring.
Breast cancer is now the most common cancer in females all over the world [bib_ref] Global cancer statistics, Jemal [/bib_ref] [bib_ref] Estimates of worldwide burden of cancer in 2008: GLOBOCAN, Ferlay [/bib_ref]. With the development of the economy and the improvement of living standards, the incidence of breast cancer is increasing year by year in China [bib_ref] Increasing breast cancer incidence in China: the numbers add up, Ziegler [/bib_ref]. In Qidong, breast cancer has been the fourth most common cancer in females over the past 40 years, with an aggregate crude incidence rate of 14.94 per 100 000, accounting for 9.95% of all cancer sites combined in women. The annual percentage change (APC) of the world age-standardized incidence rate between 1972 and 2011 was 1.31%, with a rapid increase during the latter stages of the past four decades. Thus, further research on breast cancer etiology, prevention and treatment hence is necessary.
This study presents current survival data and their trends during the period of 1972-2011. The data collected over this long-term period and the results accurately derived from them reflect a profile for the prevalence and management of female breast cancer in Qidong. This information on survival provides clinicians and researchers with opportunities to explore breast cancer trends, to measure progress against breast cancer, and to examine implications for breast cancer control in the context of associations with risk factors, prevention interventions, and the dissemination of advances in treatment.
# Methods
The data in this study are from the QCR, which at its founding was designated as the cancer registration repository by the local health authority with compulsory reporting by health care workers. Several years later cancer registration was mandated to be compulsory by the provincial health authority. Now this cancer registry is a member of the national monitoring program (or the National Cancer Registration Network) [bib_ref] Cancer registration in the Peoples Republic of China, Wei [/bib_ref] [bib_ref] Cancer burden in China: a Bayesian approach, Chen [/bib_ref] The Qidong All-death Cause Registration System has been an official vital statistics source approved by the Ministry of Health of China since 1974 [bib_ref] Feasibility of a prospective study of smoking and mortality in Qidong, China...., Chen [/bib_ref] , with death from any cause reported by death certification notifications (DCN). Meeting International Agency for Research on Cancer/International Association of Cancer Registries (IARC/IACR) standards for quality, completeness, timeliness and unresolved duplicate records, the QCR is a member of the IACR. QCR data have been included in "Cancer Incidence in Five Continents" (CI5)and other publications [bib_ref] Cancer survival in Qidong, China, Chen [/bib_ref]. The data reliability has earned recognition in domestic and international studies [bib_ref] Cancer survival in Qidong, China, Chen [/bib_ref]. For the use of these cancer statistics, there is no further need for approval by an Institutional Review Board and no need for informed consent from any of the patients involved.
The QCR uses both active and passive methods for cancer data collection. All data files received from lower-level registries and all other hospitals are checked with cancer report lists and DCNs in order to track down missing cases and to exclude duplicate registrations. At present, there are 12 town-level registries, each with one full-time or part-time health worker doing the registration and collection of DCNs. All new cancer patients in the catchment area are therefore registered, checked, and then reported to the QCR. When the patient dies, whether at home or in hospital, the registration official adds the date of death to the record, and reports it again, together with a DCN card. If the registry personnel receive the death notification first, the patient's medical records are reviewed or a home visit is carried out to obtain further information. Hence, the collection of high-quality follow-up data has been possible in Qidong.
According to Union for International Cancer Control (UICC), breast cancer refers to the malignant tumor originating in the mammary gland epithelial tissue. In QCR, cancer cases were classified and coded according to the International Classification of Diseases, the 10 th revision (ICD-10), and the International Classification of Diseases for Oncology 3 rd editionas well. This study included all patients with female breast cancer reported to the QCR from 1972 to 2011; codes of ICD-10 C50.0-C50.9 and topography codes C50.0-C50.9 of ICD-O-3 with behaviour codes 3 were included. Excluded were all death certificate-only, autopsy, and individual cases with malignant secondary (metastatic to the breast), benign, in situ and uncertain or unknown diagnosis.
A total of 3,452 cases with breast cancer were registered from January 1, 1972 to December 31, 2011. Fifty-four (1.56%) male breast cancer cases were excluded, hence 3,398 female breast cancers were included for analysis. The deadline for the last follow-up for survival status was April, 2012. The proportion of cases with morphological verification was 93.11% (3,164/3,398), with no breast cancer cases recognized by death certificate only. The survival duration of each case was determined as the time difference from the date of initial diagnosis to the date of death due to breast cancer (1,687/3,398 = 49.65%), date of death due to other diseases (3/3,398 = 0.09%), date of loss to follow-up (381/3,398 = 11.21%), and the closing date for those still alive (1,327/3,398 = 39.05%).
Cumulative observed survival (OS) and relative survival (RS) rates were calculated. RS was defined as the ratio of the OS rate to the expected rate, which was estimated from the general gender and calendar period-specific life tables for Qidong residents according to Hakulinen's method [bib_ref] Cancer survival corrected for heterogeneity in patient withdrawal, Hakulinen [/bib_ref]. The survival experience of patients were adjusted for normal life expectancy of the general population of the same age, which makes RS rate an estimate of the chance of surviving the effects of cancer [bib_ref] Analysis of survival, Parkin [/bib_ref]. The OS and RS rates were computed within six age groups (15-34, 35-44, 45-54, 55-64, 65-74, 75+) and nine calendar periods , using Hakulinen's method performed by the SURV3.01 software developed at the Finnish Cancer Registry. Survival trends could be shown through comparing different calendar periods. Statistical tests are included in this software. Relative rates among different age groups were tested using H 0 -H 2 equal vs. unequal hazards tests.
# Results
Observed survival and relative survival rates [fig_ref] Table 1: Observed survival [/fig_ref] shows OS and RS rates by the survival year. The one-, three-, five-, ten-, fifteen, twenty, thirty, and forty-year OS rates of female breast cancer in Qidong during [fig_ref] Figure 1: Trends for the observed survival [/fig_ref] indicates OS and RS trends of female breast cancer in Qidong during 1972-2011 by the survival year. Significant disparities between OS and RS rates could be found in female breast cancer patients surviving over 10 years. For survivors more than 20 years after diagnosis, RS rates increased with the survival year, implying that these women with breast cancer have a relatively higher survival chance compared to the general population with the same demographical characteristics.
Survival rate by age group
## Survival rate by period
# Discussion
This study provides survival estimates of female patients diagnosed with breast cancer in Qidong, China from 1972 through 2011. We compare the results available from other countries or areas. For example, reports from Korea, Japan, United States, and Germany have shown better relative survival rates of female breast cancer than observed in Qidong. The 5-year RS rate (63.12% in 1972-2011) of female breast cancer in Qidong was lower than those in Korea (79.6% in 1993-1997, 85.0% in 1998-2002) [bib_ref] Cancer survival in Korea 1993-2002: a population-based study, Jung [/bib_ref] , Japan (84.6% in 1993-1996) [bib_ref] Survival of cancer patients diagnosed between 1993 and 1996: a collaborative study..., Tsukuma [/bib_ref] , United States (89.0% in 1996-2002) [bib_ref] Cancer statistics, trends, and multiple primary cancer analyses from the Surveillance, Epidemiology,..., Hayat [/bib_ref] , Germany (80.6% in 2000-2002) [bib_ref] Long-term survival of cancer patients in Germany achieved by the beginning of..., Brenner [/bib_ref] , and most of other Asia cities or areas (58.6%-88.2% in 1999) [bib_ref] Proposal for a cooperative study on population-based cancer survival in selected registries..., Tanaka [/bib_ref] , but higher than that of Kampala, Uganda (45.4% in 1993-1997) [bib_ref] Cancer survival in Kampala, Uganda, Gondos [/bib_ref] , and Mumbai, India (46.2% in 1992-1994) [bib_ref] Population-based survival from cancers of breast, cervix and ovary in women in..., Yeole [/bib_ref]. As Bradley et al. [bib_ref] Cancer, medicaid enrollment, and survival disparities, Bradley [/bib_ref] have reported, there are disparities in cancer survival between subjects enrolled in Medicaid and subjects not enrolled in Medicaid, a health insurance program in the United States. Verdecchia et al. [bib_ref] Patient survival for all cancers combined as indicator of cancer control in..., Verdecchia [/bib_ref] indicated that the most direct way for poorer European countries to improve all-cancer survival would be to get richer; for richer countries investment in health technology is important. Hayat et al. [bib_ref] Cancer statistics, trends, and multiple primary cancer analyses from the Surveillance, Epidemiology,..., Hayat [/bib_ref] suggested these disparities may reflect variations in the prevalence of risk factors, the use of screening tests for early detection, access to health care services, and/or social and demographic factors. In a comparative study of Stockholm and Singapore women with breast cancer, there appear to be differences in the 5-year OS rates of 72% vs. 64%, and 5-year RS rates of 80% vs. 70%, respectively. In Qidong, the data highlight an improvement in prognosis over the calendar periods, which were likely influenced by marked economic improvement leading to better medical facilities and management of patients from diagnosis through treatment, as well as improved treatment options [bib_ref] Do Asian breast cancer patients have poorer survival than their western counterparts?..., Tan [/bib_ref]. At the molecular level, Guo et al. [bib_ref] The relationship between estrogen receptor α expression and 5-year survival in Chinese..., Guo [/bib_ref] reported that the 5-year survival in female patients with ERα (estrogen receptor α +) is higher than that with ERα (−) in Chinese women. But no matter how poor the survival shown in this series, compared to results from other parts of the world or from big cities in China, the data indicate that survival rates for breast cancer patients over this 40 year period in Qidong have been improving. Significant upward trends for the 5-year RS rate were observed, increasing from 52.08% in 1972 to 69.26% in 2003-2007, and the 10-year RS rates from 43.16% in 1972 to 60.85% in 1998-2002. The improvement in survival rate between 1972 and 2011 may be attributed to social and economic development, as well as improvement in early detection, such as by increasing the use of mammography, and therapy. Patients also received better quality care, as shown in a pay-for-performance program for breast cancer care in Taiwan [bib_ref] Effect of the pay-for-performance program for breast cancer care in Taiwan, Kuo [/bib_ref]. Last century, however, breast cancer screening programs were almost nonexistent in Qidong. It seems reasonable to assume that many of these patients had no chance to receive early detection for the diagnosis of this cancer, and hence, effective treatment was rarely possible. In recently years, screening as well as substantive improvements in diagnosis and treatment have become possible, through which breast cancers could be detected at earlier stages and perhaps more suitable treatment options could be obtained, in turn resulting in improvements in prognosis. Our 40-year results indicate that 58.75% (2*SE: 1.81%) of female patients with breast cancer survived 5 years or more, with a 5-year RS rate of 63.12% (2*SE: 1.95%). Statistical differences were noted among the different age groups. Higher 5-, 10-year RS rates were observed from the groups of age 35-54 years in our series, which is consistent with a report from a rural area in China by Chen and his colleagues [bib_ref] Relative survival rate analysis of cancer patients in Changle city, Chen [/bib_ref]. Ballard-Barbash [fig_ref] Table 3: shows the RS rates by survival year and by period [/fig_ref] Relative survival rate of female breast cancer by period in Qidong, 1972-2011 (%) [bib_ref] Physical activity, biomarkers, and disease outcomes in cancer survivors: a systematic review, Ballard-Barbash [/bib_ref] in a recent systematic review concludes that physical activity is associated with a reduction in breast cancer-specific mortality as well as all-cause mortality; and there is evidence for a dose-response effect of decreasing mortality risk with increasing activity in roughly half of the 27 reported observational studies. This association may be one of the reasons that younger patients had better survival in our series. Besides a variety of known factors including tumor size, nodal status and grade, histological type, improved treatment modalities and screening, and racial differences [bib_ref] An overview of prognostic factors for long-term survivors of breast cancer, Soerjomataram [/bib_ref] [bib_ref] Breast cancer racial disparities: unanswered questions, Ademuyiwa [/bib_ref] , there may be other factors that affect the prognosis of breast cancer, although their mechanisms are unclear. In a meta-analysis which included 43 studies of women diagnosed with breast cancer between 1963 and 2005, Protani et al. [bib_ref] Effect of obesity on survival of women with breast cancer: systematic review..., Protani [/bib_ref] illustrated that women with breast cancer who were obese, had poorer survival than women with breast cancer who were not obese, with a HR of 1.33 (95% CI: 1.21-1.47). Some suggest [bib_ref] Weight management and its role in breast cancer rehabilitation, Demark-Wahnefried [/bib_ref] that weight management is a key to controlling prevalent co-morbid conditions in breast cancer patients, although it is currently unknown whether post-diagnosis weight loss can improve prognosis and disease-free survival. Stage is often considered to be the most important factor determining survival [bib_ref] Do Asian breast cancer patients have poorer survival than their western counterparts?..., Tan [/bib_ref]. Hayat et al. [bib_ref] Cancer statistics, trends, and multiple primary cancer analyses from the Surveillance, Epidemiology,..., Hayat [/bib_ref] reported that the 5-year relative survival for those diagnosed at early stage (stage I) is very good (100% for all races combined), but if metastatic disease is diagnosed (stage IV), survival drops to 21% for all races combined. A study in a Finnish population comparing screening-detected and non-screening-detected breast cancer found that breast cancer detected by mammography screening is an independent prognostic factor in breast cancer that is associated with a more favorable survival rate: a 15-year survival of 86% for patients screening-detected vs. 68% for patients diagnosed by other methods [bib_ref] Long-term prognosis of breast cancer detected by mammography screening or other methods, Lehtimäki [/bib_ref]. Unfortunately, for our population-based cancer registration series, one limitation of our study was that stage information was available sporadically, a common problem in populationbased cancer registries worldwide [bib_ref] Imputation of missing values of tumour stage in population-based cancer registration, Eisemann [/bib_ref]. This limitation does not hinder the assessment of the general evolution of better survival from breast cancer in this area. We believe, as the literature [bib_ref] Breast cancer racial disparities: unanswered questions, Ademuyiwa [/bib_ref] has suggested, that disparities in access and quality of care may be eliminated by understanding and addressing cultural and economic barriers.
# Conclusions
The survival outcomes in women with breast cancer in Qidong, China have shown gradual progress during the past 40 years. There remain disparities of survival rates of female breast cancer comparing Qidong to more developed regions, yet the gaps are getting narrower, and there is continuing opportunity for improving breast cancer survival in Qidong.
[fig] Figure 1: Trends for the observed survival (OS) and relative survival (RS) rates of female breast cancer in Qidong, China, 1972-2011. (−Ο−) OS; (−Δ−) RS. [/fig]
[table] Table 2: shows the 5-, and 10-year OS and RS rates by age group. The 5-year OS rates of age groups 15-34, 35-44, 45-54, 55-64, 65-74, and 75+ were 59.81%, 67.42%, 66.26%, 53.44%, 47.82%, and 30.42%, while the 10-year OS rates were 53.36%, 57.82%, 58.09%, 42.11%, 32.05%, and 14.78%, respectively. The 5-year RS rates were 60.17% 68.27%, 67.79%, 56.03%, 55.50%, and 55.28%, and 10-year RS rates were 54.16%, 59.59%, 61.34%, 47.78%, 51.30%, 59.28%, respectively. The patients at 35-44 or 45-54 years of age experienced higher 5-, and 10-year OS and RS rates. Statistical differences could be found among the different age groups (RS: χ 2 = 152.15, P = 0.000). [/table]
[table] Table 3: shows the RS rates by survival year and by period. An increasing trend for the RS rates of female breast cancer could be observed through the nine intervals during 1972-2011 period. Remarkable improvement could be seen for the 5-year RS rates of 52.08% in 1972 to that of 69.26% in 2003-2007, and for the 10-year RS rates of 43.16% in 1972 to that of 60.85% in 1998-2002. Little improvement was observed for the 1-year RS rate from 87.91% in 1972 to 90.30% in 2008-2011, and for the 15-year RS from 45.81% in 1972 to 48.22% in 1993-1997. [/table]
[table] Table 1: Observed survival (OS) and relative survival (RS) rates of female breast cancer in Qidong, 1972-2011 (%) SE OS , Twice standard error of OS; 2*SE RS , Twice standard error of RS. [/table]
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Perioperative Pain Management in Bariatric Anesthesia
Weight loss (bariatric) surgery is the most commonly performed elective surgical procedure in patients with morbid obesity. In this review, we provide an evidence-based update on perioperative pain management in bariatric anesthesia. We mention some newer preoperative aspects-medical optimization, physical preparation, patient education, and psychosocial factorsthat can all improve pain management. In the intraoperative period, with bariatric surgery being almost universally performed laparoscopically, we emphasize the use of non-opioid adjuvant infusions (ketamine, lidocaine, and dexmedetomidine) and suggest some novel regional anesthesia techniques to reduce pain, opioid requirements, and side effects. We discuss some postoperative strategies that additionally focus on patient safety and identify patients at risk of persistent pain and opioid use after bariatric surgery. This review suggests that the use of a structured, step-wise, severity-based, opioid-sparing multimodal analgesic protocol within an enhanced recovery after surgery (ERAS) framework can improve postoperative pain management. Overall, by incorporating all these aspects throughout the perioperative journey ensures improved patient safety and outcomes from pain management in bariatric anesthesia.
# Introduction
W i t h i t s r i s i n g g l o b a l p re v a l e n c e , i n c re a s i n g n u m b e r s o f p a t i e n t s w i t h m o r b i d o b e s i t y a re undergoing a wide variety of elective and emergency surgery. Morbid obesity is a chronic systemic illness that reduces the quality of life and life expectancy and simultaneously increases patients' perioperative morbidity and mortality. We have previously reviewed the management of acute and perioperative pain for patients with morbid obesity. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] Bariatric surgery is now almost universally performed laparoscopically and is the most commonly performed elective surgery in patients with morbid obesity. Bariatric anesthesia has provided the opportunity to study and standardize perioperative pain management protocols. [bib_ref] Assessing the feasibility of a randomized, double-blinded, placebo-controlled trial to investigate the..., Wu [/bib_ref] [bib_ref] Effect of intraperitoneal local anesthesia on enhanced recovery outcomes after bariatric surgery:..., Jarrar [/bib_ref] The recently enhanced recovery after surgery (ERAS) society guidelines for bariatric anesthesia have also emphasized the need for protocol standardization and implementation. [bib_ref] Guidelines for perioperative care in bariatric surgery: Enhanced recovery after surgery (ERAS)..., Stenberg [/bib_ref] The importance of bariatric anesthesia and analgesia research cannot be overstated. These bariatric studies can inform Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022 and improve the perioperative protocols for other patients with morbid obesity irrespective of the surgical procedures they are undergoing. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] This review aims to provide an evidence-based update for perioperative pain management in bariatric anesthesia with a separate focus on preoperative preparation, intraoperative management, and postoperative care.
## Preoperative preparation
As with all other elective surgery, patients with morbid obesity require careful screening and evaluation for co-morbidities before bariatric surgery. It must be emphasized here that preoperative co-morbidity optimization can significantly impact postoperative pain management after bariatric surgery. [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] [bib_ref] Guidelines for perioperative care in bariatric surgery: Enhanced recovery after surgery (ERAS)..., Stenberg [/bib_ref] Though the perioperative implications of all the individual medical conditions in bariatric anesthesia are beyond the scope of this review, there are certain aspects of preoperative preparation directed specifically towards improving perioperative pain that merit further discussion [ [fig_ref] Figure 1: Preoperative preparation for improving postoperative pain management in bariatric surgery [/fig_ref] ].
First and foremost, medical optimization should focus on the diagnosis and treatment of sleep-disordered breathing (SDB) including obstructive sleep apnea (OSA). This is not only an important modifiable risk factor for morbidity and mortality in bariatric anesthesia, but the diagnosis and treatment of OSA can specifically improve the safety and outcomes of postoperative pain management. [bib_ref] Opioids for acute pain management in patients with obstructive sleep apnea: A..., Cozowicz [/bib_ref] The management of acute pain and the tenuous relationship of opioids with OSA in patients with morbid obesity has been described elsewhere. [bib_ref] Obstructive sleep apnea, pain, and opioids: Is the riddle solved?, Lam [/bib_ref] Especially in moderate to severe OSA that remains undiagnosed or untreated, an opioid-centric approach to pain management in the perioperative period should be avoided. In a dose-dependent manner, opioids impair the arousal response, worsen the degree of airway obstruction, and reduce respiratory rate and ventilatory effort with increasing periods of apnea that can trigger cascades of complex physiological derangements. This is the well-known opioid-induced ventilatory impairment (OIVI) that has been identified as an important preceding or triggering event in serious postoperative respiratory adverse events (PRAEs) in patients with morbid obesity. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] Additionally, certain patients have pre-existing risk factors for poorly controlled acute pain -chronic pain, chronic opioid use, substance abuse, complex psychiatric history, or previous experience of poorly controlled pain [ [fig_ref] Table 1: Risk factors for poorly controlled acute postoperative pain, persistent postoperative pain, and... [/fig_ref] ]. [bib_ref] An enhanced recovery program for bariatric surgical patients significantly reduces perioperative opioid..., King [/bib_ref] [bib_ref] Outcomes of enhanced recovery after surgery protocols versus conventional management in patients..., Díaz-Vico [/bib_ref] [bib_ref] Preoperative patient education and patient preparedness are associated with less postoperative use..., Khorfan [/bib_ref] [bib_ref] Hospital opioid use predicts the need for discharge opioid prescriptions following laparoscopic..., Diaz [/bib_ref] [bib_ref] Chronic abdominal pain and persistent opioid use after bariatric surgery, Simoni [/bib_ref] [bib_ref] Risk factors for moderate to severe pain during the first 24 hours..., Iamaroon [/bib_ref] These subsets of patients often have a perioperative escalation of their opioid requirements and hence should be meticulously screened and tested for OSA. Many of these patients will need specialist consultations for their pain before elective surgery and plan for extended postoperative stay with appropriate level and duration of monitoring.
The second aspect of the preoperative period that can improve pain management after bariatric surgery is the recent emphasis on the better physical preparation of patients. Within the ERAS paradigm, active deep breathing and coughing with early ambulation all contribute to early discharge and return to baseline function. Therefore, bariatric programs, including our center, have reported the use of functional prehabilitation to improve these outcomes after surgery. These interventions, including the 6-min walk test and peak expiratory flow rate, are intended to concurrently prepare patients, engage them in their own care, and improve their functional status before bariatric surgery. [bib_ref] Assessing the feasibility of a randomized, double-blinded, placebo-controlled trial to investigate the..., Wu [/bib_ref] The third aspect of preoperative pain management is patient education. Ongoing work at our center and elsewhere are looking at how better education can help set realistic expectations for the postoperative period with regards to reducing pain, analgesic requirements, and expected side effects. [bib_ref] Assessing the feasibility of a randomized, double-blinded, placebo-controlled trial to investigate the..., Wu [/bib_ref] [bib_ref] Effect of intraperitoneal local anesthesia on enhanced recovery outcomes after bariatric surgery:..., Jarrar [/bib_ref] For this, we have seen the evolution of patient education from hard copy booklets and web-based programs to app-based interactive modules providing patients in contemporary bariatric programs with the necessary tools to have the best possible experience and outcomes.
Fourth and finally, special attention should be made towards addressing psychosocial factors. Among these, optimizing anxiety, depression, and catastrophizing can contribute to reductions in postoperative pain. [bib_ref] Opioid requirements after laparoscopic bariatric surgery, Weingarten [/bib_ref] Patients with chronic pain or chronic opioid use should be referred preoperatively to appropriate pain specialists to better optimize their pain management and followed up after their elective surgery.
While further studies are required to confirm the importance of all these preoperative interventions, initial experience in bariatric anesthesia suggests significant benefits in terms of pain management from preparation, prehabilitation, and education [ [fig_ref] Figure 1: Preoperative preparation for improving postoperative pain management in bariatric surgery [/fig_ref] ].
## Intraoperative management
The perioperative pharmacology of acute pain management can be discussed using the framework of the WHO pain ladder. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] In this approach, the pain ladder divides analgesics that are directed towards "nociceptive pain" into three steps -foundational analgesics (acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs)), weak opioids, and strong opioids. We briefly discuss their individual roles in pain management for patients undergoing bariatric surgery.
For most medications and as a general guideline, the use of ideal body weight (IBW, Height cms-100) is the most widely applicable scalar to bariatric anesthesia. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] This standardization of drug dosing to IBW is important to ensure the safety of pain management protocols in patients with morbid obesity with various oral drugs, intravenous infusions, and other parenteral therapies.
## Acetaminophen
Acetaminophen is widely considered the cornerstone of perioperative analgesia in the general surgical population.
In patients with morbid obesity, especially those with or at risk of having OSA, routinely administered acetaminophen reduces pain, opioid requirements, and side effects when compared to the more traditional opioid-centric approach. The benefits of using the later introduced intravenous formulation of acetaminophen have been supported by studies and experience in bariatric centers. [bib_ref] Intravenous acetaminophen versus placebo in post-bariatric surgery multimodal pain management: A meta-analysis..., Lee [/bib_ref] Additional benefits of parenteral acetaminophen include administration to patients unable to tolerate oral formulations -in the early postoperative period or postanesthesia care unit (PACU), those with significant postoperative nausea and vomiting (PONV), remaining strict nil per os (NPO) due to surgical reasons, and also those who are intubated and ventilated in intensive care unit (ICU). [bib_ref] Intravenous versus oral acetaminophen for pain: Systematic review of current evidence to..., Jibril [/bib_ref] Irrespective of the route of delivery, acetaminophen 1 g should be started at or 2 h before surgery and then continued every 6 h postoperatively for the first few days.
## Non-steroidal anti-inflammatory drugs
The other components of foundational analgesia for nociceptive pain are the NSAIDs, which can contribute to additional excellent opioid-sparing analgesia. When not contraindicated, these drugs should be routinely scheduled for pain after bariatric surgery. The well-known perioperative concerns with NSAIDs (nephrotoxicity, gastrointestinal (GI) ulceration, and increased bleeding risk) are less likely with cyclooxygenase-2 (COX-2) inhibitors. In the bariatric surgical population, additional concerns include the risk of marginal ulcers, anastomotic leaks, and potentiation of acute renal failure in patients at risk or prolonged procedures where the risk of rhabdomyolysis increases. Ketorolac is a widely available parenteral anti-inflammatory that has been widely incorporated into many bariatric protocols. [bib_ref] Ketorolac use shortens hospital length of stay after bariatric surgery: A single-center..., Hariri [/bib_ref] While best given as a scheduled analgesic, in patients at risk for adverse effects (older, diabetics, and those with borderline renal function), the ketorolac dose and duration of use can be effectively reduced to "on demand" (prn) as a rescue analgesic in the early postoperative period. We have observed an interesting conundrum (yet unpublished) with the use of NSAIDs in bariatric anesthesia. Especially seen in patients with undiagnosed and/or untreated OSA, profound
Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022 respiratory depression can be seen after the administration of ketorolac in the PACU. This phenomenon is explained by the pain relief from the NSAIDs that "unmasks" the respiratory depressant effects of previously administered opioids. It is therefore important clinically that NSAIDs should be scheduled round the clock or, when used "on demand," precede the administration of opioids. This caveat further emphasizes the need for following a step-wise, severity-based, opioid-sparing approach, where foundational analgesics are administered on schedule and before the more potent opioids.
## Tramadol
The second step on the nociceptive side of the WHO ladder was "reserved" for weak opioids. [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] While codeine and tramadol were previously considered here, their perioperative use remains controversial. Tramadol is a unique agent as it is "inherently multimodal" -weak mu-opioid receptor agonist with serotonergic and noradrenergic properties. These properties make tramadol especially effective not only for moderate nociceptive pain; it also has modest anti-hyperalgesic (see below) activity. Perioperative tramadol side effects in the bariatric surgical population include increased PONV, serotonin syndrome-inducing interactions (with anti-depressants), and more non-specific intolerance. Despite these, tramadol has been found useful in postoperative pain management after bariatric surgery. [bib_ref] Perioperative analgesia for fast-track laparoscopic bariatric surgery, Bamgbade [/bib_ref] Tapentadol is a newer molecule, approximately twice as potent as tramadol and devoid of the serotonin-related side effect profile. [bib_ref] Tapentadol versus tramadol: A narrative and comparative review of their pharmacological, efficacy..., Roulet [/bib_ref] Despite these promising advantages over tramadol, the evidence for tapentadol use in bariatric anesthesia is currently limited. As with any other pure mu-opioid receptor agonist, all these second-step "weak opioids" also have the potential for serious sedation and respiratory depression, especially in bariatric anesthesia and particularly in patients who have undiagnosed or untreated OSA. If encountered, these PRAEs should be treated with standard opioid reversal protocols.
## Opioids
The "strong opioids" remain on the third step of the WHO nociceptive ladder. Opioid naïve patients with morbid obesity can be given titrated doses of opioid analgesics for moderate to severe pain that is not adequately controlled with step 1 and step 2 analgesics. Before initiating opioid analgesic therapy, the presence of hyperalgesia should ideally be sought using the DN4 (Douleur Neuropathique 4 Questions) questionnaire (see below) and appropriately treated. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] In the postoperative period, if prolonged parenteral opioid therapy is required, the use of intravenous patient-controlled analgesia (IVPCA), with similar settings as in non-obese patients, has been described. [bib_ref] Risk factors for moderate to severe pain during the first 24 hours..., Iamaroon [/bib_ref] The use of continuous opioid infusions is generally contraindicated after bariatric surgery; patients who remain intubated and ventilated in ICU may be the only exception to this rule.
Opioid-dependent and tolerant patients should continue to receive their baseline opioid drugs, possibly with reduced doses, throughout the perioperative period. Opioid conversions and rotations may allow for improved analgesia with reduced doses in the chronic pain setting, but perioperatively these require both expertise and individualization of care and are generally not recommended. Similarly, the perioperative management of more complex pain patients undergoing bariatric surgery with methadone maintenance therapy, opioid antagonist therapy, and transdermal opioid therapy should always involve consultations with specialists with expertise in their use. The risk of poorly controlled acute pain has to be balanced against the risk of opioid overdose and acute withdrawal, both of which are most likely in these sub-groups of surgical patients with chronic opioid use. Therefore, for patients undergoing bariatric surgery with chronic opioid use, extended postoperative monitoring will be required -same-day discharge, ambulatory, or outpatient bariatric surgery should be avoided. [bib_ref] Opioids for acute pain management in patients with obstructive sleep apnea: A..., Cozowicz [/bib_ref] [bib_ref] Obstructive sleep apnea, pain, and opioids: Is the riddle solved?, Lam [/bib_ref]
## Non-opioid adjuvants
Over the past decade, perioperative pain management has seen a paradigm shift away from the traditional opioid-centric approach towards the use of more non-opioid analgesic adjuvants for acute pain. [bib_ref] Acute pain management in morbid obesity-an evidence based clinical update, Budiansky [/bib_ref] We have integrated the non-opioid anti-hyperalgesic drugs into a modification of the WHO Pain Ladder and used a novel concept of the "Ottawa
Step Ladder" to better understand this paradigm. [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] The Ottawa
Step Ladder integrates the perioperative use of anti-hyperalgesic drugs (gabapentinoids, ketamine, lidocaine, and dexmedetomidine) into acute pain management. The use of these non-opioid adjuvants should be based on the identification of pro-nociception or acute neuropathic pain, which presents as hyperalgesia. Published evidence has described acute hyperalgesia or "pro-nociception" occurring more frequently in some patients undergoing certain surgical procedures. [bib_ref] Postoperative respiratory depression with pregabalin: A case series and a preoperative decision..., Eipe [/bib_ref] This diagnosis of acute neuropathic pain can be objectively made with the validated DN4 questionnaire. [bib_ref] Perioperative pain management in morbid obesity, Belcaid [/bib_ref] In our opinion, the presence of pronociception or hyperalgesia connects poorly controlled acute pain with the progression to chronic post-surgical pain and persistent opioid use after elective surgery. We believe an appreciation of this concept will improve immediate patient safety and long-term Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022 outcomes in these patients, especially since there is a small but not insignificant risk of persistent opioid use after bariatric surgery [ [fig_ref] Table 1: Risk factors for poorly controlled acute postoperative pain, persistent postoperative pain, and... [/fig_ref] ].
## Gabapentinoids
These drugs have been used in a wide variety of surgical procedures with mixed results-some improvements in acute pain management and potential for prevention of progression to chronic post-surgical pain. Pregabalin has been studied and shown to reduce pain scores and analgesic consumption, but more so in patients undergoing painful procedures. Sedation and respiratory depression are well-known side effects when gabapentinoids are prescribed as routine premedication or in less painful procedures. [bib_ref] Postoperative respiratory depression with pregabalin: A case series and a preoperative decision..., Eipe [/bib_ref] This may not be desirable or safe in patients with morbid obesity, with or without OSA, who are already at increased risk of PRAE. As mentioned previously, bariatric surgery is almost universally done laparoscopically with limited risk in progression to chronic pain or needing persistent opioid use. These latter two are other proposed benefits of perioperative gabapentinoids. Pregabalin is more widely studied as an adjuvant to improve perioperative pain management in bariatric anesthesia with mixed results. [bib_ref] Pregabalin to improve postoperative recovery in bariatric surgery: A parallel, randomized, double-blinded,..., Martins [/bib_ref] Even if started in the postoperative period, gabapentin and pregabalin may increase sedation and potentiate opioid-induced respiratory depression, adversely impact sleep architecture, and impair balance and vision. Their use in the perioperative period should require a very careful assessment of the risk-benefit in patients after bariatric surgery, especially those with OSA. [bib_ref] Postoperative respiratory depression with pregabalin: A case series and a preoperative decision..., Eipe [/bib_ref] [bib_ref] Pregabalin to improve postoperative recovery in bariatric surgery: A parallel, randomized, double-blinded,..., Martins [/bib_ref] Hence, the routine use of the gabapentinoids is not useful or justified in bariatric anesthesia unless the patient is already on them or develops acute hyperalgesia postoperatively.
## Ketamine
Despite decades of use and other recent advances in the pharmacotherapy of acute pain, the NMDA antagonist ketamine remains the most widely available, easily administered, and relatively inexpensive non-opioid analgesic with a wide range of doses and applications. The popularity of ketamine in the perioperative period can be attributed to its analgesic, anti-hyperalgesic, and opioid-sparing effects. In contrast to opioids, ketamine provides additional cardiorespiratory stability and improvements in mood without sedation or respiratory depression. It should be noted that the beneficial effects and side effects of ketamine are primarily dependent on the dose administered and these can be considered as "high or anesthetic," "low or analgesic," and "ultra-low or anti-hyperalgesic" doses.
"High or anesthetic" dose of ketamine (bolus >1 mg/kg IBW and/or infusion of >1 mg/kg IBW/h) will result in profound analgesia but an unpredictable loss of consciousness and significant emergence phenomena. This should be reserved for use in trauma and/or anesthetic management where the patient's airway has been secured and prolonged controlled ICU ventilation is planned. "Low or analgesic" dose ketamine (bolus 0.5 mg/kg IBW and/or infusion of 0.5 mg/kg IBW/h) also provides excellent analgesia but can often be accompanied by psychomimetic effects, emergence reactions, and prolonged sedation. This dose range is widely used as an anesthetic adjunct, especially when regional anesthesia techniques and/or opioids have not been used. "Ultra-low" dose ketamine refers to the use of sub-analgesic doses (bolus <0.1 mg/kg IBW and/or infusion of <0.1 mg/kg IBW/h) and is almost always devoid of any neuropsychiatric effects. This is our recommended dose as a component of multimodal analgesia for laparoscopic bariatric surgery. [bib_ref] Effect of intraperitoneal local anesthesia on enhanced recovery outcomes after bariatric surgery:..., Jarrar [/bib_ref] While studies support the use of ketamine in patients with morbid obesity as an analgesic, pragmatically it is almost always described in conjunction with other modalities. [bib_ref] Impact of intraoperative ketamine on postoperative analgesic requirement following bariatric surgery: A..., Hung [/bib_ref] In our experience, this allows for significant opioid-sparing analgesia without using larger doses that are associated with unpredictable emergence phenomena.
## Dexmedetomidine
This novel short-acting intravenous α2-agonist is closely related to its predecessor, clonidine. Clonidine was also used as oral premedication in patients with morbid obesity and studied for the reduction of postoperative pain. The popularity and use of clonidine are limited by intraoperative bradycardia, hypotension, and postoperative sedation. These cannot be adjusted for, within the small clinically relevant dose range, especially when clonidine is given as a single oral dose before surgery.
Dexmedetomidine is a highly selective centrally acting intravenous α2-receptor agonist with fewer cardiovascular and CNS side effects than clonidine and was introduced as a "sedative without respiratory depression." [bib_ref] Benefits of dexmedetomidine on postoperative analgesia after bariatric surgery: A systematic review..., Tian [/bib_ref] This clinical profile of dexmedetomidine was thought to be uniquely beneficial for use in patients with morbid obesity. Though tempered by the initial higher cost considerations, the excellent safety of dexmedetomidine in patients with SDB and OSA has led to its current popularity and widespread use in bariatric anesthesia. Dexmedetomidine significantly reduces opioid consumption and PONV in patients undergoing bariatric surgery, with earlier discharge from the recovery room and the hospital. [bib_ref] Opioid-free total intravenous anaesthesia reduces postoperative nausea and vomiting in bariatric surgery..., Ziemann-Gimmel [/bib_ref] The administration of dexmedetomidine during laparoscopic surgery still requires Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022 careful consideration. In our center, we use the IBW, avoid giving a bolus at induction, and initiate the infusion at a fixed rate (0.5 mcg/kg IBW/h) only after the pneumoperitoneum has been established. These three measures significantly reduce the otherwise frequent and sometimes problematic bradycardia, which has the potential to progress rapidly to asystole and cardiac arrest. Additional benefits of intraoperative dexmedetomidine include reductions in emergence phenomena and shivering in the PACU.
## Lidocaine
This amide-type sodium channel blocker, when administered intravenously, is an anti-inflammatory and anti-hyperalgesic agent with additional opioid-sparing analgesic and anti-stress effects. Through direct and indirect effects on the gut, it promotes motility and prevents the development of postoperative ileus. [bib_ref] Intravenous lidocaine for acute pain: An evidence-based clinical update, Eipe [/bib_ref] In the general surgical population, lidocaine has shown significant improvements in postoperative pain management and outcomes, especially after abdominal surgery. [bib_ref] The use of intravenous lidocaine for postoperative pain and recovery: International consensus..., Foo [/bib_ref] The most commonly reported dose is 1-2 mg/kg IBW given as a bolus at induction of anesthesia and followed by an infusion of 1-2 mg/kg IBW/h continued till emergence. We have previously described the indications for the perioperative use of IV lidocaine and broadly divided it into two categories -as an alternative to regional anesthesia and in patients whose pain is expected to be difficult to treat. [bib_ref] Intravenous lidocaine for acute pain: An evidence-based clinical update, Eipe [/bib_ref] In bariatric anesthesia, if any other regional block is planned (see below), the use of intraoperative intravenous lidocaine is to be avoided as it can increase the incidence of local anesthetic systemic toxicity (LAST). If, however, no regional technique is planned, intraoperative lidocaine infusions can be used to decrease pain and postoperative opioid requirements. [bib_ref] Systemic lidocaine to improve quality of recovery after laparoscopic bariatric surgery: A..., Oliveira [/bib_ref] In our center, intravenous lidocaine is reserved only for revision bariatric surgery, previous intra-abdominal surgery, some patients with chronic pain, and those who have had previous prolonged ileus. In these situations, the intraoperative lidocaine infusion is stopped 20 to 40 min before the end of surgery to allow for wound infiltration or truncal blocks with other longer-acting local anesthetics (ropivacaine or bupivacaine). If a patient who has received a large volume local anesthetic block with these longer-acting agents develops an ileus and requires postoperative lidocaine, then lidocaine infusion can be started (without a bolus) 2 to 4 h after the block.
## Regional anesthesia
As mentioned previously, bariatric surgery is almost universally now performed laparoscopically. This has significantly diminished the role of neuraxial techniques. In patients with chronic pain or opioid use, those undergoing revision surgery with the possibility of conversion to open, there may be a role for epidural analgesia. [bib_ref] Roux-in-y gastrojejunal bypass: Which anesthetic technique has best results?, Ruzzon [/bib_ref] Akin to the evolution in laparoscopic colorectal pain management, intrathecal morphine may be useful in a small subset of bariatric surgical patients, especially when there are multiple or significant risk factors for poorly controlled acute pain or risk of conversion to open surgery. [bib_ref] A prospective observational study of high-dose intrathecal diamorphine in laparoscopic bariatric surgery:..., Wojcikiewicz [/bib_ref] Currently, as different regional techniques are being investigated, overall, it appears that truncal blocks may provide additional or extended analgesia when compared to simple wound infiltration. Our group continues to study novel techniques in bariatric anesthesia -for example, intraperitoneal local anesthetic (IPLA) instillation and surgical laparoscopic transversus abdominus plane (LapTAP) block. [bib_ref] Assessing the feasibility of a randomized, double-blinded, placebo-controlled trial to investigate the..., Wu [/bib_ref] [bib_ref] Effect of intraperitoneal local anesthesia on enhanced recovery outcomes after bariatric surgery:..., Jarrar [/bib_ref] Overall, regional techniques have significant opioid-sparing benefits, high safety, and efficacy with the additional advantage of a minimal patient-to-patient variability in terms of clinical efficacy. Further techniques and research are needed to establish these as the standard of care in bariatric anesthesia.
## Postoperative pain control
In postoperative pain, a scheduled step-wise, severity-based, opioid-sparing approach should be continued. Most patients recovering from bariatric anesthesia should be able to tolerate analgesics by the oral route immediately after surgery. With meticulous preoperative screening and testing, suspected or untreated OSA should be exceedingly rare.
In patients with diagnosed and treated OSA, resumption of continuous positive airway pressure (CPAP) therapy can be initiated, especially if they are experiencing pain that is requiring opioid analgesia. In keeping with this cautious approach, poorly controlled pain in patients should be identified early and treated appropriately with the same step-wise approach. Opioids can be used, but in smaller and more frequent doses that are carefully titrated within the monitored setting, as there can be an exaggerated and delayed CNS depression in some patients recovering from bariatric anesthesia. Additional increased sensitivity to sedative effects of premedication, residual anesthetics, and inadequate neuromuscular blockade reversal can significantly potentiate the risk of PRAE, respiratory failure, and the need for either non-invasive ventilation or re-intubation. If left undetected, this "deadly triad of obesity, opioids, and OSA" can contribute significantly to postoperative mortality where OIVI results in severe hypercarbia and hypoxia and presents as sudden cardiac death -also referred to as the "dead in bed" syndrome.
Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022
A bariatric surgical patient with an elevated serum bicarbonate level is at high risk for sedation and/or serious respiratory depression from opioids. We recommend avoiding or decreasing doses of opioids and increasing the frequency of (or continuous) monitoring. Early and prompt reversal of CNS depression with naloxone (40-80 mcg IV bolus repeated every 5 min, up to 400 mcg) should be part of all standard bariatric anesthesia postoperative protocols. While postoperative monitoring should be directed towards detecting hypoxia, pulse oximetry alone may not detect hypoventilation and brief apneic episodes, especially when supplemental oxygen is administered. Despite its drawbacks, respiratory rate monitoring should be considered after bariatric surgery -using continuous electrocardiogram (ECG) monitoring to measure respiratory rate may provide important information about patients at risk. More accurate postoperative respiratory monitoring can be achieved by measuring exhaled CO 2 (capnography) and newer monitors available have been adapted to nasal prongs and face masks. All these monitoring techniques will improve the safety of acute pain management in bariatric surgical patients.
If an IVPCA is required after bariatric surgery, a premixed solution containing opioids with ketamine can be considered. The combination of ketamine with morphine (1:1) can be prescribed for delivery via a standard IVPCA device. This combination has significant benefits -decreases opioid consumption, pain scores, and improves the quality of analgesia (mood and patient satisfaction) with reductions in PRAE, sedation-respiratory depression, and PONV. [bib_ref] Benefit and harm of adding ketamine to an opioid in a patient-controlled..., Assouline [/bib_ref] In the immediate and delayed postoperative period, continuing analgesic medications after bariatric surgery may require dose adjustments due to changes in pharmacokinetics due to altered gastric motility, volume and acid content, surface area for absorption, and first-pass metabolism. [bib_ref] Medication management and pharmacokinetic changes after bariatric surgery, Lorico [/bib_ref] Some patients will require follow-up for pain management after bariatric surgery as readmissions for poorly controlled pain have been reported. [bib_ref] Analysis of emergency department visits and unplanned readmission after bariatric surgery: An..., Makki [/bib_ref] Postoperative in-hospital opioid use following laparoscopic bariatric surgery may also predict opioid use after discharge. [bib_ref] Use of opioid analgesics before and after gastric bypass surgery in Sweden:..., Wallén [/bib_ref] Studies continue to evaluate the prediction and prevention of chronic post-surgical pain, persistent opioid use, and long-term pain outcomes after bariatric surgery [ [fig_ref] Figure 1: Preoperative preparation for improving postoperative pain management in bariatric surgery [/fig_ref] and [fig_ref] Table 1: Risk factors for poorly controlled acute postoperative pain, persistent postoperative pain, and... [/fig_ref] ]. The evidence thus far supports preoperative education and prehabilitation, implementation of ERAS programs, and the use of structured pain management protocols. [bib_ref] An enhanced recovery program for bariatric surgical patients significantly reduces perioperative opioid..., King [/bib_ref] [bib_ref] Outcomes of enhanced recovery after surgery protocols versus conventional management in patients..., Díaz-Vico [/bib_ref] [bib_ref] Preoperative patient education and patient preparedness are associated with less postoperative use..., Khorfan [/bib_ref] [bib_ref] Hospital opioid use predicts the need for discharge opioid prescriptions following laparoscopic..., Diaz [/bib_ref] [bib_ref] Chronic abdominal pain and persistent opioid use after bariatric surgery, Simoni [/bib_ref] [bib_ref] Risk factors for moderate to severe pain during the first 24 hours..., Iamaroon [/bib_ref] Therefore, perioperative pain-related interventions have benefits beyond early discharge and enhanced recovery. We believe that this focus on long-term reductions in pain and persistent opioid use will allow for these patients to achieve all the benefits of undergoing weight loss surgery.
# Conclusions
Laparoscopic bariatric surgery has become the standard of care for surgical management of obesity and morbid obesity. Standardization of perioperative pain management has benefitted from pragmatic and practical ERAS pathways that ensure excellent analgesia with perioperative opioids. Patients with morbid obesity who have OSA continue to be a challenge with regard to PRAE and require extended monitoring. Regional techniques continue to evolve and standardized integration of non-opioid adjuvants shows promising improvements. As rapid advances and further improvements in perioperative pain management and general care of the bariatric surgical population continue, it is hoped that other surgical patients with morbid obesity would benefit from being included in these processes, protocols, and programs.
## Financial support and sponsorship
Nil.
[fig] Figure 1: Preoperative preparation for improving postoperative pain management in bariatric surgery. OSA: Obstructive sleep apnea, SDB: Sleep-disordered breathing, PRAE: Postoperative respiratory adverse events, PEFR: Peak expiratory flow rate, 6MWT: 6-min walk test Saudi Journal of Anesthesia / Volume 16 / Issue 3 / July-September 2022 [/fig]
[table] Table 1: Risk factors for poorly controlled acute postoperative pain, persistent postoperative pain, and persistent opioid use identified in bariatric surgical patients [/table]
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Neurofibromatosis
# Introduction
In 1882 the German pathologist Friedrich Daniel von Recklinghausen for the first time described a series of patients with a combination of cutaneous lesions and tumors of the peripheral and central nervous system. Only in the 20th century neurofibromatosis type 1 (NF1), namely Recklinghausen's disease, and neurofibromatosis type 2 (NF2), previously referred to as central neurofibromatosis, were distinguished from each other as two different autosomal dominantly inherited genetic disorders with common features [1-3]. Briefly, NF1 exposes a characteristical cutaneous phenotype including benign neurofibromas, which are mixed tumors composed of all cell types found in the peripheral nerves, hyperpigmented macules, termed café-au-lait macules, the so called axillary/inguinal freckling, as well as pigmented hamartomas of the iris, called Lisch nodules. NF2 on the other hand is mainly restricted to tumors of the central and peripheral nervous system, which are only seldomly accompanied by cutaneous disorders [4].
## Nf1 clinical features
NF1 is considered as one of the most common genetic disorders in human with an incidence of 1/3500 individuals. In 1997, Gutmann and colleagues updated the diagnostic criteria for NF1 and NF2 [bib_ref] The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2, Gutmann [/bib_ref]. Usually cutaneous manifestations are the first symptoms observed in NF1 patients [bib_ref] Neurofibromatosis type 1, North [/bib_ref]. Café-au-lait macules (CALMs) mainly develop in childhood and are found in almost all patients. CALMs present as light brown macules of about 10 to 40 mm in diameter with an ovoid shape and poorly circumscribed borders [fig_ref] Figure 1: A 9-year old boy with NF 1 and multiple [/fig_ref]. Whereas the presence of ≥ 6 CALMs is defined as strong diagnostic criterion for NF1, additional features are mandatory for a definite diagnosis. A common feature is a characteristic axillary and/or inguinal freckling, which usually develops subsequently to CALMs and which is observed in 90% of all patients [fig_ref] Figure 2: Characteristic axillary freckling of a 51-year old woman with NF 1 [/fig_ref] [bib_ref] A child with axillary freckling and cafe au lait spots, Wainer [/bib_ref]. The development of neurofibromas around or on peripheral nerves is a distinct symptom of NF 1 but is observed to a lesser extent also in NF2 patients . Neurofibromas occur as either encapsulated dermal and subcutaneous tumors or as plexiform neurofibromas . Dermal and subcutaneous neurofibromas may cause little or no clinical symptoms but can be very disfiguring. Plexiform neurofibromas on the other hand, which are often congenital and can develop near nerve roots deep within the body, bear a 10 % probability of malignant transformation. In cases of transformation the arising malignant peripheral nerve sheath tumors (MPNSTs) have been shown to have a high metastatic potential [2, [bib_ref] Images in clinical medicine. Von Recklinghausen's disease and breast cancer, Posada [/bib_ref] [bib_ref] Constitutional NF1 mutations in neurofibromatosis 1 patients with malignant peripheral nerve sheath..., Kluwe [/bib_ref] [bib_ref] Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve..., Frahm [/bib_ref] [bib_ref] Histopathology and clinical outcome of NF1-associated vs. sporadic malignant peripheral nerve sheath..., Hagel [/bib_ref]. Additional complications of plexiform neurofibromas may manifest as diffuse appearance and/or a tendency to expand along large segments of affected nerves, causing disfigurement and nerve dysfunction. Finally pigmented hamartomas of the iris, so called Lisch nodules, have to be mentioned as a characteristic ophthalmologic feature of NF1 [bib_ref] Constitutional NF1 mutations in neurofibromatosis 1 patients with malignant peripheral nerve sheath..., Kluwe [/bib_ref].
## Nf2 clinical features
Whereas the clinical features of NF2 were initially described in the late 1800 s , NF2 was first considered as a subtype of NF1. It took almost a hundred years for NF2 to be recognized as a self-contained entity. With an incidence of 1/25.000 it occurs much less frequently than NF1 [1]. Often the first clinical sign of NF2 is a sudden loss of hearing due to the development of bi-or unilateral vestibular schwannomas [fig_ref] Figure 4: A 63-year old man with a known history of NF 2 presented... [/fig_ref] [4]. These tumors occur on or around the vestibular branches of both auditory nerves. Unlike in NF1 patients, tumors in NF2 patients are uniformly benign. Nevertheless, these tumors can compress associated nerves and can cause considerable pain, nerve dysfunction and intracranial pressure. Further symptoms include a sudden onset of tinnitus or imbalance [bib_ref] Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve..., Frahm [/bib_ref]. In addition, patients suffering from NF2 tend to develop further nervous tissue tumors such as meningiomas or gliomas. Dermal manifestations of schwannomas in NF2 patients are rare and can easily be confused with cutaneous neurofibromas [bib_ref] Histopathology and clinical outcome of NF1-associated vs. sporadic malignant peripheral nerve sheath..., Hagel [/bib_ref]. In these cases, histopathological analysis is needed to reveal that these tumors consists of Schwann cells only. According to the 1991 NIH consensus criteria, the diagnosis of NF2 requires either the presence of bilateral vestibular schwannomas or a positive family history and an unilateral vestibular schwannoma in combination with either a menigioma, a glioma, a neurofibroma, a schwannoma, or posterior subcapsular opacities [bib_ref] Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve..., Frahm [/bib_ref].
## Pathogenesis of nf1 and nf2
Inactivating mutations in the NF 1 and NF 2 genes, both, inherited and/or new germline mutations, are responsible for the development of the phenotypes of the diseases. The NF1 gene, located on 17q11.2, encodes for a protein also known as neurofibromin. Neurofibromin functions as a tumor suppressor by negatively regulating mitogenic Ras signalling through a GTPase activating protein (GAP), which is essential for NF 1-associated tumorigenesis. Particularly in neurocutaneous tissues, loss-of-function mutations result in increased Ras activity, causing increased proliferation and tumorigenesis [1, [bib_ref] Neurofibromatosis type 1, North [/bib_ref] [bib_ref] Neurofibromatosis type I: genetics and clinical manifestations, Savar [/bib_ref] [bib_ref] Audiologic and radiographic response of NF2-related vestibular schwannoma to erlotinib therapy, Plotkin [/bib_ref] [bib_ref] Plexiform schwannoma: a clinicopathologic overview with emphasis on the head and neck..., Berg [/bib_ref]. The NF2 gene is located on 22q12.2 and encodes for merlin (schwannomin). The gene is much smaller than neurofibromin, which explains the much lower mutation rate of NF2 as compared to NF1. Merlin is considered to act as a regulator of growth, motility, and cellular remodeling by inhibiting the transduction of extracellular mitogenic signals such as CD44-mediated contact-dependent inhibition of proliferation. Correspondingly, Merlin has been shown to prevent epidermal growth factor receptor (EGFR) signalling, known to induce cellular proliferation [1, [bib_ref] Neurofibromatosis types 1 and 2, Yohay [/bib_ref] [bib_ref] NF2: the wizardry of merlin, Xiao [/bib_ref] [bib_ref] The merlin interacting proteins reveal multiple targets for NF2 therapy, Scoles [/bib_ref] [bib_ref] Contact-dependent inhibition of EGFR signaling by Nf2/Merlin, Curto [/bib_ref].
## Treatment and management
Due to the variety and complexities of the manifestations of NF 1 and NF 2, an interdisciplinary approach is inevitable. Biannual screening examinations in childhood and then yearly thereafter are strongly recom-mended. Examinations should include the measurement of the head circumference as well as regular checks of blood pressure due to the risk for pheochromocytoma and renal abnormalities [bib_ref] NF2: the wizardry of merlin, Xiao [/bib_ref]. In childhood, behavioural and developmental parameters should be evaluated carefully for signs of learning disability and attention deficit hyperactivity disorder (ADHD) [bib_ref] Neurofibromatosis type 1, North [/bib_ref] [bib_ref] The merlin interacting proteins reveal multiple targets for NF2 therapy, Scoles [/bib_ref]. In general, patients with NF 1 and NF 2 should undergo yearly neurologic and ophthalmologic examinations [bib_ref] The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2, Gutmann [/bib_ref]. Treatment of severe and disfiguring tumors is usually performed by surgery. Notably, in 2008 Lantieri and colleagues reported the first face transplantation with a composite tissue allograft in a 29-year-old man with NF1 suffering from a massive, disfiguring plexiform neurofibroma diffusely infiltrating the middle and lower part of his face [bib_ref] Contact-dependent inhibition of EGFR signaling by Nf2/Merlin, Curto [/bib_ref]. Although benign, the complete excision of tumors often remains a therapeutical endeavour due to their close association to nerves. It has to be noted that tumor-excision bears a high risk of recurrence [bib_ref] Neurofibromatosis type I: genetics and clinical manifestations, Savar [/bib_ref]. Regarding NF2, the total surgical resection of vestibular schwannomas is a suitable therapeutic option and may result in definite tumor control. However, due to the frequently multilobulating and infiltrating character of the tumors, consecutive damage to the cochlear nerve or facial nerves is associated with a high risk of permanent hearing loss and other malfunctions [bib_ref] Multiple gastrointestinal stromal tumors and bilateral pheochromocytoma in neurofibromatosis, Kramer [/bib_ref]. Interestingly, a recent study by Phi and colleagues demonstrated a good 5-year tumor control and preserved serviceable hearing in approximately one-third of 30 NF2 patients with vestibular schwannomas treated with gamma knife radiosurgery [bib_ref] Behavioral phenotype of neurofibromatosis, type 1, Kayl [/bib_ref]. Comparable studies report local control rates of 85% and 81% after 5 or 10 years, respectively, with hearing preservations of about 40%. In patients with a serviceable hearing before therapy the estimated risk of deafness is about 20% [bib_ref] Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve..., Frahm [/bib_ref]. The authors promote radiosurgery as an effective and less invasive treatment option compared to standard surgical techniques [bib_ref] Behavioral phenotype of neurofibromatosis, type 1, Kayl [/bib_ref]. Another notable therapeutic approach was reported by Plotkin and colleagues in 2008. The authors demonstrated the first successful laboratory-to-clinic translation of research by treating a 48-year-old NF2 patient with the EGFR-inhibitor erlotinib. Application of this novel targeted cancer drug caused a significant decrease in tumor volume and a re-establishment of useful hearing [bib_ref] Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve..., Frahm [/bib_ref]. Taken together, this variety of novel and evidence-based therapeutic options raises new hope for affected patients.
[fig] Figure 1: A 9-year old boy with NF 1 and multiple (n ≥ 6) caféau-lait macules. [/fig]
[fig] Figure 2: Characteristic axillary freckling of a 51-year old woman with NF 1. [/fig]
[fig] Figure 3a: Multiple neurofibromas with a maximum diameter of 4 cm on the back and gluteal region of a 46-year old male with NF1. 3b: A 6 cm in diameter measuring, disabling tumor in the gluteal region of a 51-year old woman with NF1. [/fig]
[fig] Figure 4: A 63-year old man with a known history of NF 2 presented with a progredient right-sided tinnitus and mild deafness. Magnetic resonance imaging showed a schwannoma of the vestibular portion (VS) of the right VIIIth cranial nerve. [/fig]
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Regulation of the expression of proinflammatory cytokines induced by SARS-CoV-2
An outbreak of a novel coronavirus was reported in Wuhan, China, in late 2019. It has spread rapidly through China and many other countries, causing a global pandemic. Since February 2020, over 28 countries/regions have reported confirmed cases. Individuals with the infection known as coronavirus disease-19 (COVID-19) have similar clinical features as severe acute respiratory syndrome first encountered 17 years ago, with fever, cough, and upper airway congestion, along with high production of proinflammatory cytokines (PICs), which form a cytokine storm. PICs induced by COVID-19 include interleukin (IL)-6, IL-17, and monocyte chemoattractant protein-1. The production of cytokines is regulated by activated nuclear factor-kB and involves downstream pathways such as Janus kinase/signal transducers and activators transcription. Protein expression is also regulated by post-translational modification of chromosomal markers. Lysine residues in the peptide tails stretching out from the core of histones bind the sequence upstream of the coding portion of genomic DNA. Covalent modification, particularly methylation, activates or represses gene transcription. PICs have been reported to be induced by histone modification and stimulate exudation of hyaluronic acid, which is implicated in the occurrence of COVID-19. These findings indicate the impact of the expression of PICs on the pathogenesis and therapeutic targeting of COVID-19.
# Introduction
In December 2019, an infection manifesting as atypical pneumonia was discovered in Wuhan, China , and its rapid spread has been noted in China and many other countries . So far, the 2019 novel coronavirus (2019-nCoV) has affected > 20 countries and has become a major global health problem. As of February 2020, approximately 51000 cases of the disease known as coronavirus disease 19 (COVID-19) have been documented globally, resulting in at least 1600 deaths since it first appeared in Central China's Hubei Province .
The pathogen, initially called 2019-nCoV is a beta coronavirus, and was later named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It forms a clade among the subgenus Sarbecovirus . Serious human infections have been caused by Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV, and SARS-CoV-2 is one of the seven members in the family of coronaviruses capable of infecting humans . On confirming its origin from the bat coronavirus (BatCoV RaTG13), SARS-CoV-2 has been assigned to the subgenus Sarbecovirus, of the genus Betacoronavirus . Evidence suggests that it is related to the bat and pangolin coronaviruses, as well as SARS-CoV, the SARS pathogen .
Clinically COVID-19 is characterized by fever, severe respiratory illness, and pneumonia similar to those produced by SARS-CoV, which led to an outbreak in Southern China in 2003 . The two biologically related coronaviruses contribute to a clinical picture attributed to the high production of proinflammatory cytokines (PICs), due to the interactions of the viral and host proteins when the virus enters the cell . An acute respiratory illness and increased PICs have been described in COVID-19 patients; the cytokines include interleukin (IL)-2, IL-6, IL-10, granulocyte colonystimulating factor (G-CSF), interferon (IFN)-g-induced protein-10 (IP-10), monocyte chemoattractant protein factor-1 (MCP-1), macrophage inflammatory protein 1 (MIP-1) and tumor necrosis factor-αα) .
Like most proteins, the expression of PICs is triggered by activation of transcription factors and post-translational modification (PTM) of the factors and histones, the basic units of the nucleosome where genomic DNA is housed. The expression of PICs is regulated by the association of transcription factors and chromatin markers in the promoter and enhancer sequences upstream of the coding portion of genes. The activation or repression of these elements is modulated by PTM. In this review, we discuss the impact of histone and nonhistone transcription factor PTM in the increased production of PICs.
## Impact of the nonhistone transcription factors nuclear factor kappa-b, signal transducers and activators, and janus kinases on pic synthesis
Similar to the pathogenesis of SARS and MERS, higher levels of cytokines in plasma, including IL-2, IL-6, IL-7, IL-10, G-CSF, IFN-gα . The production of PICs is induced by SARS-CoV-2, which forms a cytokine storm, leading to a fatal syndrome involving multiple organ dysfunction . PICs induced during COVID-19 originate from inflammatory cells that constitute the innate immune defense, including monocytes, macrophages, neutrophils, and pulmonary and renal epithelial cells.
The major PICs induced by the novel coronavirus include IL-1β, IL-6 and MCP-1; they promote the pathogenesis of COVID-19 by triggering an inflammatory response . Among these, elevated IL-6 levels are closely correlated with disease progression . The production of these factors involves a complicated regulatory network of transcriptional activation by transcription factors, PTM of histones, and nonhistone transcription factors. IL-6 production depends on activated transcription factors, in particular, nuclear factor kappa-B (NF-kB) and the aggregated IL-6 receptor when ligated by IL-6 stimulates a number of downstream intracellular signaling pathways, the most common being Janus kinase (JAK)/signal transducers and activators transcription (STATs). Cytokine-coding gene promoter occupancy with chromatin markers has also been identified .
After being ligated by cytokines released by inflammatory cells, activated cognate receptors stimulate downstream signaling pathways, mediated by JAK . IL-6, a PIC that has been shown to act as a critical part of the pathogenic process of COVID-19, activates the JAK/STAT signaling pathway to exert a variety of biological effects, including immune regulation, proliferation, and differentiation of lymphocytes and oxidative stress .
The JAK/STAT pathway that transduces growth signals transmitted by surface receptors has also been shown to contribute significantly to the pathogenesis of COVID-19. To date, four proteins of the JAK non-receptor family have been identified in mammalian cells; namely, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). JAK1/2 and TYK2 are expressed in different cell types, while JAK3 appears to play an essential role in the function of lymphocytes. The human STAT family comprises seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 .
The novel coronavirus-encoded spike (S) protein is expressed on the viral surface. Virus entry to host cells is mediated by S protein, which interacts with angiotensinconverting enzyme 2 (ACE2) that acts as the viral receptor.
ACE2 is an enzyme that cleaves angiotensin II into angiotensin 1-7, which possesses vasodilatory and anti-inflammatory effects. In patients with cardiac disease, upregulation of ACE2 may increase susceptibility to COVID-19 .
ACE inhibitors or angiotensin receptor blockers may enhance immune cell infiltration, and they also increase local production of PICs such as TNF-α, IL-1, IL-6, and IFN-g .
S protein has been shown to activate NF-kB like some other viral proteins . Latent membrane protein 1(LMP1), encoded by a human lymphotropic herpesvirus, Epstein-Barr virus, is a viral homolog of mammalian CD40 and activates NF-kB behaving like an aggregated TNF-α . The molecular interaction involving activation of NF-kB by S protein remains to be characterized.
In patients with severe COVID-19, elevated IL-6 has been frequently detected in the serum and correlated with nonsurvival . Consequently, clinical trials have been initiated to evaluate the therapeutic effects of IL-6 inhibitors in COVID-19 patients . The JAK/STAT pathway has also been explored for use as a new therapeutic target against SARS-CoV-2 infection . Available data provide a rationale for administering licensed JAK inhibitors for improving the clinical management of COVID-19 and meeting the urgent global demand to mitigate the infection.
Both constitutively active and noncanonical STAT functions are involved in the molecular processes of tumorigenesis and viral pathogenesis. STAT3 and STAT5 have been investigated due to their activities in controlling proliferation, survival, cellular adhesion, intercellular communication, and angiogenesis when triggered by a wide range of stimuli .
The activity of STAT3 is also modified by methylation at Lys 49 and Lys 180 by the enhancer of Zester homolog 2 (EZH2), a component with lysine methyltransferase activity in the polycomb repressive complex 2 (PRC2), which regulates gene transcription. EZH2 methylates STAT3 downstream of AKT signaling and increases the transcriptional activity of STAT3, which has been shown to be activated by modification with methylation . We discuss the role of histone modification by the methylation involved in the control of gene expression in the next section.
## Impacts of histone modification on the production of pics
The activation and repression of gene transcription are regulated by chromatin modification. The basic units of the chromosome are the nucleosome, which is composed of a nucleus of histone octamer (H), heteromerized with four dimers of H1, H2A, H3 and H4, and the octamer is wrapped with strands of genomic DNA . Extended peptide tails contain a series of modifiable amino acid residues called chromatin marks. The main modification pattern for H3 is methylation, and for H4 it is acetylation, of lysine residues. They are important in the activation or repression of genes . The methylation of lysine residues in histones, mainly H3, is catalyzed by a group of enzymes called histone lysine methyltransferases (KMTs). The protein clusters are driven by the Suv39H1, G9a and SETDB1/SETDB2 families of enzymes known as lysine methyltransferase 1 (KMT1), and they function to catalyze mono-, bi-and trimethylation of lysine residues 4 and 9 (H3K4 and H3K9) .
Much remains to be elucidated with regard to the role of epigenetic modification of histones played by different modifiers in the regulation of inflammatory responses.
As mentioned above, methylation at various lysine residues on histone H3 leads to the activation or repression of gene transcription. While methylation at H3K9 or H3K27 represses gene expression, methylation at H3K4, H3K36 or H3K79 activates gene expression. The disruptor of telomeric silencing-1-like is a modifier that specifically catalyzes methylation of H3K79 to regulate proliferation and differentiation of tumor cells.
## Reading of chromatin markers
Chromosomal marks, for example, H3K4 and H3K9, with different modifications bind to the target sequence, in particular to regulatory elements such as promoters or enhancers, to modulate gene expression. The marks are recognized and incorporated into the target sequence in a process called reading . Chromatin marks are modified by adding chemical groups with methylation, acetylation and SUMOylation, and ubiquitination, called writing. The modification can alter the ability of the marks to regulate gene expression. The modification is reversible, and the added groups are removed in a process called erasing. Chromatin marks such as H3K4 bind to the promoter upstream of the IL-6 coding sequence and methylated H3K4 is demethylated by a demethylase, lysine-specific demethylase 1A/lysine-specific demethylase 1 .
The methyltransferase catalyzes adding different numbers of methyl groups to lysine, termed mono-, bi-and trimethylation. As mentioned above, six lysine residues on H3, i.e., K4, K9, K27, K36, K79 and K36, together with K20 from histone H4 and K26 from histone 1 have been recognized as the canonical sites of methylation.
Except for H3K79, all of these are found in the N-terminal tails of histone proteins. It has been identified that some proteins or the domain contained within the molecules serve as readers of methyllysine. Ankyrin, double chromodomain, tudor tandem domain, tudor and zinc finger, as well as the known motifs found in transcription regulators, malignant brain tumor, plant homeodomain and Pro-Trp-Trp-Pro.
The initiation of viral genome transcription, virion production, and spread of human immunodeficiency virus (HIV)-1 is triggered when host cell replication is stimulated by cytokines, drugs, or activation of T cell receptors.
The reactivation of provirus depends on the binding of NF-kB and/or nuclear factor of activated T cells with the specific target sequences on the viral long terminal repeat (LTR). The factors which initiate transcription of viral genome act by recruiting histone acetyltransferase p300, CBP-associated factor to the LTR of HIV; the modifying enzymes acetylate histones adjacent to the viral promoters .
## Enzymes that catalyze histone methylation and demethylation and their effect on target gene expression
The acetylation modifications on histones H3 and H4, like H3 lysine 4 acetylation (H3K4ac) and H3 lysine 27 acetylation (H3K27ac) create an open chromatin configuration, favoring binding of transcription factors and forming complexes of initiation and elongation. Trimethylation of H3 lysine 4, H3K4me3 and lysine H3 K27, H3K27me3 bind to gene promoters, activating and repressing gene transcription, respectively. Dimethylated H3K4 (H3K4me2) and di-and trimethylated H3K36 March 6, 2021
Volume 9 Issue 7 The scheme of modification of peptide tails of histones H3 and H4. "M" and "A" denote modification by methylation and acetylation, respectively; the letter "K" on the H3 tail presents activities of activation (blue; K4 and K36), and repression (red; K9 and K27). "C" and "N" represent carboxyl terminus and amino terminus, respectively.
(H3K36 me2, me3) have been identified on chromatin sites being transcriptionally activated.
Modification of methylation is catalyzed by a panel of enzymes to add the chemical group to the lysine residue, and some of them are listed in.
Members of the KMT3 family, SET and MYND domain containing (SMYD)2 and SMYD3, function to catalyze methylation at the H3K4 or H3K36 marks, to activate gene transcription. SMYD2 has also been shown to regulate latency of HIV-1, and is associated with latent HIV-1 promoter in H4K20me1-enriched chromatin-harboring DNA, H4K20me1 enrichment is known as a mark lost in cells defected with SMYD2. A reader protein recognizing H4K20me1 Lethal 3 malignant brain tumor 1 (L3MBTL1), is recruited to the HIV latent promoter. The data suggest that an axis of SMYD2-H4K20me1-L3MBTL1 is implicated in the regulation of HIV-1 latency and may be targeted by small molecule inhibitors of SMYD2 .
SMYD3, another member of the KMT3 family has been reported to be recruited to the inclusion bodies of Ebolavirus (EBOV) by interacting with the virally encoded nucleoprotein. A significant suppression of EBOV mRNA synthesis has been noted on deletion of SMYD3. A nonphosphorylated VP30 mimic, which is a transcription activator, can partially rescue viral mRNA production. Whether methyltransferase activity plays a role in this process remains to be determined, and nucleoprotein encoded by SARS-CoV-2 also interacts with SMYD3 .
Methyltransferase SET7 generates H3K4me by monomethylation of H3K4. H3K4me represents a tag specific for epigenetic gene activation. It transcriptionally activates genes like collagenase or insulin on recruitment to the promoter sequence of the target genes. SET7 also methylates nonhistone proteins p53/TP53 and Transcription initiation factor TFIID subunit 10 (TAF10). When monomethylated on Lys/K-189, TAF10 has an enhanced affinity for RNA polymerase. Lys/K-372 monomethylation of p53/TP53 stabilizes p53/TP53 and increases p53/TP53-mediated transcriptional activation. The relationship between methylation and demethylation and coronavirus infection has not been investigated. Human papillomavirus E6 has been reported to attenuate the transactivation function of p53 by decreasing the enzymatic activities of SET7 . SET7 was found to promote HIV transcription through monomethylated Tat protein . Recent studies have demonstrated that the antiviral function of IFN-induced transmembrane protein 3 for vesicular stomatitis virus and influenza A virus could be attenuated by SET7. Han et al observed that SET7 expression in Huh7.5.1 cells was upregulated by hepatitis C virus (HCV) infection, and high levels of SET7 expression were also found in serum, peripheral blood mononuclear cells, and liver tissue of HCV patients compared with healthy individuals. Subsequent research showed that SET7 enhanced HCV replication in an enzyme-dependent manner. Furthermore, the data showed that SET7 decreased the expression of virus-induced IFN and IFN-related effectors.
As mentioned above, the production of proinflammatory cytokines is regulated by histone modification, and the modification also affects virus-host interaction. The contribution of KMT histones to maintaining HIV latency remains to be elucidated . EZH2/KMT6, which specifically methylates lysine residues H3K9 and H3K27, as repressive markers for gene transcription, is abundant on the LTR of the silenced proviruses of HIV, as demonstrated by chromatin immunoprecipitation in latently infected Jurkat T cells.
In proviral reactivation, it was rapidly displaced. The latent HIV-1 proviruses carried by methylated lysine residues on H3 are mainly trimethylated repressive genomic markers H3k9me3 and H3K27me3 or H3K9me2 . A histone KMT, SUV39H1, responsible for generation of H3K9me3, interacts with COUP-Transcription Factor-interacting protein 2 (CTIP-2) and Heterochromatin protein 1 homolog gamma (HP1γ) to maintain HIV-1 latency in microglial cells . Removal of the two proteins contributes to HIV-1 activation. It has been proposed that HKMT G9a, which catalyzes the dimethylation of H3K9, may also contribute to the maintenance of HIV-1 latency.
## Role of cytokines in pathogenesis as related to production of hyaluronic acid
The lungs of some patients with COVID-19 are filled with a clear liquid with jelly-like appearance. One of the components identified in this fluid is hyaluronic acid (HA), a polysaccharide seen in most connective tissues. In water, HA can trap its weight up to 1000 times. It is present as a high-molecular-weight polymer in airway epithelial cells covering the apical surface. Depolymerization of HA initiates a cascade leading to the production of quinine and processing of growth factors. HA synthesis and degradation are mediated by integral membrane proteins HA synthase (HAS), including HAS1, HAS2 and HAS3, and hyaluronidases (HAases), respectively. HYAL1, HYAL2 and PH20 are among six HAases that have been well characterized. HYAL1-like HAase was initially isolated in specimens of human urine and plasma. HYAL1, alternatively termed Luca1 (lung cancer 1) is known as the main tumor-derived HAase expressed in bladder and prostate cancer tissues. HYAL2 was originally identified in lysosomes, and it cleaves high-molecular-mass HA with release of a 20-kDa fragment. HYAL3 is the third HAase, whose coding gene is mapped on the chromosomal region 3p21.3. Its transcripts have been detected in different tissues, and the sequence of the HYAL3-encoded product has only been predicted.
An enzyme that catalyzes the degradation of the jelly-like fluid HA, HYAL1, is not expressed in the bronchial alveolar lavage fluid (BALF) of COVID-19 patients and control individuals. This suggests that the amount of HA in the bronchoalveolar space of the lungs would be induced by the high PICs production during COVID-19 pathogenesis. In combination with the induced increase in vascular hyperpermeability due to the elevated PICs, a viscous hydrogel is generated to frustrate gas exchange . The genesis of acute respiratory distress syndrome (ARDS) is associated with HA in BALF; the concentration of HA is significantly negatively correlated with the index of pulmonary oxygenation. HA is also responsible for pulmonary thrombosis, and ground-glass opacities; a characteristic finding of interstitial pneumonia on radiography.
In certain cell types, the effects of TNF-α -3 is present. Their expression and activity are augmented by IL-1 in synergy with TNF-α, HYALs are localized intracellularly, while HYAL2 is expressed in plasma-membrane-associated apical poles and in apical secretions in soluble form.
Also, increased HYAL expression and activity are noted in tracheal secretions and BALF derived from individuals with asthma in comparison with healthy controls with normal lungs. HYALs 1-3 expressed by airway epithelium coordinate to depolymerize HA during inflammation mediated by elevated TNF-α and IL-1 .
Symptoms in patients with ARDS include rapid and exhaustive respiration and cyanosis. Severe cases of COVID-19 admitted to intensive care units normally require mechanical ventilation, and patients who are unable to breathe can be maintained with extracorporeal membrane oxygenation.
COVID-19 is characteristic on computed tomography images, with white spots known as ground glass opacities, with fluid in the lungs. Autopsy confirms that the lungs are filled with clear liquid gelatin, similar to the lungs after drowning. While further validation is needed, an association between HA and ARDS has been suggested, and the production and regulation of HA have been noted in SARS.
In the lungs of patients with COVID-19, the levels of PICs, IL-1 and TNF-α are elevated and they strongly induce HAS2 in epithelial-cell-adhesion-molecule-positive pulmonary alveolar epithelial cells and fibroblasts. HA can absorb water up to 1000 times its molecular weight. Therefore, inhibition of HA production would greatly contribute to improvement of breathing in COVID-19 patients.
## Implication of ptm on drug development
At present, specific therapy for COVID-19 has not been available, and the drugs administered mainly target viral infection and inflammation triggered by viral invasion, accompanied by supportive therapy. The HIV protease inhibitors ritonavir and lopinavir have been used in anti-COVID-19 treatment . High production of PICs such as IL-6 and helper T cytokine IL-17, is triggered, and the JAK/STAT signaling pathway is activated. The United States FDA and the European Medical Association have approved the use of JAK inhibitors, e.g. ruxolitiniband baricitinib, for therapy of COVID-19.
More candidate JAK inhibitors are being tested in clinical trials. A JAK1/2 inhibitor, ABT-494, has the potential to inhibit the regulation of endocytosis and may possess the potential to prevent entry to susceptible cells by SARS-CoV-2. In addition, its interaction with cytochrome enzymes is minimal and it has low serum binding. ABT-494 is active in combination with other drugs such as remdesivir in COVID-19 therapy .
IL-6 is a cytokine implicated in the cytokine release syndrome (CRS) seen in COVID-19. In its presence, immature T helper cells (Th0) differentiate into the Th17 subtype, and JAK2 is utilized by IL-6 to activate downstream signals.
As a characteristic disease of CRS, markedly high numbers of Th17 cells have been identified in COVID-19 patients, suggesting that CRS of Th17 type is involved in severe immune damage. A highly selective JAK2 inhibitor, fedratinib, approved for myelofibrosis therapy, has been shown to inhibit expression of IL-17 in murine Th17 cells . The data suggest that inhibition of JAK could be a therapeutic target against COVID-19 through an antagonistic effect on the activation of Th17-associated cytokines.
# Conclusion
In summary, SARS-CoV-2 enters host cells via binding of viral surface proteins to ACE-2 as a receptor. Viral entry stimulates PIC production through a complicated network involving intracellular signaling pathways and histone PTMs. Therapeutic agents targeting the various stages of the virus-host interaction could be developed. |
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