text
stringlengths 66
377k
|
|---|
Don’t judge toxic weeds on whether they are native but on their ecological effects
The sharp rise in anthropogenic activities and climate change has caused the extensive degradation of grasslands worldwide, jeopardizing ecosystem function, and threatening human well-being. Toxic weeds have been constantly spreading in recent decades; indeed, their occurrence is considered to provide an early sign of land degeneration. Policymakers and scientific researchers often focus on the negative effects of toxic weeds, such as how they inhibit forage growth, kill livestock, and cause economic losses. However, toxic weeds can have several potentially positive ecological impacts on grasslands, such as promoting soil and water conservation, improving nutrient cycling and biodiversity conservation, and protecting pastures from excessive damage by livestock. We reviewed the literature to detail the adaptive mechanisms underlying toxic weeds and to provide new insight into their roles in degraded grassland ecosystems. The findings highlight that the establishment of toxic weeds may provide a self-protective strategy of degenerated pastures that do not require special interventions. Consequently, policymakers, managers, and other personnel responsible for managing grasslands need to take appropriate actions to assess the long-term trade-offs between the development of animal husbandry and the maintenance of ecological services provided by grasslands.K E Y W O R D Sadaptive strategy, degraded grassland, ecological function, grassland management, toxic weed | 9015 ZHANG et Al.
## | fore word
Toxic weeds refer to plants of secondary compounds which are toxic to livestock, wild herbivores, and human. Some toxic weeds accumulate toxins at high levels whose concentration can be influence by the inhabiting conditions. The toxic principles mainly include toxic proteins, terpenoids, glycosides, alkaloids, polyphenols, and photosensitive substances , which can be extracted and used as pesticides with remarkable pesticidal and antimicrobial activities. As an indicator of grassland health, toxic weeds have become increasingly global in their distribution in recent decades indicating that the widespread land degradation is a serious issue that threatens the sustainable developmental goal of "no poverty, zero hunger" of the Food and Agriculture Organization of the United Nations.
Furthermore, a longer growing season and warming induced by climate change will intensify the increases in the occurrence and production of toxic weeds.
There are approximately 1,300 toxic species in over 140 families covering approximately 33.3 million hm 2 in China's natural grasslands . They have been traditionally thought that the wide distribution of toxic weeds leads to pasture degeneration and thereby reductions of grassland forage availability. Additionally, poisonous weeds not only damage livestock breedingbut also poison-or even killdomestic animals if they are ingested by accident or if the pollen is inadvertently inhaled , potentially resulting in substantial economic losses and hindering the sustainable development of the livestock industry. About 300 of the 1,300 species of poisonous plants found in China exhibited negative effects on livestock. It is estimated that toxic weed poisoning results in direct or indirect economic losses of billions of CNY in China each year. The reduced grazing capacity and economic losses induced by toxic weed lead to lower resilience and increase in vulnerability of livelihoods that depend on livestock. Therefore, numerous approaches have been employed to control the spread of toxic weeds.
However, most techniques have done little to eradicate established plants, and some approaches may even have negative environmental effects.
In fact, the spread of toxic weeds is not the reason for grassland degradation but a consequence of their strong adaptive capacity.
Toxic weeds often have long and well-developed root systems to facilitate the capture of water and nutrients from deep soil profiles, inhibit the growth of co-occurring plants via allelopathy, form intraspecific aggregations that enhance their ability to compete with heterospecific competitors, and are not exposed to selection by livestock and small rodents . From an ecological perspective, the colonization of toxic weeds might be more beneficial than harmful by promoting the process of succession in degraded grasslands by excluding excessive disturbance from livestock . An improved understanding of the potential role of toxic weeds in grassland conservation will challenge the traditional view that toxic weeds are uniformly deleterious and will enable pasture managers and policymakers to modify and design more flexible strategies for addressing global change and promoting sustainability. Here, we conduct a review of the literature to detail the fitness and potential effects of toxic weeds. These findings provide novel insight into the adaptive management of weed-dominated grasslands.
## | adap tati on s of toxic weeds
In addition to the effects of natural factors, such as soil physiochemical properties and topographical conditions, toxic weeds are most commonly a product of overgrazing and grassland degeneration. Previous studies have revealed that the population gradually increases and becomes dominant in plant communities as grassland degradation and grazing intensity increase. This pattern is mostly due to that toxic weed has various strategies including higher genetic variation, well-developed roots, allelopathy effect, and poisonous for herbivores adapting to environmental stress and anthropogenic disturbance.
## | adaptive strategies to the environment
A large number of toxic weeds are long-lived perennial species with self-incompatible mating systems and therefore generally have high genetic variation, which facilitates adaptive evolution to various environmental conditions and contributes to their wide geographic distribution. For example, Stellera chamaejasme inhabits a wide range of altitudes from 130 to 4,200 m, including a broad area from southern Russia to southwest China and the western Himalayas, which is suggestive of high adaptability . The various morphological and physiological traits of toxic weeds promote increases in the fitness to harsh environmental conditions, such as drought, cold, or barren soils. As shown in , leaves of these weeds are often lanceolate with thick waxy layers that tolerate prolonged drought conditions. Moreover, many toxic weeds can capture water and nutrients from deeper soil profiles via their long and deeply distributed roots. Additionally, rhizobacteria has been found to stimulate the growth of these weeds by optimizing nutrient supplies and promoting plant metabolism and systemic resistance under unsuitable growth conditions. Endophytic bacteria also make some toxic weeds more tolerant to abiotic stress.
Toxic weeds follow the optimal partitioning rule wherein plants partition photosynthate among their various organs to maximize growth rate in different habitats. For example, some toxic weeds have been observed to allocate more biomass to hydrotropic roots under drought stress . In addition, plant body size decreases at higher elevations to reduce nutritional needs in less resource-rich environments; however, more photosynthetic products are allocated to flowers at higher elevations to enhance reproductive success . High altitudes make some toxic weeds produce fewer, but larger, flowers with color polymorphisms to attract pollinators in adverse environmentswhere low temperatures and strong winds discourage insect activity. Also, the number of branches on toxic weeds is reduced and plant height is increased in north-facing compared with south-facing slopes, suggesting that toxic weeds allocate more photosynthate to vertical growth than to horizontal growth in response to competition for light. The physiological responses of toxic weeds also show signatures of adaptation to resource-constrained conditions. For example, toxic weeds have higher rates of water use and proline concentrations which is conducive to a stronger resistance against adversity stress in south-facing slopes with arid environments. However, those in north-facing slopes with weaker light intensities have higher chlorophyll contents and photosynthetic efficiencies.
## | interspecific relationships
Owing to their wide niche breadth, toxic weeds can successfully coexist with several other plant species. Unlike the shallow-rooted graminoids whose roots horizontally extend in the surface soil, toxic weeds are mostly axialroot species which deeply root, and thus can absorb water and nutrients from much deeper in the soil compared to forages. Such interspecific differentiation in the acquisition of soil resources alleviates competition and permits co-existence with heterospecific plants F I G U R E 1 Global distribution of S. chamaejasme based on previously published records, primarily including southern Russia, North Korea, Mongolia, Nepal, and northern and southwestern China. Nevertheless, perennial toxic weeds are usually tall and thus superior competitors for light resources relative to shorter plant species. In addition, individuals often aggregate to form patches that facilitate intraspecific cooperation, enhance their competitive ability, and promote their expansion. As a consequence, patches of heterospecific plants that are separated by toxic weeds often are not able to survive in the presence of competitively superior toxic weeds.
The allelopathy is an important competitive behavior of some toxic weeds that inhibits the growth of their surrounding receptor plants . Most studies on allelopathy were done under laboratory conditions which is a serious caveat in allelopathy research.
Here, we sorted the studies done under both laboratory and field conditions, and found that the primary phytotoxic mechanisms were regulated via the following two pathways. First, allelochemicals (e.g., flavonoids, coumarins, and phenolic compounds) can inhibit mitosis, reduce chlorophyll content, disrupt root development , promote the overproduction of proline, inhibit germination, reduce endogenous auxin content, and promote reactive oxygen species accumulation.The second pathway is the arrest of sexual multiplication by pollen allelopathy. Interestingly, phytotoxic effects increase with age; that is, older plants are superior competitors compared with younger plants .
Notably, the allelopathy effects of toxic weeds exhibit species specificity; for example, S. chamaejasme has strong inhibitive effects on some species including Setaria viridis, Amaranthus retroflexus, Pedicularis kansuensis, Festuca rubra L., Medicago sativa ,
## Melilotus suaveolens ledeb
## , and
Onobrychis viciifolia, while other species such as Agropyron mongolicum, Psathyrostachys juncea, Elymus dahuricus, and Lolium perenne show resistance against the allelopathy effect of S. chamaejasme. Therefore, these species can be used to restore degraded grasslands inhabited by toxic weeds.
## | weed-animal interactions
Toxic weeds are more resistant to grazing than grasses favored by herbivores, especially when available forage is limited. They also exhibit superior tolerance to physical breakdown because of their tenacious capacity to regenerate once damaged. Endophytic fungi can protect . The content of toxic substances is highest in leaves, which is the vegetative organ most likely to be consumed by herbivores. Furthermore, the content of toxic substances dramatically increases in response to trampling and consumption by livestock, which reduces the grazing intensity on toxic weeds. The texture and color of toxic weeds are also striking , which likely aid the identification, recognition, and classification of toxic weeds by animals as distasteful and indigestible food items.
In response to long-term overgrazing and selective foraging, palatable grasses would exhibit a dwarfing tendency, restricting their ability to utilize natural resources. In addition to grazing duration, grazing intensity also affects the distribution of toxic weeds, which often aggregate when grazing is intense but are randomly distributed when grazing is especially intense. Thus, the intraspecific relationship shifts from being mutualistic to competitive depending on the intensity of grazing .
Reproductive strategies of toxic weeds with high survival rates include floral traits, such as the brilliant terminal flower head F I G U R E 3 Conceptual graph of the adaptive strategies of toxic weeds for environmental stress (yellow background), competition from other plants (blue background), and animal disturbance (orange background). Fine dotted arrow = impacts of environmental conditions; thick blue dotted arrow = intraspecific and interspecific relationships; thick orange dotted arrow = interactions between plant and animals . For instance, the flower colors of Iris lacteal, Gentiana sino-ornata, Consolida ajacis, Anaphalis sinica are in sequence lavender, purple, blue, and while, which increase reproductive success by attracting pollinators.
Additionally, the seeds are hard and long-lived and the seedlings are capable of exploiting grazed areas with reduced competition from palatable grasses . The proportion of old plants in grasslands increases with grazing intensity. In addition, old individuals have a higher fecundity and produce larger quantities of seeds compared with younger plants. Thus, the breadth and density of the soil seed bank increases as the intensity of grassland degradation rises, enhancing the ability of the population to regenerate.
## | p otential ecolog i c al effec ts of toxi c weeds
Traditionally, toxic weeds are not only thought to cause economic losses to livestock production but are also thought to do great harm to grasslands and lead to their degradation. However, this parochial view may neglect the manifold ecological roles that toxic weeds can play as important natural components of grassland ecosystems. For instance, toxic weeds can provide a number of ecological, social, and economic benefits by improving soil quality, protecting forage resources, and promoting the sustainable development of grasslands.
## | effects on soils
Regarding soil and water conservation, the well-developed root systems of toxic weeds can fix sand and capture nutrients from soils with coarser textures. Grazing and grassland degradation induce reversed vegetation succession with deterioration of plant community structure from palatable grasses to toxic weeds. Even so, compared to bare land, grassland covered by toxic weeds is more susceptible to erosion from strong wind and rain . On the other hand, toxic weeds significantly increase the water content of the soil surface under drought conditions . The higher coverage of plants shields topsoil from solar radiation and decreases evaporation; moreover, the soil infiltration rate is relatively high as a result of a well-developed root system, stimulating rainfall storage.
In addition to the physical protection that they provide to grasslands, toxic weeds have remarkable effects on soil nutrient pools and can create fertile islands .
Toxic weeds produce more litter as a consequence of their increased growth and because they lose less tissue through grazing.
Toxic weeds are also more labile and have higher tissue nitro-
## | effects on co-occurring plants
It is commonly assumed that toxic weeds have negative effects on the quantity of forage via allelopathy, thereby decreasing grassland productivity. However, toxic weeds actually provide biotic refuges and keep surrounding herbaceous species away from livestock in overgrazed grasslands .found that the number of species and the coverage of neighboring plants are noticeably higher in plots with toxic weeds than in those in open grasslands. There are two principal means by which toxic weeds can facilitate the proliferation of neighboring plants in overgrazed pastures. First, the toxic smell could repel livestock and thus reduce the ingestion and trampling of edible forage surrounded by toxic weeds. Second, toxic weeds alter the surrounding micro-environmental conditions. For example, toxic weeds can redistribute soil nutrients, form fertility islands, and create a cool environment that promotes soil moisture retention via the height of the plant canopy. All of these micro-environmental changes provide better soil conditions and microclimates for plant growth.
Additionally, the niche overlap between toxic weeds and fine herbage is smaller than that between toxic weeds and unpalatable weeds, reflecting the lower degree of competition between toxic weeds and edible forage .
## | potential ecological roles in degraded grasslands
From a successional perspective, the spread of toxic weeds is a consequence of their high adaptability rather than a cause of grassland degeneration. As an important part of the grassland ecosystem, toxic weeds improve plant community structure in degraded pasturesand play a crucial role in preventing further desertification of degraded grasslands . Animals usually avoid poisonous toxic weeds, which inherently suppresses excessive disturbance by livestock when overgrazing occurs. The unfounded removal of toxic weeds might lead to ecosystem collapse because grazing pressure on pasture is greater without the protection that toxic grasses provide. This hypothesis is potentially consistent with previous studies that report that the degree of degradation of mowed grasslands was greater than that of grazed grasslands inhabited by toxic weeds.
## F i g u r e 5
The processes of grassland succession. ① Grassland degrades as a result of climate change and human activities; ② Toxic weeds invade as a consequence of their many adaptations to disturbed environments; ③ Degraded grassland recovers under the protection of toxic weeds from excessive destruction; ④ Livestock and rats destroy degraded grasslands by the excessive removal of toxic weeds; ⑤ The grassland ecosystem collapses and desertification occurs as a consequence of the excessive damage. Red solid arrows indicate the positive feedback loop with toxic weeds. Yellow dotted arrows indicate the negative feedback direction that occurs in the absence of toxic weeds Furthermore, the presence of toxic weeds provides an essential means by which the coverage of vegetation can be maintained and the ecological functions of degraded grassland can be preserved , although these should be considered some of their "better-than-nothing" effects. Toxic weeds provide an important gene pool, and their invasion increases the diversity of insects and invertebrates, facilitating the maintenance of biodiversity. Consequently, degraded grassland with toxic weeds does not require any special interventions aside from controlling grazing intensity or limiting the overgrowth of toxic weeds. In support of these effects, the occurrence of toxic weeds is inhibited by the absence of grazing.
The potential process and underlying mechanism are as follows:
First, residual yak dung deposition accelerates the proportional increase in graminoids and promotes the transformation of grasslands to gramineous communities following the exclusion of grazing. Moreover, grasses will recolonize and regain prevalence due to the maintenance of local genetic variation and because they can regenerate rapidly through the production of a large number of seeds. We hypothesize that degraded grassland ecosystems will eventually be restored and become prosperous again following a long period of self-healing .
## | con clus i on s and future pros pec ts
An improved understanding of toxic weeds is valuable for the sus- The limitation of this paper is that we focused on the potential positive effects of toxic weeds which have been largely neglected by conventional wisdom. Notably, an objective justification to treat these poisonous species differently must be based on the trade-off of their positive and negative effects considering many aspects.
However, due to the limited availability of studies, we were unable to make a quantitative assessment of the negative and positive effects of toxic weeds. Subsequent studies should allocate more efforts to quantify and assess the trade-off between positive and negative effects of poisonous species, so as to adopt adaptive grassland management dealing with the presence of toxic weeds.
## Ack n owled g m ents
## Co n fli c t o f i nte r e s t
The authors declare no conflict of interest.
## Auth o r co ntr i b uti o n
## Data ava i l a b i l i t y s tat e m e n t
All data included in this study are available upon request by contact with the corresponding author.
## O rci d
## Jian sun
https://orcid.org/0000-0001-8765-5015
## R e fe r e n c e s
An, D. Y., Han, L., Ju-Ying, W. U., Chen, J., Jiang, Y. Y., . Effects of Stellera chamaejasme on soil properties of grassland in farming-pastoral zone in north China. Acta Agrestia |
The Identification of Diabetes Mellitus Subtypes Applying Cluster Analysis Techniques: A Systematic Review
Diabetes Mellitus is a chronic and lifelong disease that incurs a huge burden to healthcare systems. Its prevalence is on the rise worldwide. Diabetes is more complex than the classification of Type 1 and 2 may suggest. The purpose of this systematic review was to identify the research studies that tried to find new sub-groups of diabetes patients by using unsupervised learning methods. The search was conducted on Pubmed and Medline databases by two independent researchers. All time publications on cluster analysis of diabetes patients were selected and analysed. Among fourteen studies that were included in the final review, five studies found five identical clusters: Severe Autoimmune Diabetes; Severe Insulin-Deficient Diabetes; Severe Insulin-Resistant Diabetes; Mild Obesity-Related Diabetes; and Mild Age-Related Diabetes. In addition, two studies found the same clusters, except Severe Autoimmune Diabetes cluster. Results of other studies differed from one to another and were less consistent. Cluster analysis enabled finding non-classic heterogeneity in diabetes, but there is still a necessity to explore and validate the capabilities of cluster analysis in more diverse and wider populations.
# Introduction
Diabetes Mellitus (DM) is a chronic and lifelong metabolic disorder characterized by elevated levels of glucose circulating in the blood that occurs either when the pancreas does not secrete enough insulin, due to destruction of pancreatic β-cells; when the body's cells do not respond to insulin effectively; or by a combination of both mechanisms. The prevalence of DM has increased across the globe and is expected to rise to 592 million by 2035, incurring tremendous human, economic and social costs [bib_ref] Global estimates of diabetes prevalence for 2013 and projections for 2035, Guariguata [/bib_ref].
DM imposes a considerable burden on society in the form of low productivity, poor quality of life, increased healthcare expenditures, and premature mortality. The global cost of DM is overwhelming: US $1.31 trillion or 1.8% of global GDP. Notably, indirect costs accounted for 34.7% of the total burden [bib_ref] The global economic burden of diabetes in adults aged 20-79 years: A..., Bommer [/bib_ref].
DM significantly increases the risk of mortality: 1 in 12 of all-cause deaths may be attributable to DM [bib_ref] Mortality in type 2 diabetes mellitus: Magnitude of the evidence from a..., Nwaneri [/bib_ref] [bib_ref] Update of mortality attributable to diabetes for the IDF Diabetes Atlas: Estimates..., Group [/bib_ref] [bib_ref] Risk factors, mortality, and cardiovascular outcomes in patients with type 2 diabetes, Rawshani [/bib_ref]. Regardless of existence of effective treatments, DM outcomes are poor: DM patients show high frequency of serious and life-threatening micro-and macrovascular complications (strokes, acute coronary events, blindness, amputations, renal disease, heart failure) and premature mortality exceeding the general population [bib_ref] Understanding heterogeneity in response to antidiabetes treatment: A post hoc analysis using..., Hardin [/bib_ref]. DM management is challenging because of the heterogeneity in individual patient responses, which vary due to factors such as illness severity, sociodemographic characteristics, and specific clinical factors (e.g., glycated hemoglobin (HbA1c), insulin sensitivity, body composition, and duration of disease) [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref]. DM is much more complex than the classification into Type 1 and Type 2 suggests. Recently, Alhqvist and colleagues using K-means cluster analysis (CA) has proposed a novel classification of adult onset DM into five subgroups: Severe Autoimmune Diabetes (SAID); Severe Insulin-Deficient Diabetes (SIDD); Severe Insulin-Resistant Diabetes (SIRD); Mild Obesity-Related Diabetes (MOD); and Mild Age-Related Diabetes (MARD) [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref]. This classification is based on six measures that are commonly collected in clinical practice: body mass index (BMI); age at DM diagnosis; HbA1C; β-cell functioning; insulin resistance; and the presence of DM-related autoantibodies. The five subgroups differ in their patterns of progression and risk of complications. Currently, there is a rising interest in identifying more homogeneous groups of DM patients so therapeutic plans could be applied in a more targeted manner. New analytic techniques, namely unsupervised learning methods, such as CA, have been used in a variety of settings, with various sources and information and including different types of variables for proposing subtypes of DM patients.
The objective of this work is to systematically review the scientific literature to identify publications that have applied CA to generate homogeneous groups of DM patients, describe the main features of the analytic techniques that have been applied, as well as the variables included to propose DM subgroups.
# Methods
## Search strategy and selection criteria
We systematically searched Medline Complete (from 1978 until August 2020) and PubMed August 2020) databases on 7 August 2020 following PRISMA guidelines. Additionally, the reference lists of the selected articles from the above-mentioned databases were hand-searched.
In the databases we searched studies published on the area of unsupervised CA of DM patients. The search strategy applying Medical Subject Headings (MeSH) was used in the Medline Complete database with the following keywords: "Diabetes Mellitus" or "Diabetes Mellitus, Type 2" or "Diabetes Mellitus, Type 1" or "Diabetes" AND "Cluster analysis" or "Cluster". In the Pubmed database papers were searched applying "Diabetes" and "Cluster" keywords. The results were limited to articles in the English language and which had humans as a research subject. All database-specific technical variations were taken into account during the search.
## Methods of the review
The selection process was performed by two independent researchers. Search results from two databases were combined to remove duplicates, after which all unique results were screened based on the title and abstract. In the next stage, full-text articles of potentially suitable articles were obtained and assessed for eligibility criteria: (1) the study population consisted of diabetic patients (Type 1 and/or Type 2 DM); (2) clusters were identified through one of the unsupervised clustering algorithms; (3) clustering was based on the patients' clinical data. Studies with specific aims were excluded to provide comparability within clusters.
## Data extraction
The information was retrieved by two authors from selected articles to the a priori prepared tables, with the following columns: study design, source of the data taken for exploration, size and characteristics of targeted population, diagnostic criteria of DM, variables chosen for cluster analysis, and the number of clusters and their characteristics, as well as the data standardization, chosen clustering algorithm, methods for the determination on the number of clusters, and validation of clusters on an independent sample (please, see Appendices A and B).
# Results
The search identified 6319 publications from two databases. After removing duplicates and screening the papers, 75 full-text articles were reviewed and 65 were excluded for the following reasons: 6 were review articles, 9 papers focused on exploring clusters of diabetic patients with specific comorbidities at baseline, 32 studies pursued other aims than finding subgroups of DM, 7 studies used other methodologies than unsupervised learning techniques, 9 studies conducted a similar analysis but with other specific aims (clustering of genetic data etc.), and 2 studies were conducted on mice. As a result, 14 papers were found to be eligible: 10 articles were included in the review [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] and an additional 4 eligible papers were found after hand-searching of the reference lists of selected articles [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref]. The selection process is presented in [fig_ref] Figure 1: PRISMA Flow Diagram. [/fig_ref].
## Sample characteristics
The sample size ranged between 33 and 85,783 participants within studies constituting a total 130,353 diabetic patients: 33 type 1 diabetes (T1DM) patients, 238 latent autoimmune diabetes patients (LADA) and 130,082 type 2 DM (T2DM) patients. The largest sample size was in the study of Karpati et al. from Israel, constituting 85,783 patients of whom 60,423 were considered eligible for cluster analysis [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref]. The second largest was the study of , followed by 8980 individuals from the ANDIS cohort in the study of Ahlgvist et al. [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] The study with the smallest sample of 33 T1DM patients from several university hospitals was conducted in the UK [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref].
The variability in population size could be explained by the source of the data, as data were taken from electronic medical records, healthcare databases, from previously conducted longitudinal observational studies and surveys. Disease duration among target populations of reviewed publications, along with newly diagnosed diabetic patients, ranged from 40 days after diagnosis to 12 years or longer [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref]. The age of the participants varied depending on the type of DM: 5-16 years among T1DM patients, LADA patients were 35 years and older, the age of T2DM patients were between 18-96 years. Different criteria were used for the diagnosis of DM in the studies: American Diabetes Association Criteria [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] , 1999 World Health Organization criteria [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] , International Diabetes Federation diagnostic guidelines [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref]. When data were extracted from health records or healthcare databases, diagnosis was based on specific ICD-10 codes for DM or antidiabetic medications [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref]. Some studies used different diagnostic methods using biochemical indicators (fasting plasma glucose/HbA1c levels/blood test for autoimmune responses) with or without restrictions on the duration of treatment [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] , while one study used self-reported DM cases [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref].
The 14 selected studies, published between 2003 and 2020 years, were observational retrospective studies, 7 studies with follow-up periods [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] and 7 cross-sectional studies [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref]. Reviewed studies were originated from different countries (Japan [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] , Germany [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] , USA [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] , China [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] , UK [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] , Sweden [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] , Italy [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] , Israel [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] , Australia [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] and Denmark [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref].
# Cluster analysis
## Data standardization
Seven studies did not perform data standardization before doing the CA [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref]. Two studies reported a presentation of the mean and standard deviation for values [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] , three studies reported centering the values [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] , and two studies reported calculating the mean of 0 standard deviation of 1 [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref].
## Variables selected for cluster analysis
Eight studies had almost similar variables for CA and the difference was trivial [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The following variables were, mainly, included in the CA of those eight studies: age at diagnosis; BMI; glutamic acid decarboxylase antibody (GADA) level; HbA1c level; homoeostasis model assessment 2 estimates of β-cell function (HOMA-2b); homoeostasis model assessment 2 estimates of insulin resistance (HOMA-IR).
Amato and colleagues used measurements of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), ghrelin for clustering [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref].
Arif and colleagues included the following variables: interferon-g, interleukin 10 (Il-10), antigenspecific autoantibodies (Aabs), proinsulin, insulin, islet antigen antibodies (IA-2Ab), glutamic acid decarboxylase 65 antibody, zinc transporter 8 antibody [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref].
Pes and colleagues also had quite distinct variables for clustering: gender, BMI, total cholesterol, triglycerides, systolic blood pressure, diastolic blood pressure, anti-glutamic acid decarboxylase (GAD) autoantibody, anti-islet antigen-2, anti-thyroid peroxidase, cumulative genetic score, insulin-free period [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref].
Hammer and colleagues tried to cluster participants with DM according to self-reported symptoms, including, upper GI/dysmotility, diarrhea, constipation, nausea/vomiting [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref].
Karpati and colleagues focused on clustering based on HbA1c levels. Thus, changes in HbA1c levels during the 3 year period, mean of the absolute first differences in HbA1c, and the ratio of the maximum absolute second difference to mean absolute first difference of HbA1c have been measured and included in the CA [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref].
Li and colleagues had the highest number of variables included for CA among studies included in this systematic review, 73 variables [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref].
Methods for determining the number of clusters varied from one study to another. Seven papers used the direct silhouette width method [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] , one paper had a fixed number of clusters [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] , one study determined the number of clusters based on hierarchical clustering with Ward's method [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref].
In addition, two publications determined the number of clusters based on principal component analysis (PCA) [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] , one publication performed a "NbClust" algorithm that selected an optimal method for the determination of number of clusters [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] , one study was based within the cluster sums of squares against the number of clusters [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] , one study was based on a cosine distance metric [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref].
## Methods of clustering and dimensionality reduction
Only two studies have indicated reducing the dimensionality of the data prior to CA [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref]. The widespread method for clustering among included publications was k-means clustering [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. Several studies performed k-means analysis only for GADA-negative individuals [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The second widespread method of clustering was hierarchical CA: six studies reported performing hierarchical clustering [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref]. The least frequent methods for clustering were PCA [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] and topology-based analysis (TBA) [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref].
## Cluster validation on an independent sample
Only five studies performed validation of results of CA on an independent sample [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] , while Karpati et al. split the database to train and test datasets to replicate findings [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref].
# Main results
Two [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] , three [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] , four [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] and five [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] different clusters were identified in the reviewed papers. The majority of studies revealed the same 5 clusters: SAID, SIDD, SIRD, MOD, and MARD [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. Two additional studies identified the same four clusters except SAID, due to the unavailability of GADA measurements [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref]. The proportion of SAID cluster varied between 4% and 22.3% in the studies with the same applied cluster name, while the Autoimmune β-cell failure cluster described by Safai et al. was identical to SAID with GAD-positive antibodies comprising 2.8% of the total sample [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The proportion of the SIDD cluster was between 2.5% and 20% within studies, while the non-autoimmune β-cell failure cluster identified by Safai et al. shared similar characteristics to SIDD and composed 22.3% of the total sample [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The proportion of the SIRD cluster ranged within 7.2% and 23.7% among studies, while 2 similar clusters were revealed by Safai et al. such as insulin resistance with short disease duration (21.4%) and insulin resistance with long disease duration (31.7%) [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The next most frequent cluster was MOD with varying percentages between studies from 20.4% to 34% [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. The MARD cluster was the most prevalent among the mentioned five clusters in each study, falling within 34% and 45.4% [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. Additionally, Safai et al. reported a cluster based on the presence of metabolic syndrome, which had the highest BMI and constituted 21.7%, but differed by clinical characteristics from the aforementioned MOD and MARD clusters [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref].
The main five clusters identified across studies shared similar phenotypic characteristics. All of the patients in the first SAID cluster were GADA-positive, were younger compared to other cluster members, had low BMI and insulin deficiency characterized by low HOMA-2b and higher HbA1c levels. The patients with DM in the SIDD cluster had the same characteristics but were GADA-negative. At the same time, participants from SIRD differed with high BMI, whole-body and/or adipose-tissue insulin resistance characterized by high HOMA-IR and were at a relatively younger age. Individuals in the MOD cluster were slightly younger and had obesity and moderate insulin resistance compared to the SIRD cluster. The oldest age of diabetic patients and moderate metabolic dysregulations were inherent to the MARD cluster. Authors in the reviewed papers identified several complications associated with each cluster, which were also observed in the replicated studies. The major conditions were diabetic or chronic kidney diseases (DKD, CKD), liver diseases (non-alcoholic fatty liver disease (NAFLD) or hepatic fibrosis), retinopathy, polyneuropathies, and cardiovascular diseases (CVDs). Thus, in studies of Zaharia et al. and Ahlgvist et al., the SIRD cluster and in the study of Tanabe et al., both SIRD and SAID clusters were associated with a higher risk for CKD and DKD [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref]. The cluster with the presence of metabolic syndrome in the study conducted by Safai et al. reported the same association with nephropathies [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref]. However, Dennis et al. did not find an increased risk for CKD complications among clusters after adjustment for baseline estimated glomerular filtration rate (eGFR) [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. SAID and SIDD in the study of Tanabe et al., but only the SIDD cluster in the studies of Ahlgvist et al., were associated with the increased risk for retinopathy [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref]. Along with them, the similar non-autoimmune b-cell failure cluster to SIDD in the study of Safai et al. demonstrated the same association with retinopathy [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref]. Liver diseases such as NAFLD and hepatic fibrosis were found to be associated with the SIRD cluster in studies of Zaharia et al. and both studies of Ahlgvist et al. [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] At the same time, neuropathies identified in the Zaharia et al. study among SIDD individuals, were not associated with any cluster after adjustment for disease duration or age at onset in the study of Safai et al. [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] In the study of Kahkoska et al., unadjusted analysis showed that CVDs were associated with the SIDD cluster, which is characterized by low BMI and insulin deficiency [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref]. However, CVDs did not differ among clusters after adjustment for known modifiable and non-modifiable risk factors in the studies of Safai et al. and Tanabe et al. [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] Amato et al. phenotyped diabetic patients based on fasting incretin levels into two independent clusters: cluster 1 (65.6%) with lower incretin levels and cluster 2 (34.4%) with higher incretin levels [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref]. Thus, cluster 1 differed by a lower glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and, consequently, with higher levels of HbA1c and fasting plasma glucose (FPG) compared to cluster 2, which was explained by possible increased a-cell activity and its effect on the reduction in b-cell function. However, there were no differences in the clinical-anthropometric characteristics between clusters.
Based on the data from electronic medical records, Li et al. clustered T2DM patients applying TBA and came up with three different subtypes with inherent clinical characteristics and comorbidities [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref]. Individuals in subtype 1 had higher weight and serum glucose levels and were associated with diabetic nephropathy and retinopathies, patients in subtype 2 had lower weight and were associated with cancer malignancy and CVDs, while subtype 3 was characterized by neurological diseases, allergies, HIV and CVDs.
Karpati et al. found ascending (14.4%, mean HbA1c 8.7% (1.9)), descending (10.0%, mean HbA1c 7.8% (1.8)) and stable (75.6%, mean HbA1c 7.1% (1.2)) subtypes of T2DM patients, with the duration of 3-7 years, based on their HbA1c levels' trajectories and their five-year risk of complications [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref]. Diabetic patients in the ascending cluster were the youngest compared to the representatives of other clusters, and were taking mostly non-insulin medications, while insulin medications were often prescribed to patients in the descending cluster. However, micro-and macrovascular complications were prevalent in both ascending and descending clusters. The mortality rate was higher in the descending cluster.
Hammer et al., based on gastro-intestinal symptoms of T2DM patients, found four4 such clusters as Upper GI/Dysmotility (44.8% of the total variance), Diarrhea (10.4% of the total variance), Constipation (7.8% of the total variance), and Nausea/Vomiting (6.3% of the total variance) [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref]. Analysis in the given study has shown that oral medications taken by diabetic patients were associated with the Nausea/Vomiting cluster. After adjustment for the type of treatment (insulin or oral medication), gender, and age, members of Upper GI/Dysmotility cluster were heavily linked with use of insulin in conjunction with hypoglycemic medication, while Nausea/Vomiting cluster members had a strong relationship with the intake of insulin, oral hypoglycemic medication, and with the combination of both. Diarrhea and Constipation clusters have not shown any significant linkages.
Arif et al. found two clusters of T1DM patients by assessment of different parameters of autoimmunity of CD4 T-cell and B-lymphocyte responses [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref]. Thus, T1DM patients in the later stages are differentiated with (AAb++ and IFN-g. IL-10) and (AAb6 and IFN-g, IL-10), as well as other non-diabetic individuals with high AAbs who had an increased risk for T1DM development. Overall, cluster 1 was dominated for IL-10 response to GAD, insulin, and proinsulin compared to cluster 2.
Pes et al. found four different clusters of LADA patients. Each cluster had a special set of important characteristics extracted based on the PCA. One of the main findings related to the disease progression was the association of b-cell function with four clusters (PCs) [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref]. The fastest b-cell failure was observed among members of PC 2, which was characterized by genetic profile, while mild and slower b-cell activity was seen among PC 1, as well as gender and TGs predominated PC 3 with cholesterol predominated PC 4, respectively.
# Discussion
The main finding of this systematic review is that data-driven algorithms reflect a larger heterogeneity in DM subtypes that the classical division into T1DM and T2DM or solely based on glycemic or HbA1c levels may reflect. Another finding is that a significant number of studies with data from a diversity of patient origins receiving the same five clusters of DM patients, which shared similar physiological and clinical characteristics across studies and were associated at most with analogous comorbidities, although having a different prevalence as well as variations across them in the frequency of the variables included in each of them. However, there were also six papers that provided clusters of DM patients based on different types of variables shown also to be appropriate in terms of statistical significance as well as clinical meaning. Another relevant finding is that there is significant variability in terms of the use of specific analytic techniques to generate those clusters of DM patients.
Overall, those findings confirm that the process of using clustering techniques, although not exempt from certain limitations, may be applied for monitoring the progression and control of patients with DM, but there is still uncertainty on the variables that should be used for generating subtypes of patients, as well as for what is the most appropriate clustering method.
As for the studies that proposed the same five clusters, the proportions of individuals in each cluster varied from one study to another. Several factors may influence those disproportionate distributions. First, the source of the data applied for CA in the studies varied based on the availability and may explain some variations in the sample size, as well as the type of diabetic patients participating in the analysis. For instance, the extreme proportion of SAID patients in the study of Zaharia et al. could be explained by active recruitment of T1DM patients, while studies that have utilized data from other cohorts showed consistent results [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref]. Second, some cohorts used for CA were focused specifically on the studies with DM patients at onset [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] , while others recruited patients with a longer [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] or not defined [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref]. This is an important aspect, since the clustering results of the Japanese population showed that Asian diabetic patients, due to their inherent lower b-cell activity and insulin secretion, showed a higher proportion of SIRD cluster with a comparatively lower BMI than the studies from western cohorts, meaning there is a potential earlier onset of DM in their population [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Ethnic differences in the relationship between insulin sensitivity and insulin response: A..., Kodama [/bib_ref].
Overall, five main clusters were reproducible in the studies which used databases from cross-sectional, longitudinal observational and trial studies. All the aforementioned papers, revealing meaningful complications specific for clusters, used data from longitudinal observational studies. The cross-sectional study of Zou et al. and of Kahkoska et al., which selected patients with a baseline high risk for CVDs and long-lasting DM, were not able to estimate risks for complications [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] , while Dennis et al., who did the study with protocol-driven follow-up, were able to find out several complications adjusting to different treatments [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref].
The range of associated comorbidities is not limited to the aforementioned conditions. There might be other complications of diabetic patients that would eventually need to be considered in the further clustering studies. Li et al., in their study, observed a wider range of associated comorbidities applying TBA [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref]. Adjustment for known modifiable and non-modifiable risk factors are also suggested to determine their true effect, as some studies showed no association with CVDs, indicating the importance of sticking to a healthy lifestyle to reduce the risk of complications [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref].
Another relevant issue still in need of further investigation is the optimal number of variables which provide the balance between validity and economic efficiency of clustering diabetic patients: Kahkoska et al., using only three variables (age, BMI, and HbA1c), obtained the four clusters with very similar characteristics to the original clusters proposed by Ahlgvist et al. with six variables.
Other studies which found clusters of diabetic patients with different GI symptoms [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] , fasting incretin tone [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] , trajectories of HbA1c levels [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] , clusters among T1DM [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] and LADA [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] patients, as well as clusters identified through novel TBA [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] , were unique and not replicated and therefore should be considered as a call for future research initiatives.
However, the study of Karpati et al., with a sufficient sample size of 60,423 patients, identified interesting findings by clustering based on HbA1c levels: the ascending cluster had complications only in the extremely high levels, which could possibly suggest other risk factors among this group, while the highest risk for complications among DM patients were found in the stable cluster with HbA1c < 6.0%, which contradicts the guideline recommendations and is consistent with J-shaped risk [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref] [bib_ref] The HbA1c and all-cause mortality relationship in patients with type 2 diabetes..., Arnold [/bib_ref].
Moreover, the only study of clustering T1DM identified patients with different immunological responses and could be implicated in the clinical practice by tailoring immune-based therapies, raising issues about the underlying basis for the different phenotypes observed if they reflect the different immunological pathways of the disease.
Overall, results of all studies indicated the need to pay attention to symptoms and clinical characteristics of the diabetic patients, which previously were underestimated and may have an impact on their disease progression, as well as on the need to incorporate the wealth of information of unstructured data from the free text of patient records [bib_ref] Groningen initiative to analyse type 2 diabetes treatment. Computerized extraction of information..., Voorham [/bib_ref]. Genetic information is another critical domain that will be necessary to explore in order to identify subgroups of DM patients [bib_ref] Type 2 diabetes genetic loci informed by multi-trait associations point to disease..., Udler [/bib_ref].
Review studies applied different methodological approaches of CA. Each step before and during the running CA in different ways may affect the clustering outputs. It is critical not to violate the reproducibility of unsupervised learning techniques, therefore, validation in different datasets is required to provide robustness of the results. Second, the type of data (observational/longitudinal) is also critical in cluster analysis to give a chance to observe temporal patterns of disease progression, as cluster analysis does not explain the aetiology of the disease. Third, the number of clusters depends on the specific methodology applied as well as the proportions of populations among clusters that could vary based on the chosen sample size and the presence/absence of scaling the dataset (preprocessing) [bib_ref] Unsupervised analysis of classical biomedical markers: Robustness and medical relevance of patient..., Gordon [/bib_ref].
Among the issues related to methods for determining the number of clusters, one study has chosen to limit the number of clusters to two [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref]. Manually limiting the number of clusters could lead to error as there might be more clusters within the data.
Regarding the methods for clustering, seven out of fourteen studies have performed a k-means clustering. Several studies relied solely on k-means, other studies have performed it only to confirm the results from the hierarchical clustering or to cluster only GADA-negative individuals. In k-means clusters, the presence of outliers could distort the results of clusterization [bib_ref] Outlier detection and removal algorithm in k-means and hierarchical clustering, Barai [/bib_ref]. Among seven studies, only two reported excluding outliers prior to clustering [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref]. Performing k-means requires running the clustering multiple times to obtain optimal results, but it also increases the risk of ending in a local optimum. The local optima is characterized by poorer quality of clusters that might affect the number of clusters [bib_ref] Towards enhancement of performance of K-means clustering using nature-inspired optimization algorithms, Fong [/bib_ref]. None of the studies reported minimizing the local optima. The next widespread method after k-means was hierarchical clustering. The distance metric and linkage criteria choices ranged among six studies that performed hierarchical clustering. Those choices could affect the result of clustering as, currently, there is no sturdy theoretical justification for such decisions. Another issue with hierarchical clustering is the treatment of missing values. Most software does not work if this is the case. Four studies have not reported the presence or absence of missing data variables [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref]. The third widespread method for clustering was PCA [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref]. Pes and colleagues have not reported standardizing the data standardization prior to PCA, which is essential to enable the PCA with the search of optimal principal components [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref]. The last method to discuss is TBA. Li and colleagues performed TBA, which is quite new in machine learning and it has a strong theoretical basis [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref].
The next aspect to discuss is the validation of clustering results. Nine studies have not reported validating clustering results [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref]. The validation of the results by external validation on an independent sample or cross-validation within a dataset is vital to obtain the information on the quality of performed CA [bib_ref] Survey of clustering algorithms, Xu [/bib_ref].
The data standardization process is also an important step to enable comparison of variables that could have units at different scales. Without standardization, variables with different scales would unequally contribute to the results of analysis [bib_ref] The identification of Parkinson's disease subtypes using cluster analysis: A systematic review, Van Rooden [/bib_ref]. Only seven studies out of fourteen have reported standardizing the data prior to CA.
Some common limitations among the included studies were: the lack of some variables in their data that would affect the clustering results [bib_ref] Novel subgroups of adult-onset diabetes and their association with outcomes: A data-driven..., Ahlqvist [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Stratification of type 2 diabetes based on routine clinical markers, Safai [/bib_ref] [bib_ref] Novel subgroups of patients with adult-onset diabetes in Chinese and US populations, Zou [/bib_ref] [bib_ref] Disease progression and treatment response in data-driven subgroups of type 2 diabetes..., Dennis [/bib_ref] ; having small or relatively small sample sizes for doing clustering [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Phenotyping of type 2 diabetes mellitus at onset on the basis of..., Amato [/bib_ref] [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Identification of type 2 diabetes subgroups through topological analysis of patient similarity, Li [/bib_ref] ; issues that may affect the generalizability of the results [bib_ref] Factors Associated with Risk of Diabetic Complications in Novel Cluster-Based Diabetes Subgroups:..., Tanabe [/bib_ref] [bib_ref] Risk of diabetes-associated diseases in subgroups of patients with recent-onset diabetes: A..., Zaharia [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] ; and having a relatively short follow-up of participants [bib_ref] Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Arif [/bib_ref] [bib_ref] Clustering of immunological, metabolic and genetic features in latent autoimmune diabetes in..., Pes [/bib_ref] [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref] [bib_ref] Validation of distinct type 2 diabetes clusters and their association with diabetes..., Kahkoska [/bib_ref] [bib_ref] Patient clusters based on HbA1c trajectories: A step toward individualized medicine in..., Karpati [/bib_ref]. Last but not least, Hammer et al. had reported grouping all oral medications into one group, while some drugs, such as metformin, could have significantly different effects on controlling the high blood sugar than other drugs [bib_ref] Symptom clustering in subjects with and without diabetes mellitus: A population-based study..., Hammer [/bib_ref]. Thus, it might have affected the results of clustering.
# Conclusions
This systematic review has explored the research publications that utilized clustering algorithms to identify non-classic heterogeneity in DM. DM is a complex condition and clustering analysis is showing to be an effective method for finding clinically meaningful subgroups. Identifying homogeneous subgroups of patients with potential disease progression at an onset, based on routinely collected measurements, could be useful to apply therapeutic and prevention measures, targeting patients that will be benefitted the most. There is a significant number of effective therapeutic alternatives to treat DM, including insulin and oral medications, the latter having quite diverse mechanisms of action. It will be necessary to identify which sub-groups of patients with DM benefit most of those available therapies and advance towards more targeted treatments. Nevertheless, there are still some methodological aspects that must be clarified as well as what may be the metabolic pathways affected in each subgroup of patients. There is also a need for studies that would explore and validate the capabilities of CA in more diverse and wider populations, combining variables that have already shown statistical and clinical relevance to generate homogeneous groups of DM patients.
## Conflicts of interest:
The authors declare no conflict of interest. Cluster 1 (SAID, n = 577): characterized by early-onset disease, relatively low BMI, poor metabolic control, insulin deficiency, and presence of GADA; Cluster 2 (SIDD, n = 1575): GADA negative but otherwise similar to cluster 1: low age at onset, relatively low BMI, low insulin secretion (low HOMA2-B index), and poor metabolic control. 3.BMI; 4.HbA1c; 5. HOMA2-B; 6.HOMA2-IR cluster 1 (SAID, 68 (5.4%)): was positive for islet-related autoantibodies and was young at onset, had an increased risk of diabetic retinopathy, after adjusting for modifiable risk factors; cluster 2 (SIDD, 238 (19.0%)): had a severe insulin deficiency and the highest A1c; cluster 3 (SIRD, 90 (7.2%)): was the highest in BMI, HOMA 2-IR, and HOMA2-B and had an increased risk of DKD; cluster 4 (MOD, 363 (28.9%)):had a higher BMI and was slightly younger than the MARD subgroup; cluster 5 (MARD, 496 (39.5%).
# Appendix a
## 3.
Zaharia et al. cluster 1 SAID (N = 247): GADA positive, were more likely to be of a younger age, had relatively low BMI, poor glycemic control and overt insulin deficiency. 158 (67.0%) received insulin on diagnosis cluster 2 SIDD (N = 28): showed similarities with patients with SAID, but GADA negative; had the highest prevalence of confirmed diabetic sensorimotor polyneuropathy and cardiac autonomic neuropathy; 12 (44.0%) were treated with insulin on diagnosis; cluster 3 SIRD (N = 121): had high BMI and whole-body adipose-tissue insulin resistance, had the highest sensitivity for C-reactive protein, high hepatocellular lipid content and fatty liver index, low eGFR levels; cluster 4 MOD (N = 323): had obesity and substantial adipose tissue insulin resistance, high sensitivity for C-reactive protein, but they had moderate whole-body insulin resistance; cluster 5 MARD (N = 386): older than those in other clusters and showed only minor metabolic abnormalities. Health records about known type 2 diabetes for <6 months and in stable treatment for the last 3 months with metformin
[formula] (1) glucagon-like peptide-1 (GLP-1) (2) glucose-dependent insulinotropic polypeptide (GIP) (3) ghrelin [/formula]
Cluster 1 (n = 63): significantly lower levels of GLP-1, GIP and ghrelin compared to cluster 2 (n = 33), and higher levels of HbA1c and fasting plasma glucose.
Regarding the clinical and anamnestic characteristics of the patients, there were not any significant differences between the two clusters, except for a greater prevalence of patients practicing physical activity in Cluster 2.
## Arif et al. (2014) [11] uk cross-sectional study
Several university and regional hospitals in UK took part in the research N = 33. Children with newly diagnosed type 1 diabetes (5-16 years), unaffected siblings of patients with type 1 diabetes (6-16 years).
Test of blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses For Autoimmune Inflammatory Phenotypes in Children With Newly Diagnosed Type 1 Diabetes group:
1. interferon-g 2. interleukin 10 (Il-10)
3. antigen-specific autoantibodies (Aabs) 4. proinsulin 5. insulin 6. Islet antigen antibodies (IA-2Ab) 7. GAD65 antibody 8. zinc transporter 8 antibody Cluster 1 (n = 15): a combination of islet AAbs and IFN-g responses to all antigens. Have a significantly higher frequency of IL-10 response to GAD, insulin, proinsulin. There are also differences in the frequency of islet AAbs between clusters. AAbs against IA-2 and ZnT8 are significantly less frequent in the IL10-dominated cluster-1. Two children had no islet AAbs present at diagnosis, five had only a single AAb, and eight had two or more AAbs. Cluster 2 (n = 18): The frequency of multiple AAbs was significantly higher, all 18 children had two or more IL-10 responses to all antigens. 1. Autoimmune β-cell failure cluster (n = 65), characterized by patients with a positive GAD65 autoantibody titer. They also had the lowest TG level.
2. Insulin resistance with short disease duration cluster (n = 490), characterized by patients being diagnosed with type 2 diabetes relatively recently and having the highest HOMA2-β. 3. Non-autoimmune βcell failure cluster (n = 510), patients in sub-group 3 were the youngest at diabetes diagnosis but otherwise resembled sub-group 1 apart from the lack of positive GAD65 autoantibody titer.
Increased risk for retinopathy. 4. Insulin resistance with long disease duration cluster (n = 727). Cluster 4 and 2 were very alike with a high age at diagnosis, similar BMI, better glycemic regulation, a relatively preserved β-cell function and at the same time a relatively high HOMA2-IR. The most important variable separating these two subgroups was the duration of diabetes. 5. Presence of metabolic syndrome cluster (n = 498), characterized by having the highest BMI compared to the other groups. It also consisted of those with the highest fasting glucose, HbA1c, C-peptide, HOMA2-IR and TG level. only cluster 4 (MOD) in RECORD trial. After adjustment to baseline UACR, time to albuminuria was shorter for cluster 3 (SIRD) vs. cluster 2 (SIDD) in ADOPT, but not RECORD.
## 13.
Li Patients in subtype 1 (762) were the youngest (59.76 ± 0.45 years) and were notable for features classically associated with T2DM, such as the highest BMI (33.07 ± 0.29 kg/m 2 ) and highest serum glucose concentrations at point-of-care testing (POCT) (193.69 ± 11.45 mM). Although these patients had better kidney function compared to those in the other two subtypes. They were characterized by T2DM complications as diabetic nephropathy and diabetic retinopathy and ACE gene. Patients in subtype 2 (617) had the lowest weight (85.17 ± 1.14 kg) compared with those in the other subtypes. Subtype 2 was enriched for cancer malignancy and cardiovascular diseases. Patients in subtype 3 (1096) had the highest SBP (135.7 ± 0.7 mmHg), serum chloride levels (102.03 ± 0.11 mEq/liter), and troponin I levels (0.36 ± 0.09 mg/liter) and were more often prescribed ARB/ACEI (62.96%) for the treatment of hypertension and statins (56.0%) for cholesterol reduction. They were associated most strongly with cardiovascular diseases, neurological diseases, allergies, and HIV infections and FHIT gene. Cluster 1 (SAID, 6.4%); was characterized by early onset, relatively low BMI, poor metabolic control, insulin deficiency, and presence of GADA, frequent ketoacidosis (30.5%); Cluster 2 (SIDD, 17.5%): was GADA negative but otherwise similar to SAID, frequent ketoacidosis (25.1%) and early signs of diabetic retinopathy; Cluster 3 (SIRD, 15.3%): was characterized by insulin resistance (high HOMA2-IR) and high BMI, had the highest prevalence of non-alcoholic fatty liver disease and high risk for CKDs; Cluster 4 (MODD, 21.6%): was characterized by obesity but not by insulin resistance; Cluster 5 (MARD, 39.1%): were older, but showed, as cluster 4, only modest metabolic derangements.
BMI-body mass index. GADA-glutamic acid decarboxylase antibody; GAD65 antibody-glutamic acid decarboxylase 65 antibody; HbA1c-glycated hemoglobin; HOMA-2b-homoeostasis model assessment 2 estimates of β-cell function; HOMA-IR-homoeostasis model assessment 2 estimates of insulin resistance; FPG-fasting plasma glucose.
[fig] Figure 1: PRISMA Flow Diagram. [/fig]
[fig] Author: Contributions: Conceptualization, A.S.-S.; methodology, A.S.-S., B.O., T.M.; software, B.O. and T.M.; validation, A.G. and K.A.; formal analysis, T.M. and B.O.; investigation, A.S.-S., B.O. and T.M.; resources, A.S.-S.; data curation, B.O. and T.M.; writing-original draft preparation, B.O. and T.M.; writing-review and editing, A.S.-S., T.M. and B.O.; visualization, B.O. and T.M.; supervision, A.S.-S.; project administration, A.S.-S., A.G. and K.A. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This systematic review was part of the research grant funded by the Nazarbayev University, #080420FD1916. [/fig]
[fig] Cluster 3: SIRD, n = 1373): characterized by insulin resistance (high HOMA2-IR index) and high BMI. Cluster 4 (MOD, n = 1942): characterized by obesity but not by insulin resistance. Cluster 5 (MARD, n = 3513): similar to cluster 4, only modest metabolic derangements. [/fig]
[fig] PC 1: (explained 18.0% of total variance): the dominant variables were: BMI, triglycerides, systolic and diastolic blood pressure and duration of insulin-free time period, showed a mild beta-cells failure. PC 2 (explained 15.0% of total variance): genetic variables such as Class II HLA, CTLA-4 as well as anti-GAD65, anti-IA-2 and anti-TPO antibody titers, and the insulin-free time period predominated, showed a faster beta-cells failure. PC 3 (explained 12.0% of total variance): gender and triglycerides predominated, showed a slower beta-cells failure. PC 4 (explained 12.0% of total variance): cholesterol predominated, showed a slower beta-cells failure. [/fig]
[table] Table A1: Characteristics of included studies. [/table]
|
Functional characterization of human Heschl’s gyrus in response to natural speech
Heschl's gyrus (HG) is a brain area that includes the primary auditory cortex in humans. Due to the limitations in obtaining direct neural measurements from this region during naturalistic speech listening, the functional organization and the role of HG in speech perception remain uncertain. Here, we used intracranial EEG to directly record neural activity in HG in eight neurosurgical patients as they listened to continuous speech stories. We studied the spatial distribution of acoustic tuning and the organization of linguistic feature encoding. We found a main gradient of change from posteromedial to anterolateral parts of HG. We also observed a decrease in frequency and temporal modulation tuning and an increase in phonemic representation, speaker normalization, speech sensitivity, and response latency. We did not observe a difference between the two brain hemispheres. These findings reveal a functional role for HG in processing and transforming simple to complex acoustic features and inform neurophysiological models of speech processing in the human auditory cortex.This is an open access article under the CC BY license
# Introduction
Heschl's gyrus (HG), also known as the transverse temporal gyrus, is an important part of the human auditory cortex. HG has been suggested as the location of the core auditory area because the cellular structure (koniocortex) and myelination of HG in postmortem tissue indicate dense thalamic input from the medial geniculate body. While these architectonic studies suggest that the primary auditory cortex (PAC) is located in HG, this area is functionally heterogeneous, contains multiple auditory fields, and has high morphological variability across individuals and brain hemispheres . Neurophysiological models of speech processing in the human auditory cortex have postulated a limited role for HG in processing low-level acoustic features. However, due to the limitations in obtaining direct neural measurements from the human auditory cortex, the functional organization and the role of HG in speech perception remain uncertain.
Auditory field maps have been extensively studied in HG using both noninvasive (fMRI) and invasive (iEEG) neurophysiology techniques. These studies have examined the organization of temporal modulation, frequency tuning, response latency, and speech sensitivity in HG. Stimuli such as modulated tones were used to measure temporal modulation tuning in HG to reveal a medial to lateral gradient, both with fMRIand iEEG . Moreover, response latency has been shown to increase from the posteromedial to anterolateral part of HG when listening to click trains and syllables. Studies of tonotopic maps, on the other hand, have not produced consistent results. There are multiple proposed orientations of the tonotopic map in HG, which include perpendicular to HG, parallel to HG, circular, and high-low-high frequency gradient along the posteromedial to anterolateral axes. Responses to pure tones recorded with iEEG have also shown a high-to low-frequency tuning gradient within posteromedial HG. Possible causes for these discrepancies could be the limited temporal resolution of fMRI, different types of synthetic stimuli used, and the limited coverage of HG in iEEG studies. It has also been shown that the lateral part of HG responds more to speech than other soundsand the posteromedial part of HG shows different encoding of temporal modulation of speech than anterolateral HG.
While these studies each sought to isolate and study individual tuning dimensions in HG, collectively, they reveal that sound encoding in HG is multifeatured and varies across several overlapping dimensions that include frequency, temporal modulation, and response latency. In addition, these studies have shown that HG cannot be considered a generic low-level sound processor. Instead, HG contains specialized and distinct computations for processing speech sounds. As a result, synthetic and simple stimuli may only partially activate the neurons in HGand fail to fully capture the integrated and interactive encoding of simultaneously varying acoustic dimensions that coexist in a naturalistic sound such as speech. As a result, much remains unclear regarding the multifeatured functional organization of HG, the relationship between different tuning maps, and the emergence of speech-specific properties in HG. More specifically, I) what is the role of HG in the transformation of acoustic to phonetic features? Is there a change in speech sensitivity, speaker normalization and invariant phonemic representation across HG? These questions are important, as inconsistent results have been reported regarding the transformation from acoustic to phonemic features, which is reported to exist both outside of HGand extend into the core. II) Are the characteristic maps in HG orthogonal to each other as observed in mammalian auditory cortexor similar to those reported in human superior temporal gyrus? III) Is there a difference between tuning properties in the left and right HG, as suggested by recent studies showing lateralized asymmetry in temporal modulation processing?
To answer these questions, we used iEEG to directly measure neural activity in a cohort of 8 neurosurgical patients with comprehensive coverage of left and right hemisphere HG. We measured the neural responses as the patients listened to natural speech stories. We studied the multidimensional tuning properties of HG in response to various acoustic and linguistic attributes and measured the organization of neural responses along several tuning dimensions to create high-resolution maps of response tuning to individual and joint acoustic and linguistic attributes. Specifically, the characteristic maps for best frequency, response latency, temporal modulation, speaker invariance and speech sensitivity were created.
# Methods
## Intracranial recordings
Eight adults (five females) with pharmacoresistant focal epilepsy were included in this study. All subjects underwent chronic intracranial encephalography (iEEG) monitoring at North Shore University Hospital to identify epileptogenic foci in the brain for later removal. All subjects were implanted with depth electrode arrays. All subjects were between 18 and 60 years old. All subjects were fluent speakers of American English and had self-reported normal hearing. In seven subjects, the left hemisphere, and in one subject, the right hemisphere were dominant for language (as determined with the Wada test). Electrodes showing any sign of abnormal epileptiform discharges, as identified in epileptologists' clinical reports, were excluded from the analysis. iEEG time series were manually inspected for signal quality and were free of interictal spikes. All research protocols were approved and monitored by the institutional review board at the Feinstein Institute for Medical Research, and informed written consent to participate in research studies was obtained from each subject before implantation of electrodes. Bifurcations in Heschl's gyrus (HG) were detected by neurologists and neurosurgeons. Subjects who had bifurcations are shown in . The electrode locations were selected by the clinical team for the clinical needs of the patient. The age, sex, language laterality determined using the Wada test, seizure focus, and number of contacts in each HG are shown in . All subjects learned the English language before the age of 5 and have been using it for everyday interactions since then, thus making them fluent speakers. 3 subjects were bilingual, 2 monolingual and 3 had English as primary language but elementary or intermediate proficiency in an additional language.
## Data preprocessing and hardware
Intracranial EEG (iEEG) signals were acquired continuously at 3 kHz per channel (16-bit precision, range ± 8 mV, DC) with a data acquisition module (Tucker-Davis Technologies (TDT), Alachua, FL, USA). Either subdural or skull electrodes were used as references, as dictated by recording quality at the bedside after online visualization of the spectrogram of the signal. Speech signals were recorded simultaneously with the iEEG for subsequent offline analysis. All further processing steps were performed offline. The iEEG data were resampled to 500 Hz. A 1st-order Butterworth high-pass filter with a cutoff frequency at 1 Hz was used to remove DC drift. Line noise at 60 Hz and its harmonics (up to 240 Hz) were removed using 2nd-order IIR notch filters with a bandwidth of 1 Hz. A period of silence lasting two minutes was recorded before the experiments, and all the data were normalized by subtracting the mean and dividing by the standard deviation of this prestimulus period.
The envelope of high-gamma activity, which correlates with neural firing in the proximity of electrodes, was used as a measure of the neural response. To obtain the envelope of this broad-band signal, we first filtered the data into eight frequency bands between 70 and 150 Hz. Then, the envelope of each band was obtained by taking the absolute value of the Hilbert transform. We took the average of all eight frequency bands as the final envelope.
## Stimulus and auditory spectrogram
All stimuli were presented using a single Bose SoundLink Mini 2 speaker situated directly in front of the subject. To reduce the inevitable acoustic noise encountered in uncontrolled hospital environments, all electrical devices in the patients' room were unplugged except the recording devices, and the door and windows were closed during the experiment to prevent interruption.
All subjects listened to speech material containing short stories. Subjects 1 and 2 listened to stories recorded by two voice actors (one male and one female voice actor) with a duration of 25 min and a sampling rate of 16 kHz. Subjects 3 to 8 listened to different stories recorded by four voice actors (stories B: two male and two female voice actors, two of the speakers were common between the two tasks played for subjects 1 and 2 and for subjects 3-8) with a duration of 20 min and a sampling rate of 11,025 Hz. The sampling frequency of our stimulus (11,025 Hz) limits the maximum best frequency that can be measured to 5.5 kHz. However, this limitation is not a major concern as speech has relatively little power above 5 kHz. Male 1, male 2, female 1, and female 2 have average absolute pitches of 92 Hz, 104 Hz, 174 Hz, and 191 Hz, respectively. The stories were played at a comfortable volume customized for each patient. The results were consistent when the responses to the two-speaker stimuli and four-speaker stimuli were analyzed separately.
The time-frequency representation of speech sounds was estimated using a model of cochlear frequency analysisconsisting of a bank of constant 128 asymmetric filters equally spaced on a logarithmic axis. The filter bank output was subjected to nonlinear compression, followed by a first-order derivative along the spectral axis (modeling an inhibitory network) and finally an envelope estimation operation. This resulted in a two-dimensional representation simulating the pattern of activity on the auditory nerve. The output of the filter bank was then resampled to 16 bands.
## Neural spectrotemporal receptive fields
Using the speech stimulus and high-gamma activity recorded from the implanted electrodes, we measured the spectrotemporal receptive field (STRF) of each site. STRF is defined as a filter that predicts the neural responses from the stimulus spectrogram . STRFs were computed by using the normalized reverse correlation algorithm using STRFLab. Regularization and sparseness parameters were found via cross-validation. The best frequency and response latency parameters were estimated by finding the center of the excitatory region of the STRF along the frequency and time dimensions . The best frequency and response latency measured using high-gamma activity were highly correlated with those measured from LFP. The best temporal modulation parameter was estimated from the two-dimensional wavelet decomposition of the STRF. Wavelet decomposition extracts the power of the filtered STRFs at different temporal modulations (rates). The modulation model of STRFs has four dimensions: scale, rate, time, and frequency. To estimate the best temporal modulation, we first averaged the model over three dimensions of time, frequency, and scale to calculate a rate vector. Next, we found the weighted average of the rate vector, where weights are the rate values.
## Electrode inclusion criteria
The neural sites with significant STRF prediction accuracy (Pearson correlation) were included in all subsequent analyses (t -test, false discovery rate [FDR] corrected, . This selection criterion resulted in 132 electrodes in Heschl's gyrus and sulcus across all subjects (68 electrodes in the right hemisphere, 64 electrodes in the left hemisphere). Using these 132 electrodes, the STRFs showed an average prediction correlation of 0.44 ± 0.10 SD . The location of electrodes on an average brain is shown in .
## Speech sensitivity stimuli
To quantify the speech sensitivity of each neural site, six of the subjects (subjects 1, 3, 5, 6, 7, and 8) also performed a speech-nonspeech task (total of 85 electrodes). Subjects passively listened to 30 min of audio containing 69 commonly heard sounds . The sounds consisted of coughing, crying, screaming, different types of music, animal vocalization, laughing, syllables, sneezing, breathing, singing, shooting, drum playing, subway noises, and speech by different speakers. In total, we had 53 unique nonspeech and 16 speech sounds, each presented once (Figs. S3 and S4). The stimulus variety was chosen to cover a broad range of spectrotemporal features using every day sound categories , and corresponding sounds were downloaded from multiple corpora available online (e.g. BBC news speech corpus, RWC music database and freesound.org). All the 69 sounds used for this task are provided as supplementary material. The trials were on average 13.5 s long, and a silence duration of 1 s was added between consecutive trials. The neural data were preprocessed as explained in the "data preprocessing and hardware" section. To determine the speech sensitivity index, we first normalized the response of each site using the mean and variance of the neural data during the silent interval. We then averaged the normalized responses over the presentation of each sound. Finally, we performed an unpaired t-test between the averaged responses of all speech and all nonspeech sounds to obtain a t-value for each site denoting the specificity to speech over nonspeech sounds.
## Brain maps
Electrode positions were mapped to brain anatomy using registration of the postimplant computed tomography (CT) to the preimplant MRI via the postop MRI. After coregistration, electrodes were identified on the postimplantation CT scan using BioImage Suite. Following coregistration, the electrodes were snapped to the closest point on the reconstructed brain surface of the preimplantation MRI. We used FreeSurfer automated cortical parcellationto identify the anatomical regions in which each electrode contact was located within approximately 3 mm resolution (the maximum parcellation error of a given electrode to a parcellated area was < 5 voxels/mm). We used Destrieux's parcellation, which provides higher specificityin the ventral and lateral aspects of the medial temporal lobecompared to Desikan-Killiany parcellation. Automated parcellation results for each electrode were closely inspected by the neurosurgeon using the patient's coregistered postimplant MRI. We mapped each electrode from an individual subject's brain to a standard probabilistic atlas of the human brain from 152 human subjects, which captures intersubject variabilities such as bifurcations and duplications. All contacts localized in HG by FreeSurferand verified from individual patient CT by neurosurgeons were included in the analysis.
To calculate the topographic feature maps for each hemisphere , we used spatial smoothing for each tuning feature. Smoothing was performed by assigning the average of the four closest neighboring electrodes to each site using k-nearest neighbor search (KNN) (Euclidean distance). After smoothing, a piecewise linear interpolation surface was fitted to the values of sites using the MATLAB fit function. To find the combined map for both hemispheres (Figs. 2A, D, 3C, 4A and 5A), the distance of each site to the midsagittal plane was calculated using BioImage Suite. The absolute value of the distance to the midsagittal plane was used as the ML (medial to lateral) distance. For the purpose of readability, we set the minimum ML distance to zero. Spatial smoothing was only performed for visualization of the maps, but all significance tests and scatter plots were calculated using the actual raw values.
For the speech sensitivity map shown in , in addition to the above steps, we also used the method of KNN imputation to fill the values of sites using the nearest neighbor (i.e. k = 1) for the two subjects for which the speech sensitivity task was not played. This method was only used for the purpose of visualization in , and all the significance tests, correlations, and scatter plots were calculated using the actual values without KNN imputation and smoothing. The along HG distance was calculated by projecting electrode locations onto their first principal component (shown by the purple arrow in.
# Phonemes analysis
We segmented the speech material and neural responses into time-aligned sequences of phonemes using the Penn Phonetics Lab Forced Aligner Toolkit. The spectrograms were aligned to the onset of phonemes with a time window of 200 ms previously shown to encode phonetic features in HG. To minimize the preprocessing effects, we did not normalize the natural variation in phoneme length.
## Mds diagram of phonemes
To calculate the MDS (multidimensional scaling algorithm) diagram of phonemes for each speaker using acoustic spectrograms (shown in , we first found the average acoustic spectrogram of all instances of each phoneme spoken by each speaker. Next, the average phoneme spectrogram was windowed between 10 ms and 70 ms after the onset of the phoneme to restrict the time window to a smaller segment that incorporated acoustic differences in the phonetic categories. The duration of the window was chosen according to the maximum peak of the F-statistic between the categories of phonemes using all speakers. The F-statistic is a metric used to measure phonetic discriminability as a ratio of between-class variance to within class variance. Next, we calculated the pairwise Euclidean distance between phonemes, which resulted in a two-dimensional symmetric matrix reflecting a pattern of pairwise phoneme dissimilarities. To visualize this dissimilarity matrix, we used a two-dimensional MDS using Kruskal's normalized criterion to minimize stress for the two MDS dimensions. The MDS diagram of phonemes based on neural networks , H) was calculated using the same method with two differences. First, each instance of a phoneme was based on the segmented neural high gamma response to the phonemes. Second, because of the time delay between the stimuli and the response, the window was set to 90 ms to 150 ms after the onset of phoneme. This window was chosen to maximize the F-statistic of the neural responses to phoneme categories.
## Classification of phonemes and speakers
To examine the encoding of both speakers and articulation features at the population level, we trained a regularized least square (RLS) classifierto predict the speaker or articulation feature of individual instances of the time-locked evoked responses to phonemes(10% of data used for cross-validation). The input to the classifier was the concatenation of all of the neural sites in the HG area with a window of 70 ms to 180 ms after the onset of phoneme. One classifier was trained for subjects 1 and 2, and a separate classifier was trained for subjects 3 to 8 due to the different number of speakers that these subject groups heard. The results of both classifiers were consistent.
## Speaker invariance index
To study the categorical encoding of phonemes in HG, we examined the similarity of the neural response to various phonemes when uttered by different speakers. Specifically, we measured the degree of speaker normalization for each neural site using a phonetic feature classifier (RLS classifier) that was trained on three of the speakers and was tested on the unseen speaker. Because the baseline decoding accuracy depends on the signal-to-noise ratio across neural sites, we divided the phonetic feature classification accuracy for the unseen speaker by the classification accuracy when the classifiers were trained within each speaker. Within speaker phoneme classification was performed by training and testing the phoneme classifier using utterances from the same speaker (10% cross-validation). This normalized phonetic feature classification accuracy on the held-out speaker was defined as the degree of phonemic categorization, and we call it the speaker invariance index.
## Joint spatial organization of characteristic feature tuning
To establish a relationship between the five characteristic maps reported and to find the correlational structure of tuning to various characteristic attributes, we used the method of principal component analysis (PCA). PCA is a dimensionality reduction technique that combines the most correlated dimensions in the data. In our analysis, each characteristic map was considered a feature, and PCA was performed on x = (x 1 , x 2 , …, x M )′, where M (columns) is the number of characteristic maps, x i are vectors of N points, and N (rows) is the number of neural sites. Therefore, the PCA computes the weighted sum of feature maps where weights indicate the correlation of feature maps across all neural sites. To find the dominant direction of change for each PC projection on Heschl's gyrus, we used canonical correlation analysis. Therefore, the plotted best direction represents vector â, where <di>, X is the concatenation of the ML (medial to lateral) and PA (posterior to anterior) distances, and Y is the projected tuning value on the PCs. As a control, assigning random values to Y resulted in vectors (∨) with random direction without preference for any specific site, confirming that the location of neural sites did not have any effect on the calculated best direction.
# Results
We measured five tuning attributes for each neural site: acoustic frequency, temporal modulation, speaker invariance index, speech-nonspeech response difference, and response latency. In all of the analyses, we used the envelope of the high gamma frequency band (70-150 Hz) as the measure of neural response. We first present the result of the tuning analysis for each of the five tuning parameters separately and then show the properties of the joint tuning to all characteristic features.
The spatial organization of neural responses to each characteristic feature for all subsequent analyses is shown along the two directions of PA and ML of HG using the location of electrodes on the average FreeSurfer brain (FreeSurfer template brain, ICBM152). Across all subjects, there were 132 electrodes in Heschl's gyrus and sulcus. Analyzing all subjects together provided adequate coverage of different sections of HG along the axes of PA and ML . We did not observe a clear spatial organization for STRF prediction accuracy . Example STRFs are shown in six of the subjects in , illustrating the diversity of tuning to spectral and temporal characteristic features. For the subsequent analysis in this paper, we combined the left and right HG to generate the characteristic maps.
## Spatial organization of best frequency
The best frequency (BF) parameter for each cortical site is defined as the location of the excitatory peak of the STRF along the frequency dimension. The spatial map of the BF shows multiple areas with tuning to low and high frequencies, including a high-low-high gradient of frequency selective areas creating a "V"-shaped pattern across HG and a high-to low-frequency gradient on the anterolateral part of HG. Despite the mixed frequency tuning patterns in HG, we found an overall gradient of high-to low-frequency tuning that extends from the posteromedial to anterolateral region. This gradient is shown in, where the distance from the posteromedial part of HG (along HG distance) is plotted against the frequency tuning for individual electrodes. The along HG distance is calculated by projecting electrodes' location onto the HG axis (shown with a purple arrow inand measuring the distance from the most medial location on HG. The high-to low-frequency gradient along HG was similar in both left and right HG (Figs. S5 and S6), and there was no significant difference between the distribution of the best frequency between the left and right hemispheres (Wilcoxon rank-sum test, P > 0.1, N left = 64, N right = 68,.
## Spatial organization of best temporal modulation
In addition to the best frequency, the best temporal modulation can also be calculated from the STRF. Temporal modulation distinguishes between slowly and rapidly changing characteristic features. The human perception of sound is highly sensitive to a wide range of temporal modulations, and this acoustic attribute has been shown to be an organizing factor in the human auditory cortex. We defined the best temporal modulation (BTM) of a site as the peak of its STRF wavelet decomposition along the time axis, where the transformed time axis is referred to as the rate (in Hz). The differences between STRFs with different rates are shown with seven examples (slow to fast) in . The spatial organization of temporal modulation tuning in HG is shown in. The significant correlation of temporal modulation tuning with HG distance inillustrates an increase from the posteromedial to anterolateral region. There was no significant difference between the distribution of temporal modulations in the left versus right Heschl's gyrus (Wilcoxon rank-sum test, P > 0.1, N left = 64, N right = 68,. It is also worth mentioning that the values and maps shown inwere consistent when the STRFs were estimated from two nonoverlapping subsets of stimulus-response pairs (test-retest), indicating the reliability of the STRF measurements across stimuli and their robustness to varying degrees of neural noise (Figs. S7 and S8).
## Spatial organization of speaker invariance index
In the previous sections, we studied the organization of the best frequency and temporal modulation. However, understanding human speech is not only dependent on acoustic attributes but also is dependent on the successful decoding of linguistic units. Phonemes are the smallest contrastive units in a language to which the auditory cortex responses show some large-scale spatial organization. One of the major sources of acoustic variability in phones (each instance of phonemes) within the same phoneme category is the difference between different speakers' voices. Normalizing speaker variability is crucial for robust decoding of the phoneme category and therefore the spoken message. At the same time, representing speaker variability is necessary for the successful identification of speakers. Previous studies have shown that a categorical representation of phonemes appears in higher-level cortical areas where the encoding of phonemes becomes less sensitive to perceptually irrelevant acoustic variations (allophones). This emergence of phoneme categories, however, has not been studied in HG.
To examine the extent to which different phonemes and speaker identities are represented in HG, we used the phonetic transcription of speech utterances to obtain time-aligned neural responses to all instances of each phoneme. We first quantified the separability of phonemes and speakers using a linear classifier trained to decode phonetic features and speaker identities (10% cross-validation was used). We restricted the analysis to five representative phonetic attributes that fully measure encoding of the amount of phonetic information. These were the manner and place of articulation features, high-low and front-back vowel distinctions, and a voiced-unvoiced attribute. We found that we could successfully decode all five phonetic features significantly higher than chance from the population of HG responses . To estimate the encoding of speaker differences in HG, we classified the identity of the four speakers based on neural responses to individual phonemes. We found that the speaker differences were also decodable significantly above chance in the population responses . The confusion matrices for classification of both the phonetic features and the speaker identity are shown in .
To find the degree of phoneme encoding at each neural site, we defined a speaker invariance index (SI) that measures the invariance of phoneme encoding to different speakers (details in methods). We found that categorical phoneme encoding increased towards the anterolateral part of HG. The spatial organization of speaker-invariant phoneme encoding in HG is shown in This figure shows two distinct encoding schemes in the anterolateral and posteromedial parts of HG. The majority of sites in anterolateral HG (AL area in show a higher categorical and less speaker-dependent encoding of phonemes compared to the posteromedial part of HG (PM area in . This analysis shows an increase in categorical representation of phonemes towards the anterolateral part of HG. Similar to the previous maps, phoneme encoding was also significantly correlated with the along HG distance of electrodes . There was no difference between the degree of phonemic encoding and speaker normalization between left and right HG (Wilcoxon rank-sum test, P > 0.1, N left = 64, N right = 68, .
To further illustrate the population encoding of phonemes and speakers in posteromedial and anterolateral HG (shown in , we examined the relative distance between four representative phonemes of /UW/, /L/, /AO/, and /OW/ spoken by all four speakers. We used the first two multidimensional scaling (MDS) dimensions of phoneme responses to express their relative distances in the acoustic space and in the neural space . The population response in posteromedial HG shows a clear separation between the responses to different phonemes and speakers , similar to the representation of phonemes in acoustic space . The four speakers are shown with different colors in the MDS diagram. In contrast to posteromedial HG, the population responses in anterolateral HG still group the phonemes of the same category together, but the separation between the phonemes of the four speakers is no longer preserved. This effect can be quantified for all phonemes and neural sites using the discriminability of the phoneme's manners of articulation and speaker identities for acoustic spectrograms and the population of sites in the PM area and AL area (the MDS diagram for all phonemes is shown in . We observed that while the discriminability (defined as the F-ratio,of speaker identities and manner of articulation is similar in acoustic space, the discriminability of speaker identities is significantly higher than the discriminability of manner of articulation in posteromedial HG, whereas the exact opposite is true for anterolateral HG . Together, these results show that the anterolateral part of HG encodes a more categorical representation of phonemes by normalizing the difference between speaker voices. In comparison, posteromedial HG strongly encodes speaker-specific differences.
## Spatial organization of speech sensitivity
We observed that phonemic encoding increases towards the anterolateral region of HG. Since phonemes are specific to human speech, we tested whether anterolateral sites in HG also respond preferentially to speech over non-speech sounds. We defined the speech sensitivity of a neural site as the t-value of a t-test between the average response of the site to speech versus nonspeech sounds, which in our study consisted of 69 sounds (53 non-speech, 16 speech) from 14 categories . The speech sensitivity values are shown on HG in which shows the highest values on the anterolateral side of HG. We found that 39% of electrodes in HG were significantly more responsive to speech than other sounds (t-test, FDR corrected, q < 0.01). To confirm that speech sensitivity is not a consequence of simple acoustic tuning and requires nonlinear transformation of the sound, we compared the actual and predicted speech sensitivity using electrodes' STRFs. The speech sensitivity calculated from actual neural data was significantly higher than speech sensitivity calculated from STRF predictions . This shows the failure of the linear STRFs to account for the speech sensitivity of sites, and confirms that this response characteristic requires nonlinear signal processing which simpler acoustic attributes such as frequency and temporal modulation tuning cannot account for.
We examined the spatial organization of speech sensitivity using its correlation with along HG distance of electrodes. The significant correlation shown in (r = 0.21) confirms a strong gradient of speech sensitivity from the posteromedial to anterolateral part of HG. Speech sensitivity in both the left and right Heschl's gyrus was significantly higher than zero, and we did not observe any difference between speech sensitivity values in the left vs. right Heschl's gyrus (Wilcoxon rank-sum test, P > 0.1, N left = 37, N right = 48, .
## Spatial organization of response latency
The latency of the response along the auditory pathway approximately reflects the number of synapses away from the auditory periphery and hence has been used to speculate the direction of information processing in the auditory cortex (Da. We defined the response latency of neural sites as the excitatory peak of the STRF along the time dimension. The observed response latencies varied from 30 to 200 ms in different parts of HG, where it was lowest in the posteromedial part and gradually increased towards the anterolateral part . This gradient is shown in , where latency is plotted against the HG location (left vs. right is shown in Figs. S5 and S6). There was no significant difference between the distribution of latency in the two hemispheres .
## Multivariate organization of characteristic feature tuning
Neurons in the auditory cortex have multidimensional and joint tuning to different characteristic attributes. Our analysis thus far focused on the anatomical organization of tuning to individual characteristic attributes, as summarized in. This figure shows a correlated multidimensional organization of tuning maps to individual characteristic features. Therefore, a joint analysis of the individual tuning maps can offer further and complementary evidence for the organization of auditory fields and the main gradients of tuning change in HG .
We used an unsupervised approach to examine the organization of joint tuning and to determine the dominant anatomical directions of tuning changes in HG. We performed a principal component analysis (PCA) on tuning to all five acoustic attributes across the HG sites. The PCA therefore summarizes the correlation patterns among the tuning to individual acoustic attributes. We found that the first and second principal components of tuning values can account for 63% of the variance (40% and 23%, respectively; third and fourth PCs are included in. The weights of the first two PCs are shown in,C. The first PC shows that across all HG sites, a positive correlation exists between tuning to frequency and temporal modulation, which is negatively correlated with response latency, speech sensitivity, and phoneme encoding. The projected tuning values on the first PC are shown infor all HG sites, where the dominant direction of change is calculated using canonical correlation analysis. This analysis finds the linear combination of ML and PA distances that has the maximum correlation with projected tuning values on the PCs. This unsupervised method shows that the direction that best describes the joint functional maps runs along the axis of HG. Because the first PC assigns significant nonzero weights to all acoustic attributes, this direction can be interpreted as the main axis along which frequency and temporal modulation tuning increase, while latency, speech sensitivity, and speaker invariance decrease. The second PC shows the second main correlation pattern among the tuning maps and reveals a positive correlation between tuning to frequency and response latency. The projected attributes on the second PC are shown in, where the direction of maximum change is orthogonal to the HG long axis, resulting in a secondary dominant axis of the tuning gradient in HG. We further controlled the effect of intersubject variability by first creating a standard deviation map across subjects for each characteristic map to show that there is an absence of a unified direction of change. Second, a linear mixed effects model analysis showed that the characteristic maps for best frequency, response latency and temporal modulation hold when controlling for individual subject identity . The maps for speaker invariance and speech selectivity show greater dependence on particular subjects' data. Moreover, the maps are not influenced by the responsiveness of the electrodes' high gamma activity to the stimulus .
# Discussion
We examined the spatial organization of multiple tuning attributes in human HG in response to continuous speech. We found specific spatial maps for frequency, response latency, temporal modulation, speech sensitivity, and phonemic encoding in HG. Our results suggest that the best frequency and temporal modulation tuning decrease in the posteromedial to anterolateral direction in HG. In contrast, the response latency, speech sensitivity, and phoneme encoding increased along this HG direction. We also analyzed the properties of the joint tuning to all acoustic attributes and showed two prominent directions that explain the majority of correlated tuning changes, one along the PM-AL axis of HG and the other orthogonal to the axis. Compared to previous studies that either used unnaturalistic stimuli such as tones, ripples, or consonant-vowel syllable stimulior had limitations in the resolution of neural measurement methods, using direct intracranial recordings, our naturalistic speech stimuli revealed multidimensional feature tuning in HG that organizes the responses in this auditory region. Specifically, we could add insights into the characteristic maps for speaker invariance and categorical representation of phonemes in HG, the role of HG in transformation from simple to complex acoustic features of speech, and the relationship between the characteristic maps.
## Organization of characteristic frequency
Tonotopy, the spatial arrangement of frequency selectivity, is one of the fundamental organizing principles in the mammalian auditory cortex. Previous research that attempted to find the orientation of tonotopic maps in human HG is, however, inconclusive. Several fMRI studies that used tones and artificial stimuli showed multiple frequency-selective areas in HG. The cumulative evidence suggests that HG is located within a high-low-high gradient of frequency selective regions that create a "V"-shaped pattern. Beyond this main high-low high frequency gradient, the orientation of this tonotopy and the number of frequency selective areas have been the subject of scientific debate. On the one hand, studies have reported a collinear orientation of a high-low-high frequency gradient along HG, and on the other hand, studies have proposed that tonotopic progression runs perpendicularly across HG rather than parallel along HG (Da. Moreover, apart from the main high-low-high frequency gradient, an additional low-frequency region is often reported at the antero-lateral border of the main gradient on the anterior superior temporal sulcus or planum porale. Among the proposed maps of frequency tuning, our main result obtained from direct recordings mostly agrees with, which used tone pips and 7T fMRI measurements to report multiple subregions of low-and high-frequency selective areas in HG compared to the previously mentioned studies. It is worth mentioning that we do see a trend of V-shaped low-high-low frequency selective areas, but recordings from a higher number of subjects might result in increased smoothing of frequency tuning maps and can highlight these effects, similar to a previous report.
## Organization of temporal modulation tuning
Several previous studies have shown an encoding of temporal modulations in the human auditory system. Studies that used ripple stimuli showed that the preferred temporal rate was highest in medial HG. These studies, however, did not provide a precise spatial organization of temporal modulation in HG. An organized representation of temporal modulation tuning has previously been reported in the superior temporal gyrus. Here, we showed that HG also has a topographic representation of temporal modulation rates that decreases from posteromedial to anterolateral HG.
## Organization of speaker invariance index
The frequency and temporal modulation tuning measures were based on a linear model of stimuli-response relationship (the STRF model), which has been commonly used to characterize the tuning of auditory cortical neurons. The higher auditory cortical regions, however, become progressively more nonlinear. The inadequacy of linear models in such cases necessitates complementary and model-independent methods to characterize response properties. To achieve this task, we extended our linear tuning framework by examining preferential tuning to speech and phoneme and speaker encoding. By measuring speaker invariance across phonetic features, we showed that speaker-invariant encoding of phonemes increases from posteromedial to anterolateral HG. This invariant encoding of phonemes suggests a processing step in creating categorical representations of phonemes in which the acoustic variability of phones imposed by different speakers is reduced. While previous studies have shown the emergence of categorical phoneme representation in the cortical surface of the superior temporal gyrus (STG), our results from depth electrodes suggest that phonemic representations also appears on the superior temporal plane. This is congruent with the studies that show that the anterolateral Heschl's gyrus is as late in the hierarchy as the posterior parts of superior temporal gyrus. A categorical representation of phonemes involves more than just speaker normalization. Other sources of variability, such as contextual and prosodic variations in phones, should also be normalized to form phonemic categories. This is particularly true for more confusable allophonic variation of phonemes that may not be fully resolved in an early processing stage such as HG. Comparison of phoneme normalization in HG, planum temporale (PT), and STG may shed light on the progressive appearance of these linguistic units.
## Organization of speech sensitivity
Specialization of the human auditory cortex for speech processing has long been established. Previous fMRI studies have shown that the lateral part of HG responds more to speech than to other sound categories and speech-like artificial stimuli. Our results showed that 40% of sites in HG responded preferentially to speech over nonspeech sound categories, and this speech sensitivity was highest in the anterolateral part of HG. Our observation supports the possibility that anterolateral HG might be a higher auditory field than posterior STG. These findings are intriguing, particularly because the cytoarchitecture has shown the anatomical proximity and cytoarchitectonic similarity of lateral regions of Heschl's gyrus to the medial regions. Further research that allows for the joint analysis of anatomical and functional properties of human HG can result in a better definition of the core auditory cortex that is based on both functional and anatomical properties of the regions.
## Left and right hemisphere differences
Functional asymmetries in the human auditory cortex have long been debated in the field of neuroscience. In initial reports of Broca and Wernicke areas, it was shown that damage to cortical regions in the left hemisphere impaired speech comprehension, but this was not the case when the damage was on the right side. It has also been shown that a lesion of the right HG disturbs sound localization performance on both sides of space, while this is not the case for the left HG. Moreover, the neuroanatomy of the superior temporal plane shows asymmetry, where HG and PT are larger on the left side. In contrast to the historically established view of left lateralized speech comprehension, recent studies have argued for bilateral involvement of the STG in speech perception and production. While speech can be processed bilaterally, it does not rule out the possibility of functional and computational specialization in the left and right hemispheres. For example, recent studies showed differential activation of the left and right hemispheres, where temporal and spectral modulation processing was lateralized in the left and right hemispheres accordingly. It has also been shown that asymmetric processing of temporal and spectral modulation will result in an asymmetric emergence of speech and music representation in the auditory cortex. It is worth noting that these studies selectively filtered out spectral and temporal modulation of speech and music, resulting in synthetic and unnatural stimuli that may activate the auditory cortex differently. In contrast, we did not find any difference between the functional processing of left versus right HG in the processing of acoustic attributes. This lack of difference may suggest bilateral speech processing in HG. Further research is needed to clarify whether the lateralization reported in previous studiesalso occurs during naturalistic speech perception.
## Organization of response latency
The latency of the response at a neural site approximates the number of synapses that the neural response to sound has to travel before reaching that site. As such, we would expect a primary region such as the core auditory area to have shorter latencies in comparison with non-primary regions such as belt and parabelt areas. In nonhuman primates, it has been confirmed that caudal belt and parabelt areas have shorter response latencies than rostral areas. One advantage of using direct neural measurements in our study compared to fMRI is the ability to measure the response latency with a high degree of precision. We observed a wide range of response latencies in HG from 30 ms to 200 ms. Similar to the frequency map, the main orientation of latency increase runs along the PM to AL axis of HG. This result supports the notion that the primary auditory cortex is located in the posteromedial part of HG. Nevertheless, the human auditory cortex is more complex than nonhuman primateswith multiple core and noncore areas of auditory cortex receiving thalamic inputs from different subdivisions of the medial geniculate complex, and early activity observed in HG could potentially reflect direct activation from auditory thalamus as opposed to that of intracortical synapses. As such, a conclusive separation of core vs noncore areas in humans requires simultaneous anatomical and functional analysis of HG.
## Spatial organization of joint tuning properties
While the majority of previous research has examined the spatial organization of individual and isolated acoustic attributes, neurons in the mammalian auditory cortex have complex and multifeatured tuning properties. It is therefore crucial to examine the joint distribution of tuning properties to gain a more complete understanding of auditory field organization, particularly because a single tuning dimension may yield ambiguous separation of auditory fields. Here, we adopted an unbiased and unsupervised approach to find the primary and secondary correlational structure of tuning to various acoustic attributes. Our analysis uncovered two anatomical directions. The first direction runs from posterior-medial to anterior-lateral HG (labeled along HG inand shows a gradient of change characterized by tuning to progressively lower frequencies and temporal modulation rates, increased latencies, speech sensitivity, and speaker invariance. The second axis, which was orthogonal to the previous HG axis (labeled across HG in, showed a gradient of change in frequency (low to high to low) and response latency. As such, we found both directions, medial to lateral and posterior to anterior, to be important in capturing the change in multidimensional acoustic feature tuning in HG. Although these characteristic maps share the same direction of change, additional analysis revealed the characteristic maps to be independent .
Relating the functional properties of neural responses in HG to the underlying anatomy remains challenging. The direction of functional change that we observed along HG is consistent with the direction of anatomical gradients that are found using combined cytoand receptor architectonic maps. These structural studies divided HG into three areas, Te1.1, Te1.0 and Te1.2, which extend along the HG axis. The second direction of change, orthogonal to HG, is also consistent with anatomical changes, where the Te.1 region is surrounded by Te2.1 and TI on its sides. On the other hand, while a number of structural studies have shown left dominant asymmetry in the volume of HG , we did not find a difference between the functional properties of left and right HGs in processing acoustic attributes. In summary, our results provide a comprehensive view of multidimensional acoustic processing in HG and pave the way towards a more complete functional characterization of auditory fields in the human auditory cortex.
# Supplementary material
Refer to Web version on PubMed Central for supplementary material.
## Fig 1. spectrotemporal receptive fields (strfs) calculated from 132 sites in heschl's gyrus (hg).
A) Natural speech stories were played to the subjects, and the spectrotemporal receptive field (STRF) was calculated for each electrode. B) The histogram of correlation values between predicted and actual responses across electrodes. C) The locations of electrodes on Heschl's gyrus and sulcus are shown on an average FreeSurfer brain. The colors indicate the correlation values between predicted (20-fold cross validation) and actual response. D) STRFs of 32 example electrodes are shown for six subjects on the left (bottom row) and right (top row) HG. A) Spatial organization of frequency tuning (Freq), response latency (Lat), temporal modulation (TM), speech sensitivity (SS) and phonemic encoding (PE). Plots are discretized to one-third of the maximum value to maximum and one-third of the minimum value to the minimum. B) The first principal component of joint tuning maps. The weights of the first PC are shown on the left (error bars indicate standard error calculated by bootstrapping the neural sites), and projection of tuning parameters onto the first PC is shown for all sites (middle). The first PC projection versus location along the HG axis is shown on the right. Red curve is the binomial fit. C) The second principal component of characteristic maps: the weights of the second PC are shown on the left (error bars indicate standard error calculated by bootstrapping the neural sites), projection of tuning parameters onto the second PC is shown for all sites in HG (middle), and second PC projection versus location on the orthogonal HG axis is shown on the right. Red curve is the binomial fit. Table 1 Demographics, language laterality and seizure focus information.
The age, sex, language laterality, seizure focus location, number of contacts and anatomical classification of HG are shown for each subject. No subject had a seizure focus in HG. The language-laterality was based on the WADA test. The number of contacts shows the number of contacts that were in the HG area and were responsive to speech. For anatomical classification, number 1 represents anatomical structure containing one single, smooth HG. Number 2 represents partially divided HG, meaning that it has lateral sulcus intermedius, but a common stem is intact. Number 3 represents fully divided HG. No subject showed a fully divided HG. |
Performance indicators for initial regional medical response to major incidents: a possible quality control tool
Background: Timely decisions concerning mobilization and allocation of resources and distribution of casualties are crucial in medical management of major incidents. The aim of this study was to evaluate documented initial regional medical responses to major incidents by applying a set of 11 measurable performance indicators for regional medical command and control and test the feasibility of the indicators.Methods: Retrospective data were collected from documentation from regional medical command and control at major incidents that occurred in two Swedish County Councils. Each incident was assigned to one of nine different categories and 11 measurable performance indicators for initial regional medical command and control were systematically applied. Two-way analysis of variance with one observation per cell was used for statistical analysis and the post hoc Tukey test was used for pairwise comparisons.Results: The set of indicators for regional medical command and control could be applied in 102 of the130 major incidents (78%), but 36 incidents had to be excluded due to incomplete documentation. The indicators were not applicable as a set for 28 incidents (21.5%) due to different characteristics and time frames. Based on the indicators studied in 66 major incidents, the results demonstrate that the regional medical management performed according to the standard in the early phases (1-10 min after alert), but there were weaknesses in the secondary phase (10-30 min after alert). The significantly lowest scores were found for Indicator 8 (formulate general guidelines for response) and Indicator 10 (decide whether or not resources in own organization are adequate).Conclusions: Measurable performance indicators for regional medical command and control can be applied to incidents that directly or indirectly involve casualties provided there is sufficient documentation available. Measurable performance indicators can enhance follow-up and be used as a structured quality control tool as well as constitute measurable parts of a nationally based follow-up system for major incidents. Additional indicators need to be developed for hospital-related incidents such as interference with hospital infrastructure.
# Background
Despite the fact that lessons learned from major incidents and disasters in the past have resulted in many improvements, shortcomings still exist [bib_ref] Recurrent medical response problems during five recent disasters in the Netherlands, Juffermans [/bib_ref] [bib_ref] Performance indicators for major incident medical management-a possible tool for quality control, Rüter [/bib_ref]. We know from incidents involving casualties that a rapid response, accurate triage and controlled evacuation and distribution of casualties are important factors that influence the outcome for the victims [bib_ref] Distribution of casualities in a masscasuality incident with three local hospitals in..., Bloch [/bib_ref] [bib_ref] Mass casualty incident management, triage, injury distribution of casualties and rate of..., Rater [/bib_ref] [bib_ref] Reduction of critical mortality in urban mass casuality incidents:analysis of triage, surge,..., Aylwin [/bib_ref] [bib_ref] Collaborating group: Oslo government district bombing and Utoya island shooting, Sollid [/bib_ref]. Other studies have shown that regional coordination of medical resources improves patient flow, reduces time to definitive care and thereby improves patient outcome [bib_ref] A regional medical operations center improves disaster response and inter-hospital trauma transfers, Epley [/bib_ref]. Even though there are differences between countries, this level of management is often referred to as strategic management, gold level or regional medical command and control [bib_ref] Distribution of casualities in a masscasuality incident with three local hospitals in..., Bloch [/bib_ref] [bib_ref] Reduction of critical mortality in urban mass casuality incidents:analysis of triage, surge,..., Aylwin [/bib_ref].
The Swedish National Board of Health and Welfare (NBHW) has issued regulations for how the medical management of major incidents and disasters should be carried out. The term major incident is used in the Swedish system as a generic response term for different types of events including risk and threat situations, e.g. transportation accidents, spread of hazardous material, infrastructure disruptions, armed aggression, and psychosocial impact on society as a result of traumatic events. The decision to declare a major incident is made by a designated duty officer (DDO) at the regional level and is influenced by the type and magnitude of the incident, and what potential impact the event might have on health care [fig_ref] Figure 1: Schematic model of Swedish medical incident command and control system at major... [/fig_ref].
The indicators used in this study are derived from a national concept and process modelling of management in major incidents and disasters, conducted by the NBHW and have been described in two previous studies [bib_ref] Performance indicators -from theory to implementation; one method of scientific approach to..., Rüter [/bib_ref] [bib_ref] The development of a national doctrine for management of major incidents and..., Rüter [/bib_ref]. The measurable performance indicators extracted from this process have been used for many years in Swedish disaster management training. In addition, the same indicators have been used in an international study and as an evaluation tool in full-scale exercises [bib_ref] Increasing emergency medicine residents' confidence in disaster management: use of an emergency..., Franc [/bib_ref] [bib_ref] Performance indicators for prehospital command and control in training of medical first..., Rüter [/bib_ref] [bib_ref] Quality control in disaster medicine training-initial regional medical command and control as..., Nilsson [/bib_ref] [bib_ref] Evaluation of medical command and control using performance indicators in a full-scale,..., Gryth [/bib_ref].
Indicators for quality control are well-established within most areas of health care, but there is still a need for their further development and implementation in the field of disaster medicine. One way to address these issues is to study if performance indicators for initial regional medical command and control can be used as a quality control tool, and thereby could be included in regional medical response plans and constitute measurable parts of a nationally based follow-up system for major incidents.
The aim of this study was to evaluate documented initial regional medical response to major incidents by applying a set of 11 measurable performance indicators for regional medical command and control and test the feasibility of the indicators.
# Methods
This was a retrospective observational study of 130 major incidents occurring in two County Councils in Sweden between 2006 and 2009. Data in this study were collected from two County Councils who had fully implemented the national medical incident command and control system The personnel acting as DDO in these two County Councils are similar in terms of competencies and background. They have a clear regional mandate to declare major incidents and to take immediate medical decisions over all regional medical resources [fig_ref] Figure 1: Schematic model of Swedish medical incident command and control system at major... [/fig_ref]. The two County Councils are located in one of Sweden's largest metropolitan areas after Stockholm, Gothenburg and Malmö, with approximately 699 000 inhabitants living in urban and rural areas.
## Medical command at
All available documentation from the regional command and control for 130 incidents, all declared as a major incident, were studied with regard to type of incident, staff resources required for regional management, and how long the regional management body remained active. All incidents studied were classified into the following nine categories: accidents; fires; interferences with hospital infrastructure; chemical, biological, radiological, nuclear, and explosive (CBRNE) events; infectious events; weather alerts; support asked from another region; and incidents abroad with a regional impact [fig_ref] Table 1: Classification of 130 major incidents that occurred in two Swedish County Council... [/fig_ref].
A set of 11 previously developed measurable performance indicators for assessing initial regional medical command and control were systematically applied [bib_ref] Performance indicators -from theory to implementation; one method of scientific approach to..., Rüter [/bib_ref] [bib_ref] The development of a national doctrine for management of major incidents and..., Rüter [/bib_ref]. Each indicator was given a score of 0, 1 or 2 points; 0 = objective was not met at all, 1 = objective met but not within the stipulated time frame, 2 = objective completely met within stipulated time frame. The average score for each indicator was calculated.
## Statistics
A two-way analysis of variance with one observation per cell and the post hoc Tukey test for pairwise comparisons were used. A p value <0.05 was considered significant. Minitab version 16 (Minitab Inc W , www.minitab. com) was used for the statistical calculations.
# Results
# Descriptive results
During the period from 2006 to 2009, 130 incidents were declared as major incidents. Regional medical command and control was established in various types of incidents [fig_ref] Table 1: Classification of 130 major incidents that occurred in two Swedish County Council... [/fig_ref]. Approximately 1229 casualties (range 3-135/incident) were directly involved in 102 major incidents (78%) classified as accidents, fires, threats and CBRNE events. In 35 major incidents (27%), casualties were distributed to more than one hospital and in 15 major incidents (11.5%) one or more hospitals activated their hospital disaster plan. Study case flow. *Interruptions in hospital infrastructure, infectious events, incidents abroad, regional support asked from other region, weather alerts. Regional medical command and control was established by the DDO alone in 50 of the 130 major incidents (38%), and in 36 major incidents (28%), a specific regional medical officer (physician) was also alerted. In 34 major incidents (26%), one or more staff positions were called to support the management, such as experts on public information and communication, psychological trauma support or other experts in specific medical or management fields. A more comprehensive regional medical management group consisting of an increased number of staff positions was established in 10 major incidents (7%) . In 98 major incidents (75%), the regional medical command and control was active for 4 h or less (median time 60 min) [fig_ref] Figure 4: Length of time regional medical command and control were active in 130... [/fig_ref]. The documentation for 36 major incidents was incomplete for accurate evaluation.
## Performance indicators
The indicators were not applicable as a set in 28 incidents (21.5%) due to different characteristics and time frames. These incidents involved interference with hospital infrastructure requiring regional support (power failure, IT disturbance, phone interruptions), an incident occurring in another region (evacuation of in-hospital patients), an incident abroad having a regional impact (evacuation of Swedes from Lebanon), weather alerts (storms), and infectious events (suspected water contamination, mass vaccination during the H1N1 flu pandemic). The 11 measurable performance indicators assessing the initial regional medical command and control were applied in 102 major incidents (78%) in the following categories: accidents, fires, threats and CBRNE events. Thirty-six of the 102 major incidents were excluded due to incomplete documentation .
A total of 726 measurable performance indicators were collected from 66 major incidents involving accidents, fires, threats and CBRNE events. Four hundred and forty-six indicators (61%) were met completely or partly, and in 280 indicators (38%), the objective was not met at all.
The mean score for each performance indicator ranged from 0.03 to 1.63 out of a maximum score of 2. Indicator 4 and Indicator 5 had the highest mean values. Indicator 8 and Indicator 10 had the lowest mean values.
Comparison of the results shows that performance indicators measuring decisions in the early phase of an incident (1-10 min after alert) had a significantly higher mean score than indicators measuring decisions in the secondary phase (e.g. 10-40 min after alert) (p<0.05). Performance Indicator 8 (formulate general guidelines for response) and Indicator 10 (decide if resources in own organization are adequate) differed significantly from Indicators 1, 2, 3, 4, 5, 6, 7, 9, and 11 (p<0.05). There was no significant difference between Indicator 3 (decision on additional resources to the scene) and Indicator 11 (notify decision for referral to receiving hospital) [fig_ref] Figure 5: Comparison of scores from 11 different performance indicators in 66 MI [/fig_ref].
# Discussion
This was a study of the initial regional response to major incidents built on traceability and in compliance with a specific protocol of 11 measurable performance indicators. In the final systematic review of 66 major incidents, all performance indicators could be applied and used for assessing the regional medical response. The difficulty with the collection of data was mostly due to the lack of documentation. A decision may have been made or considered but unfortunately never documented. A prerequisite is that the indicators are known and accepted by the organizations and that the documentation is detailed enough.
Initial actions taken (often by the DDO and a regional medical officer-physician) within the first 10 min, such as declare major incident, alert to receiving hospitals and establish contact with medical command on scene, DDO DDO + Regional MO DDO + Regional MO + one or more staff functions* DDO + Regional MO + all staff functions* Number of major incidents Extent of regional medical command and control in 130 major incidents. DDO, designated duty officer. Regional MO, medical officer at regional level. Staff functions; experts on public information and communication, psychological trauma support, hospital infrastructure and other administrative or medical support.. Numbers that lie below the same horizontal line are not significantly different from each other. For example: Scores from Indicators 8 and 10 differ significantly from the rest of the indicators .
were often done correctly and on time. Decisions made after 10 min, usually concerning the distribution of casualties, were often somewhat delayed. The reason for this could be that the DDO had to wait for reports and additional information from the medical incident command on the scene. Our study of the regional documentation files revealed that the prehospital reports were sometimes not sent at all, or not according to standard operating procedures (e.g. first report within 3 min and a second verifying report within 10 min of arrival on the scene) resulting in the DDO being forced to obtain the information via the emergency dispatch centre instead. This unnecessary procedure could be one of the factors affecting the ability to make decisions at the right time.
Delays in decisions concerning distribution of victims might not be fatal in a minor incident, but can be crucial in situations with a more rapid course of events with a risk of overloading the nearest hospital [bib_ref] Mass casualty incident management, triage, injury distribution of casualties and rate of..., Rater [/bib_ref] [bib_ref] Field triage and patient maldistribution in a mass-casualty incident, Zoraster [/bib_ref] [bib_ref] Defining the problem, main objective, and strategies of medical management in mass-casualty..., Ashkenazi [/bib_ref] [bib_ref] Primary triage, evacuation priorities, and rapid primary distribution between adjacent hospitals-lessons learned..., Pinkert [/bib_ref]. Several studies on incidents involving casualties show that effective casualty distribution plays a vital role in disaster management, especially if the incident occurs in a rural area where resources are limited [bib_ref] Distribution of casualities in a masscasuality incident with three local hospitals in..., Bloch [/bib_ref] [bib_ref] Reduction of critical mortality in urban mass casuality incidents:analysis of triage, surge,..., Aylwin [/bib_ref] [bib_ref] Primary triage, evacuation priorities, and rapid primary distribution between adjacent hospitals-lessons learned..., Pinkert [/bib_ref] [bib_ref] lessons for the international EMS community, Lockey [/bib_ref].
Another well-known truism for disasters is that the hospital or health care facility closest to the incident site will be the one most significantly affected by a large number of casualties and when timely notification is lacking, the hospitals will need to respond with the resources on hand. In addition, not seriously injured casualties self-refer to the hospital they are most comfortable with. [bib_ref] Disaster preparedness: health care is ready, but is the bureaucracy?, Mattox [/bib_ref] [bib_ref] Emergency department impact of the Oklahoma City terrorist bombing, Hogan [/bib_ref]. In this study, we found that the casualties were distributed to more than one hospital according to a distribution key delivered by the regional medical command and control in 35 major incidents. Therefore, execution of a planned and timely distribution from a regional overall health care perspective can be beneficial, thus reducing the impact on daily activities and patient surge following an unnecessary activation of a local hospital disaster plan [bib_ref] Field triage and patient maldistribution in a mass-casualty incident, Zoraster [/bib_ref].
The regional command and control alerted a neighbouring county on one occasion only. The reason could be that the major incidents were not of such magnitude or there was no other reason to request resources from another region. It may also be that this decision was considered, but was not documented in the log file. However, there may also be a fundamental barrier such that neighbouring counties are only alerted when resources begin to run out. In a minor incident, it is probably enough to distribute casualties to the hospitals within one county, but in a major incident involving a large number of casualties, early contact with neighbouring counties can be crucial, particularly when higher levels of trauma care are required [bib_ref] Field triage and patient maldistribution in a mass-casualty incident, Zoraster [/bib_ref]. The significantly low mean score for Indicator 11 suggests that the importance of an early alert and establishment of cooperation between County Councils needs to be stressed even more in education and training.
Another weakness observed in regional management was the absence of formulating guidelines for response, or in other words, taking a set of objectives and designing a strategic plan to mitigate any consequences of the incident. In simulation exercises, this type of strategic decision making has also been shown to be one aspect of regional management that needs to be improved [bib_ref] Quality control in disaster medicine training-initial regional medical command and control as..., Nilsson [/bib_ref]. However, such a plan might have been considered but was not recorded in the log file. This may be very difficult to achieve in the early intensive phase of a major incident, but could have vital consequences for subsequent direction and evolution and could influence patient outcome. This emphasizes the need for more preparatory training and education for staff involved in strategic and goal-oriented decision making at the regional level of medical command.
The study demonstrates that a DDO at the regional level of health care has to deal with several types of major incidents, all with different characteristics and time lines. The experience from Khorram-Manesh et al. [bib_ref] Hospital-related incidents; causes and its impact on disaster preparedness and prehospital organisations, Korram-Manesh [/bib_ref] showed that incidents that interfere with the hospital infrastructure such as power or IT system failure also have an impact on regional preparedness and that the frequency of these types of hospital-related incidents has increased. The performance indicators used in this study were not applicable to these types of incidents. Even though an individual indicator (e.g. declare major incident) could be applied, most of the other indicators would have to be adjusted with regard to other objectives and time standards.
This study shows that in 77% of incidents, the regional medical command and control was active for 4 h or less and most incidents were handled by the DDO, a regional medical officer (physician) with one or two staff functions. This emphasizes the need to pay special attention to the important time perspectives when building up a regional response organization with regard to response time and the medical competencies needed to handle all types of major incidents.
## Limitations of the study
Our study has some limitations due to its retrospective design and the lack of coherent incident documentation. Although correct and relevant documentation of incident management is a prerequisite for evaluation and follow-up, lack of documentation is a common problem in disaster evaluation studies [bib_ref] Characteristics of medical surge capacity demand for sudden-impact disasters, Stratton [/bib_ref] [bib_ref] Analysis of the pre-incident education and subsequent performance of emergency medical responders..., Welling [/bib_ref]. This study was also limited to major incidents with sufficient documentation and therefore we cannot rule out the risk of selection bias. However, what we do know is that the category distribution of the dropouts does not differ from the rest and the indicators could have been applied if documentation had been more comprehensive. In the future, the implementation of digital support systems that can provide real-time data, capture information and share it along the chain of medical command might increase the efficiency and resource management and also facilitate follow-up at all levels [bib_ref] Evacuation support system for improved medical documentation and information flow in the..., Walderhaug [/bib_ref] [bib_ref] Data quality for situational awareness during mass-casualty events, Demchak [/bib_ref].
In summary, in order to implement an effective quality control of response to major incidents, specific standards for the regional medical response needs to be set. The quality control process of regional medical response at major incidents must be ongoing to ensure effective response and to early detect deficiencies that continuously leads to quality improvements.
Measurable performance indicators enable a structured and objective evaluation of incident management, can identify areas for improvement, and could facilitate a systematic follow-up of major incidents. Further prospective studies are needed to examine if the time taken for regional decisions about distribution of casualties correlates with patient time at the scene, time to definitive care and patient outcome.
# Conclusions
Measurable performance indicators for initial regional medical response are feasible to use as a quality control tool provided that there is sufficient documentation available. The indicators can be applied on major incidents that directly or indirectly involve casualties and could constitute measurable parts of regional and national follow-up systems. Modification of the present indicators and additional indicators might be needed to assess hospital-related incidents. Future introduction of digital information and support systems for incident management could provide more accurate and coherent documentation to support follow-up of major incidents at all levels.
## Competing interests
This work was performed by the Centre of Teaching in Disaster Medicine and Traumatology (KMC), Linköping, Sweden. The authors declare that they have no competing interests.
Authors' contributions HN was involved in the study design, data collection, analysis, and manuscript writing. COJ was involved in the analysis and manuscript writing. TV was involved in the study design and contributed to the finalization of the manuscript. All authors read and approved the final version of the manuscript.
[fig] Figure 1: Schematic model of Swedish medical incident command and control system at major incidents. [/fig]
[fig] Figure 4: Length of time regional medical command and control were active in 130 major incidents. Median value = 60 minutes. MI= major incident. [/fig]
[fig] Figure 5: Comparison of scores from 11 different performance indicators in 66 MI (Table 2). The mean values of the 11 indicators are on the baseline. The numbers of each performance indicator are circled ( [/fig]
[table] Table 1: Classification of 130 major incidents that occurred in two Swedish County Council regions, 2006-2009 [/table]
|
Formation of iron oxide nanoparticles for the photooxidation of water: Alteration of finite size effects from ferrihydrite to hematite
CalculationsPrimary particle diameterFrom the Scherrer equation the primary particle diameter can be estimated. Here we use the shape factor B as 0.89, λ of Mo Kα 0.7063 Å, Δθ as FWHM and θ as position of the respective reflection.Band gapsFrom the absorption spectra, the position of the indirect and direct band gap can be extrapolated. The direct band gap can be calculated with a Tauc plot, shown in the following equation, where the square of the absorbance A is plotted against the difference of photon energy hν and the band gap Eg.
Accordingly, the indirect band gap of hematite nanoparticles can be estimated with another Tauc plot as the square root of the absorbance here is directly proportional to the difference of photon energy and the band gap.
## Catalytic activity
From the peak ratio between area increments (A) of O2 (2.8 min) and N2 (3.2 min) signals obtained by the BID, the oxygen content in the headspace (xO2) is calculated.
For the oxygen evolution rate (OER), the difference between the sample's oxygen content and the oxygen content of a reference is multiplied with the headspace volume V and divided by the molar volume at standard conditions (Vm), by the mass of used catalyst (m) and by the irradiation time (t).
For the production rate of silver nanoparticles, the amount of ionic silver is measured as supernatant concentration (cs) by ICP-MS. The difference between the original amount of provided AgNO3 (c0) and the obtained supernatant concentration is used as measure for the produced silver nanoparticles as no adsorption of silver ions was observed with a reference sample. Thus the production rate rAg can be expressed as quotient of the amount of silver nanoparticles and the product of the mass of hematite catalyst and irradiation time. |
Biological Significance of Tumor Heterogeneity in Esophageal Squamous Cell Carcinoma
Esophageal squamous cell carcinoma (ESCC) is a common and aggressive malignancy, with hitherto dismal clinical outcome. Genomic analyses of patient samples reveal a complex heterogeneous landscape for ESCC, which presents in both intertumor and intratumor forms, manifests at both genomic and epigenomic levels, and contributes significantly to tumor evolution, drug resistance, and metastasis. Here, we review the important molecular characteristics underlying ESCC heterogeneity, with an emphasis on genomic aberrations and their functional contribution to cancer evolutionary trajectories. We further discuss how novel experimental tools, including single-cell sequencing and three-dimensional organoids, may advance our understanding of tumor heterogeneity. Lastly, we suggest that deciphering the mechanisms governing tumor heterogeneity holds the potential to developing precision therapeutics for ESCC patients.We also described new technologies and methodologies that might further our understanding and management of ESCC heterogeneity and shared our perspectives on the future of this field.Intertumor HeterogeneityTaxonomy of cancer subtypes by specific molecular characteristics significantly improves the conventional histopathological classification and guides subtype-specific precision medicine. As exemplified in breast cancer (e.g., luminal, basal-like, Her2+), lung cancer (e.g., EGFR+, ALK fusion+), and gastric cancer (e.g., Epstein-Barr virus+, microsatellite unstable), intertumor heterogeneity has been widely studied and successfully translated into clinical knowledge in various cancer types [21][22][23]. However, the stratification of ESCC patients based on intertumoral molecular heterogeneity remains comparatively understudied.In 2017, through an integrative multi-omics analysis, The Cancer Genome Atlas (TCGA) consortium classified 90 ESCC specimens into three subtypes, designated as ESCC1-3 [4]. ESCC1, mostly Asian samples, was enriched in genomic alterations in the NRF2 pathway (NFE2L2, KEAP1, CUL3, and ATG7) and amplifications of SOX2 and/or TP63; ESCC2, mainly Eastern European and South American samples, was characterized by higher rates of NOTCH1 and ZNF750 mutations, CDK6 amplification, and inactivation of KDM6A, KDM2D, PTEN, and PIK3R1; only four cases were classified into ESCC3, which were all from North America and featured in SMARCA4 mutation. Although these subtypes showed notable geographical trends, their associations with particular biological and/or clinical features were not extensively elucidated. In addition, because of the relatively small number of samples, these classifications need further validation in larger cohorts.In addition to the effort from TCGA consortium, several individual laboratories have attempted to subgroup ESCC based on transcriptomic data. Upon analyzing tumor samples from African patients, Liu et al. [24] reported three ESCC subtypes based on their distinct expression patterns of cell cycle and neural transcripts. In another study, ESCC specimens from 360 East Asian individuals were divided into four molecular subtypes associated with distinct clinical metrics [25]. Most recently, a new research work has categorized Asian ESCCs into two subtypes, with subtype I overexpressing genes in immune response process and subtype II linked to ectoderm development, cell proliferation, and glycolysis process [26]. Additionally, Tanaka et al. [27] reported the presence of an immune-reactive subtype of ESCC patients with cytotoxic T-lymphocyte signatures activated by chemoradiotherapy.Despite the fact that no consensus molecular subtypes of ESCC have been established, the above subtyping results are sufficient to confirm the existence of extensive intertumor heterogeneity among ESCC individuals, and further demonstrate heterogeneity amongst different ESCC ethnic groups. This is in line with the well-established dramatic geographic and demographic features of ESCC [28,29]. It should also be noted that the molecular factors and causes underlying intertumor heterogeneity are likely similar with those involved in intratumor diversity. In order to fully capture the tumor spectrum, and to further improve ESCC subclassification and treatment stratification, the molecular features of ESCC intratumor heterogeneity need to be comprehensively integrated.
# Introduction
Esophageal carcinoma is the sixth most lethal cancer type worldwide, responsible for over 400,000 deaths annually. Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype, accounting for 90% of cases. Despite noteworthy advances in both cancer diagnosis and therapy, the clinical outlook for ESCC patients remains dismal, with a five-year survival rate below 30%. A number of lines of evidence have demonstrated that this poor clinical outcome is at least partially attributed to the substantial intertumor and intratumor heterogeneity in ESCC.
The concept of tumor heterogeneity contains both intertumor and intratumor forms. Intertumor heterogeneity concerns the phenotypic and molecular differences among tumors from different patients, while intratumor heterogeneity refers to biological variations within the same tumor. Heterogeneity is an important attribute of cancer and a major contributor to tumor progression. It manifests at two major levels: genomic (somatic mutations, copy number alterations, chromosomal rearrangements, etc.) and non-genomic (epigenomic changes, microenvironmental variabilities, etc.) . The degree and complexity of tumor heterogeneity influence the strategy of tumor biopsy, cancer diagnosis, and treatment planning . Increasingly, advances in sequencing technology and analysis algorithms have substantially promoted the understanding of both intertumor and intratumor heterogeneity in many cancer types, including ESCC . However, translation of the accumulated knowledge on ESCC heterogeneity into clinical practice is still challenging. A systematic understanding of ESCC heterogeneity with respect to its composition, function, and implication is therefore urgently needed.
In this review, we summarized the evidence for both genomic and non-genomic sources of ESCC heterogeneity and discussed their biological and clinical significance in the context of tumor evolution.
## Intratumor heterogeneity
In the milestone paper published in 1976, Peter C. Nowell proposed a model for cancer development: the Darwinian clonal evolution and selection of tumor cells. Since then, this model has been widely accepted and the phenomenon of intratumor heterogeneity has been highlighted as a cancer hallmark to reflect the non-uniformity and intricacy within tumor ecosystems . To date, it is well established that intratumor heterogeneity is represented by the presence of distinct cell populations, which can occupy specific microenvironmental niches, behave as communities, and extensively interact with each other as well as with components of the tumor microenvironment. Therefore, intratumor heterogeneity arises not only from genomic and epigenomic disorders of tumor cells themselves, but also from the influence of the tumor microenvironment. Importantly, intratumor heterogeneity exists among different geographical regions of the same tumor (spatial heterogeneity), as well as between the primary tumor and subsequent local or distant recurrence in the same patient (temporal heterogeneity). As a cumulative result, tumor cells display remarkable variability in numerous phenotypic traits, including clinically important phenotypes such as the ability to seed metastases and to survive therapy[34].
## Clonal evolution of tumors
According to the clonal evolution hypothesis, cancer arises from a single founder cell, and tumor progression is accompanied by the resultant succession of clonal expansions that follow the Darwinian logic . This evolutionary perspective underlines genomic alterations as an essential substrate for fueling tumor transformation and evolution.
During each cell cycle, regardless of normal or cancer cells, DNA mutations may be acquired. Thus, the acquisition of mutations is a stochastic and random process. Consequently, innumerable rounds of cell divisions required for the formation of macroscopic tumors offer plenty of opportunities for Darwinian selection and emergence of clonal diversity in tumor cell populations. During clonal evolution, only a few "jackpot" mutations that activate oncogenic pathways and/or inactivate tumor suppressors are selectively advantageous, allowing the mutant clones to achieve selective sweeps. These functionally significant mutations are termed "drivers". In contrast, the vast majority of mutations are functionally neutral since they do not confer competitive fitness advantage. These mutations are so-called "passengers" and are mainly responsible for intratumor heterogeneity . Importantly, clonal evolution often proceeds in a branching rather than in a linear manner, further contributing to variegated tumor subclones and the complexity of tumor evolution. In fact, many neutral or mildly deleterious mutations during clonal expansion can be retained in the population, or even undergo expansions due to the genetic drift . Moreover, given the fact that the Darwinian selection is context-specific, and the evolutionary dynamics of tumor microenvironment and epigenomic events could translate into heterogeneous selective pressures experienced by tumor cells, the selective effect of given mutations (either driver or passenger) can change substantially at different stages of tumor progression.
## Spatial intratumor heterogeneity
Spatial intratumor heterogeneity has been elucidated at high resolution in many cancer types . Recently, several groups have performed multi-regional deep-sequencing, and have presented a comprehensive heterogeneous landscape of ESCC. Through analyzing 51 sub-tumor regions from 13 ESCC patients, Hao et al.proposed that approximately 40% of driver mutations were spatially heterogeneous, including oncogenes such as KIT, and members of the PI3K/MTOR (PIK3CA and MTOR) and NFE2L2 pathways (NFE2L2 and KEAP1). In addition, significant spatial heterogeneity was observed in copy number alterations, including EGFR amplification and CDKN2A/B deletions. Furthermore, taking into consideration the multi-step progression of ESCC, Zeng's teamsequenced different segments of ESCC tumors and their matched dysplasia samples in a cohort of 20 patients. Their analyses showed that esophagus dysplasia also carried high mutation load and, remarkably, more heterogeneous mutations were seen in dysplasia than in tumor samples from each patient. Moreover, through sequencing 682 micro-scale esophageal samples, Yokoyama et al.reported very recently that pervasive expansions of multiple independent clones were more commonly present within physiologically normal esophagus in comparison to ESCCs. These seemingly surprising data indicate that diversified mutational backgrounds were already established in the precursor lesion or even normal esophageal epithelia, conferring on the esophageal cells the ability to evade selection pressure during ESCC development. Moreover, the degree and complexity of spatial heterogeneity was found to be highly correlated with ESCC aggressiveness. Specifically, clinical stage of ESCC was negatively correlated with the proportion of ubiquitous mutations, and significantly more heterogeneous mutations were observed in ESCC patients with local metastasis, compared to those without.
Regionally segregated somatic mutations and copy number alterations have important clinical implications in ESCC. Firstly, they complicate pathological evaluation of tumor samples. Owing to potential sampling bias caused by spatial heterogeneity, the representability of tumor regions subject to pathological assessment is increasingly considered as a key factor. It is possible that diagnostic and therapeutic targets located in uninspected regions are missed by chance, and the heterogeneous spectrum of the tumor is inevitably underestimated. Additionally, spatial genomic heterogeneity is an important determinant for therapeutic responses. Although most cancers initially respond to treatment, they almost always relapse with the outgrowth of cancer cells that are no longer sensitive to the therapy. Many cases have demonstrated that resistance to targeted drugs may result from the preexisting heterogeneous cells. Examples include the impaired efficiency of EGFR inhibitor for lung cancer patients with heterogeneous driver status. Lung cancers initially containing rare mutations of EGFR, e.g., T790M, or low frequency of MET amplification, are capable of rendering resistance to targeted therapy . Another well-understood case is chronic myeloid leukemia, in which mutant forms of the BCR-ABL fusion protein have been implicated in the relapse of disease under imatinib treatment. In ESCC, heterogeneous amplifications of EGFR, FGFR1, and PD-L1 have been reported, accounting partially for the unsatisfactory efficacy of targeting such genomic lesions. Spatial genomic heterogeneity, therefore, greatly challenges both accurate diagnosis and efficient cancer treatment.
In addition to genomic alterations, epigenomic dysregulation also contributes to spatial diversity within a tumor. Mechanistically, epigenomic heterogeneity may arise from changes in chromatin status (e.g., DNA methylation, histone modification), deregulation of microRNAs, and transcription regulators, etc. These alterations potentially provide fitness benefit, leading to intratumor heterogeneity either independently or in conjunction with genomic alterations. For example, DNA methylation status within promoters of transcription factors SIM2 and SIX1 was strongly correlated with their heterogeneous expression pattern, which was further associated with ESCC differentiation, progression, and prognosis. Dynamic changes of mutational status and promoter DNA methylation were also observed in the SWI/SNF chromatin remodeling complex and were shown to involve in ESCC carcinogenesis. Moreover, epigenomic and genomic heterogeneity have been integratively analyzed in three ESCC patients. Noticeably, the spatial heterogeneous pattern of DNA methylation closely recapitulated that of somatic mutations, indicating functional interplay between genomic and epigenomic alterations in ESCC.
The tumor microenvironment, consisting of fibroblasts, extracellular matrix, immune cells (e.g., macrophages, infiltrating lymphocytes), etc., imposes yet another layer of heterogeneity. Tumor microenvironment can shape tumor cell phenotypes by augmenting both the intrinsic variability of cancer cells (e.g., by inducing stress responses and genomic instability) and the extrinsic diversity of microenvironmental contexts (e.g., different densities of blood and lymphatic vasculature, different numbers and types of infiltrating cells). In ESCC, the tumor microenvironment itself is indeed highly heterogeneous, as evidenced by recent reports of intratumor heterogeneity of tumor infiltrating T and B cells. Additionally, Yan et al.observed a tight association between genomic heterogeneity and variation of T cell repertoire in ESCC primary tumors. These results demonstrate that the intratumor genomic heterogeneity may have clinical relevance in ESCC through affecting tumor microenvironment. Meanwhile, ESCC cells could also benefit from the microenvironmental heterogeneity, which supports cellular diversity and influences evolutionary trajectories.
## Temporal intratumor heterogeneity
Accumulating evidence suggests that intratumor heterogeneity contributes to tumor growth through a process called branched evolution. This model suggests that tumorigenesis is analogous to a growing tree, whose trunk gives rise to numerous branches. Phylogenetic analysis is a useful approach to delineate such tree structure of cancer evolution . Accordingly, in the phylogenetic tree, truncal (ubiquitous) events shared by the entire tumor population likely reflect processes involved before and during tumor initiation and early development, whereas branched (heterogeneous) events present in only some regions of the tumor reveal factors shaping the genome during tumor maintenance and progression. Characterization of the relative timing of key somatic events with possible biological relevance is therefore essential for deciphering the evolutionary processes of tumors, as well as further improving precision medicine strategies.
In ESCC, driver mutations were significantly more truncal/clonal than passenger mutations, in accordance with findings in other tumor types. Importantly, the majority of driver mutations in tumor suppressors (including TP53, KMT2D, ZNF750, etc.) had a tendency to locate in the trunks of phylogenetic trees, indicating that tumor suppressors are lost as relatively early events during ESCC development. In contrast, half of the driver mutations in the branches were in oncogenes, including potential actionable targets, PIK3CA and MTOR, suggesting that they are late events in ESCC. This observation highlights the extra caution needed when considering inhibiting such oncogenic mutants in ESCC, given previous studies showing that suppressing subclonal drivers could otherwise lead to outgrowth of non-mutated subpopulations.
Esophageal squamous cell carcinoma evolution is a multi-step process that begins from low-grade dysplasia, high-grade dysplasia, carcinoma in situ to invasive tumor and metastasis. To further explore the genomic dynamics during this process, recent studies applied multi-region sequencing on samples covering different stages of ESCC from the same patients and constructed phylogenetic trees that mapped mutations and copy number alterations chronologically. Notably, only a small fraction of total genomic alterations was conserved from squamous dysplasia to ESCC tumors, implying the distinct evolutionary trajectories taken by precursor and neoplastic cells. Phylogenetic analysis confirmed truncal mutations of TP53 and CDKN2A and truncal copy number alterations of 11q13 (CCND1), 3q27 (SOX2), 2q31 (NFE2L2), and 9p21 (CDKN2A), validating that they are early changes during esophagus neoplastic transformation. Independently, Chen et al.also reported early emergence of copy number alterations in precursor lesions of ESCC and highlighted this phenomenon as a prominent genomic feature distinct from the development of esophageal adenocarcinoma, another pathological subtype of esophageal cancer. When considering alterations at pathway level, genes involved in cell cycle regulation (such as TP53, CCND1, CDK6, RB1, and CDKN2A) were frequently altered in the early stage of ESCC, whereas genes in RTK/RAS/PI3K tended to undergo alterations throughout the process of ESCC evolution.
Taking into consideration of the timing of metastatic outgrowth and the role of the intratumor heterogeneity, two distinct models for the derivation of ESCC metastasis have been proposed: the stepwise progression model and the parallel progression model. The stepwise progression model was characterized by tumor cells disseminated at the late stages of ESCC. Accordingly, metastases could be considered as direct descendants of the most malignant and aggressive clones that dominated primary tumors. This model was also described as the linear spread pattern by Yan et al.. By comparison, in the parallel progression model, early spread of metastases during ESCC tumor progression was highlighted. Specifically, divergent evolutionary trajectories were found between primary tumors and metastatic lesions, as well as among metastatic lesions. This model was represented as both explosive spread and metastasis-to-metastasis patterns by Yan and colleagues. More studies are required to elucidate the clonal relationship between ESCC primary and metastatic tumor cell populations, which will not only illuminate the evolutionary history of ESCC, but also create a more solid ground for therapeutic decision making. Ultimately, decoding the extent of differences between ESCC primary and metastases is crucial for the improved management of metastatic ESCC patients.
## New technologies for the investigation of intratumor heterogeneity
It is with great excitement that we are witnessing novel technologies and methodologies being developed at a fast pace for the investigation of intratumor heterogeneity. These advances will uncover the molecular and biological features of intratumor heterogeneity with unprecedented resolution and they hold the potential to revolutionize our understanding of the complex intrinsic and extrinsic heterogeneity of ESCC.
## Heterogeneity studies at the single-cell level
Technical advances in single-cell sequencing have been heralding a new era in resolving tumor heterogeneity and understanding the dynamics of subclonal architecture during tumor progression . For instance, single-cell RNA sequencing of glioblastomas revealed that cell-to-cell inherent variability was evident in regulatory axes central to glioblastoma biology, prognosis, and therapy. Single-cell DNA sequencing of a large number of breast cancer cells unraveled the punctuated evolution pattern of copy number alterations during tumor development . In ESCC, single-cell RNA sequencing has been recently performed on three specimens, and substantial intratumor heterogeneity contributed by both tumor-intrinsic and microenvironmental alterations has been observed. Nevertheless, more extensive single cell-based studies of ESCC are still lacking.
Compared to conventional bulk-tissue analysis, single-cell profiling is superior in its robust sensitivity in detecting subclonal/private genomic alterations at true single-cell resolution. Thus, single-cell sequencing is of pivotal importance in discovering subtle diversification and rare tumor clones. Looking to the future, we anticipate that the application of single-cell multi-omics technologies (e.g., transcriptome, methylome, and chromatinassays) to both cancerous cells and infiltrating stromal cells will finally provide us with a panoramic view of ESCC heterogeneity.
## Three-dimensional organoid culture
Organoid cultures are three-dimensional multicellular constructs wherein cells can self-assemble to faithfully represent the physiological states of parent tissues or organs. Derived from patients, 3D organoids are crucial tools for disease modeling, especially cancers. Briefly, the generation of patient-derived organoids involves disintegration or digestion of the tumor tissue into single-cell suspensions or cell aggregates, followed by implantation of the cells in growth factor-optimized media and 3D basement membrane matrix (Matrigel). Currently, 3D organoids have been developed for several cancer types and been proven to successfully recapitulate tumor spatiotemporal heterogeneity. Particularly, the generation of clonal organoids from different locations from the same tumors allows the study of tumor heterogeneity and evolution. Specific driver and passenger mutations could be introduced by the CRISPR-Cas9 genome-editing system with the resultant organoids valuable for dissecting tumor progression mechanisms. Importantly, organoid technology has also been leveraged to facilitate the development of personalized medicine, including drug screening and therapy response evaluation, which is in urgent need to overcome tumor heterogeneity.
In ESCC, Kijima and colleagueshave successfully generated and characterized 3D organoids from ESCC patients and provided proof-of-principle regarding their utility as a robust platform for analyzing cancer cell heterogeneity, evaluating drug sensitivity, and exploring mechanisms of drug resistance. However, given the importance of the tumor microenvironment, future work will require the investigation of ESCC-relevant niche factors and optimization of conditions for co-culturing with stromal and immune cells in the 3D organoids. It also needs to be determined whether the 3D organoids could faithfully recapitulate the entire heterogeneity of ESCC, and to what extent could the heterogeneity be maintained after extended ex vivo culture. Additionally, future applications of 3D organoids in the clinical setting should include analysis of both ESCC precancerous and metastatic lesions. With such improvements in the organoid system, we believe that 3D organoid culture will become a powerful tool to model the biological function of intratumor heterogeneity. Through this tool, we can gain more insights into ESCC evolution in time and space and develop more effective precision medicine against this deadly disease.
## Conclusions and future directions
It is now clear that heterogeneity is a hallmark of ESCC. However, a significant lag still exists between the knowledge advance of tumor heterogeneity and its clinical translation regarding to diagnostic, prognostic, and therapy. Firstly, it is worth investigating whether the heterogeneity degree by itself could serve as an important biomarker in predicting ESCC progression and guiding treatment decisions. Notably, the extent of clonal diversity predicts the probability of malignant progression in Barrett's esophagus. The quantitative evaluation of cellular diversity has also been used as a biomarker in breast cancer. These examples suggest that diagnostic value of heterogeneity may have universal applicability in cancers irrespective of tumor anatomy. Secondly, given that a heterogeneous tumor is the resulting accumulation of both genomic and non-genomic diversities, the heterogeneity of both cellular and non-cellular components (e.g., epigenome, tumor microenvironment) should be more extensively explored, together with their associations with ESCC biology and clinical outcomes. This entails chronological studies assessing heterogeneity along disease progression of the same patient, from squamous dysplasia to cancer metastases. Moreover, longitudinal studies evaluating intratumor heterogeneity changes during and following therapeutic interventions are required to support identifying early resistant disease and limiting iatrogenic impacts of therapy on ESCC evolution. Thirdly, although heterogeneity undoubtedly poses a formidable obstruction to therapeutic success, there exists a potential to reach drug-sensitive states by strategies of reducing or even exploiting tumor heterogeneity. Current promising therapeutics include targeting epigenetic modifications of cancer cells (e.g., histone deacetylase inhibitors), modulating the tumor microenvironment (e.g., anti-angiogenic therapy, immune therapy), as well as multiplex-targeted therapy and adaptive therapy. Importantly, there is also a significant progress in the development of anti-cancer stem cell (CSC) treatments, since CSCs constitute a new research focus in tumor heterogeneity. Particularly, CSCs not only contribute to the phenotypic heterogeneity of primary tumor but can persist under treatment and provoke drug-resistant recurrence and distant metastasis. In conclusion, advanced theoretical understanding of the tumor heterogeneity along with the rapid development of associated technologies will help develop more innovative and effective precision medicine against ESCC. |
Phrenic nerve palsy as a complication of superior vena caval stenting
Phrenic Nerve Palsy a b s t r a c t Superior vena cava obstruction typically results from either primary pulmonary malignancies, lymphoma, or fibrosis related to central catheters. Endovascular stenting of superior vena caval obstruction is a common first approach, due to the rapid clinical improvement typically seen. The commonest complications are recurrence of obstruction and stent migration. We present herein the case of a phrenic nerve palsy secondary to endovascular stenting in a patient with superior vena cava obstruction due to primary small cell lung cancer. (I.S. Van Der Walt).sis around the SVC following long term central catheters[5]. Given the common causes of SVC obstruction are malignant, the life expectancy of patients is typically around 6-7 months. The previously described management of SVC obstruction, where cerebral oedema is not a concern, includes chemotherapy, radiotherapy, and SVC stenting. Endovascular stenting is an excellent choice of initial management, as it does not preclude the option of chemotherapy or radiotherapy, provides the most rapid resolution of SVC obstruction, has a low complication rate, and is well tolerated by patients[6]. The efficacy of stenting approaches 95%, whereas chemotherapy and radiotherapy figures are up to 85% and may be lower depending on the particular pathology and patient characteristics[4].
# Introduction
We present a case of phrenic nerve palsy as an unusual complication of superior vena caval (SVC) stent placement. SVC obstruction is a syndrome characterized by cervico-facial, upper limb and thoracic oedema, dyspnoea, dysphagia, cough, collateral thoracic circulation, hoarseness, and occasionally, cerebral oedema manifested by cognitive deficits and visual disturbance [bib_ref] Stent placement in superior vena cava syndrome, Courtheoux [/bib_ref] [bib_ref] Endovascular treatment of superior vena cava syndrome by percutaneous venoplasty, Lauten [/bib_ref] [bib_ref] Percutaneous stenting of superior vena cava syndrome: a case report and review..., Hochrein [/bib_ref]. The commonest causes are primary pulmonary malignancies, classically small cell lung cancer (SCLC) [bib_ref] Superior vena cava syndrome, Wan [/bib_ref]. Non-Hodgkin's lymphoma, and less commonly Hodgkin's lymphoma are other causes [bib_ref] Superior vena cava syndrome, Wan [/bib_ref]. Benign causes include fibro-The most commonly reported complications of SVC stents are: recurrence of venous obstruction by either thrombosis or tumour invasion or compression, stent migration, and fracture of stent wires [bib_ref] Percutaneous stenting of superior vena cava syndrome: a case report and review..., Hochrein [/bib_ref]. The overall rate of major complications is 4%, and death is 2%. Stent migration and wire fracture are now uncommon with modern stents. Unusual complications reported include fever, cellulitis at the access site, pain during balloon inflation, and a single previously reported case of transient hemidiaphragm elevation after stent placement [bib_ref] Gianturco self-expanding stents: clinical experience in the vena cava and large veins, Irving [/bib_ref]. This is the only other reported case of phrenic nerve injury following SVC stent placement we are aware of. In the previous case, the stent was inserted after radiotherapy for bronchial carcinoma, and the chest radiograph finding of elevated right hemidiaphragm was shown to be resolved on repeat imaging 24 hours after the initial radiograph. It is not reported whether the patient experienced symptoms suggestive of the complication, and the initial radiograph was only taken 24 hours after the initial procedure [bib_ref] Gianturco self-expanding stents: clinical experience in the vena cava and large veins, Irving [/bib_ref]. The authors of this case, report the finding likely related to compression of the phrenic nerve against surrounding tumour, as opposed to damage caused directly by venoplasty and stent placement.
Various regimens of anticoagulation and antiplatelet therapies before, during and after stent placement have been described, including no anticoagulation, low dose heparin infusions, heparin followed by months-long anticoagulation, and antiplatelet therapy only. No authoritative consensus on the use or nonuse, and the particular regimens of the same, have been suggested to date.
## Case report
A 78-year-old woman initially presented to the emergency department after 10 days of symmetrical facial and neck swelling. She had a tooth extracted 7 days into the initial 10 days of swelling, as she thought it may have been related to a dental abscess. This made no difference to the swelling. The tooth extraction examination did not have any findings suggestive of an abscess or infection. She did not have systemic signs or symptoms of infection, she was afebrile (36.2), without rigors, airway or respiratory function compromise, and had a normal white cell count and CRP. Her swelling was not responsive to an outpatient course of fexofenadine (Telfast, Sanofi-aventis, Macquarie Park, NSW, Australia), or a course of oral amoxicillin (APO-amoxycillin, Apotex PTY LTD, Macquarie Park, NSW, Australia) She was admitted under the plastic surgery department and treated with amoxicillin, metronidazole, and gentamicin. A computed tomography (CT) of her facial bones showed a 6mm lucency within the maxilla posteromedial to the recent maxillary second right incisor (26) tooth extraction with the impression of a 2 mm direct communication with the underlying right maxillary antrum, as well as generalized right maxillary mucosal thickening. Generalized cellulitis within the soft tissues of the face, without focal abscess or collection was also seen on this scan. An orthopantomogram showed a tooth 16 periapical cyst which was thought to be potentially related to her symptoms, though was not removed due to the risk of oroantral fistula. She was discharged after 3 days of IV antibiotics, on oral antibiotics. The facial swelling was unchanged at this point.
She represented after 6 days, with worsening facial, neck, and now bilateral upper limb, and upper thoracic swelling with associated dyspnoea, and reduced neck mobility. She also reported a new cough, and dysphagia without odynophagia, to liquids but not solids. On examination at this point, she had areas of distended superficial veins on the upper thoracic wall.
A right suprahilar mass with mediastinal invasion was seen on chest CT, most suggestive of primary small cell bronchogenic malignancy, or less likely, lymphoma. There was severe SVC narrowing and complete compression of the azygous arch with features of SVC syndrome (see [fig_ref] Figure 1 -: CT-coronal view and axial views demonstrating SVC compression by the surrounding mediastinal... [/fig_ref]. A small nonocclusive thrombus in the right subclavian vein was also seen. There was associated right hilar lymphadenopathy.
The patient proceeded to endobronchial ultrasound, and fine needle aspirate cytology from this procedure later revealed primary small cell lung carcinoma. Later on the same sheath in the right common femoral vein. Venography performed from both brachiocephalic veins confirmed the 84% stenosis of the upper to mid SVC (see [fig_ref] Figure 2 -: A [/fig_ref]. Retrograde flow from the stenosis was evident with multiple collateral veins. A 100 cm long 10-French sheath (Optimed, Ettlingen, Baden-Wurttemburg, Germany) was advanced across the stenosis. A 20 × 60 mm sinus SL stent (Optimed, Ettlingen, Baden-Wurttemburg, Germany) was then deployed. Following deployment, the vein was conservatively venoplastied twice to an end diameter of 12 mm (see . Although the waist of the stenosis persisted, venography demonstrated markedly improved anterograde flow [fig_ref] Figure 4 -: Postballoon dilatation, the stent remains with a waist, and good flow is... [/fig_ref] , therefore the procedure was terminated. A Heparin infusion (Heparin Sodium, Pfizer, West Ryde, Australia) at 500 units/h was commenced and the patient was sent to recovery.
During the postprocedure period, the patient became hypertensive (systolic > 200 mm Hg), and experienced dyspnoea, nausea and pain in her jaw, shoulder, and neck. She was also mildly dyspnoeic. A chest radiograph at this time showed an elevated right hemidiaphragm compared with a chest radiograph performed during the emergency admission the day prior [fig_ref] Figure 5 and 6 -: On the left is the chest radiograph at presentation, prior to SVC... [/fig_ref].
The elevated right hemidiaphragm and associated clinical findings of dyspnoea and pain, were presumably related to a phrenic nerve palsy likely secondary to the plasty performed as part of the SVC filter placement. This provisional diagnosis was supported by a chest radiograph without diaphragmatic eventration less than 24 hours prior, and a new finding imaging finding which also coincided with the patient's clinical syndrome. The right phrenic nerve enters the thoracic inlet between the right subclavian artery and brachiocephalic vein, before marginating the lateral border of the SVC, making it highly vulnerable in anatomic position, to extrinsic compression from SVC venoplasty against the CT demonstrated tumour encasing it. The radiograph performed in recovery did not show other clear causes for the sudden onset dyspnoea, and cardiac monitoring patterns were not suggestive of an acute coronary syndrome. Her hypertension and pain were treated with clonidine (APO-clonidine, Apotex PTY LTD, Macquarie Park, NSW, Australia) and fentanyl (Fentanyl, Teva Pharma, Macquarie Park, NSW, Australia), respectively. The patient was admitted to to a monitored bed in the HDU. She remained stable and was saturating at 97% on 4 L oxygen via nasal prongs.
Subsequent CT study of her brain and abdomen and an Fludeoxyglucose Positron Emission Tomography (FDG PET) scan, did not demonstrate any evidence of metastatic disease, and she was commenced on carboplatin and etoposide for the new diagnosis of SCLC. She remained well, hemodynamically stable and had ongoing clinical improvement of the facial, upper limb, and thoracic oedema and dyspnoea.
A repeat chest radiograph on day 1 postprocedure showed persistent elevation of the right hemidiaphragm, though this did not manifest as any obvious clinical finding. It also persisted on the FDG PET acquired a month later.
She unfortunately experienced heparin induced thrombotic thrombocytopenic syndrome secondary to the heparin she received as part of her management. This was incidentally found in her pathology investigations. On 3 day postdischarge follow-up, her clinical syndrome related to SVC syndrome had resolved to premorbid levels, and she remained well from a respiratory perspective.
# Discussion
Management of SVC obstruction in patients with malignancy as a cause can be achieved with stenting, chemotherapy, radiotherapy, or a combination of these. SVC stenting is a highly effective and rapid method achieving relief of what is a distressing syndrome, in patients with a relatively short life expectancy, and thus has been suggested as an initial step in management [bib_ref] Stenting as first option for endovascular treatment of malignant superior vena cava..., Lanciego [/bib_ref]. In the present case, we experienced a rare complication of the procedure, a neuropraxia resulting from either the stretching of the nerve or compression between the stent, and the tumour.
Given the ongoing debate regarding stenting as a first option, the possibility of awaiting a cell diagnosis after endobronchial ultrasound and fine needle aspiration, with a view to chemotherapy as a first step, was discussed during the case. In the present case, there was evidence of thrombosis superior to the obstruction on initial imaging (thrombus within the right subclavian vein). The patient also had onset of dysp- noea and hoarseness on presentation, which were concerning for early signs of airway compromise. There was no concern for cerebral oedema, and hence options such as hyperventilation, mannitol, and other supportive measures were not implemented. Given that chemotherapy can typically provide symptom relief in 7-15 days [bib_ref] Superior vena cava syndrome, Wan [/bib_ref] , stenting has probably been the most sensible option in this case, and the patient did experience rapid and persisting relief of the syndrome as expected. She was also able to commence chemotherapy without any delay.
In the previously described phrenic nerve palsy related to stenting, the hemidiaphragm elevation resolved on chest radiograph 24 hours after being demonstrated. In the current case, the palsy presented itself as a syndrome of neck and jaw pain, dyspnoea (though hard to distinguish from the confounding cause), and a subsequent chest radiograph demonstrating the associated anatomy. The followup chest radiograph and FDG PET showed persistent elevation of the diaphragm, however the patient's symptomatology resolved
The distribution of shoulder, neck, and jaw pain, may have been due to extrinsic compression of the phrenic nerve. The phrenic nerve contains motor (around 70% of fibers), and sensory fibers to the central diaphragm, pericardium, and pleura. Although abdominal diaphragmatic irritation is well known to cause C4 dermatomal distribution pain, irritation, or damage to thoracic structures such as the pericardium and pleura are less well established to demonstrate shoulder tip pain.
We reaffirm the notion that SVC stenting is a reasonable first management option in patients presenting with SVC obstruction. Our case demonstrates that the technique is highly effective in providing relief of the acute and subacute results of obstruction, and that definitive tissue diagnosis and initiation of therapy need not be delayed. Further discussion surrounding the use of anticoagulants and antiplatelets is warranted, given that previously reported complications of the procedure have been related in part to the use of these, and we have experienced the same in the current case. To our knowledge, no formal retrospective or prospective evaluation has been completed looking specifically at outcomes in cohorts who have received anticoagulation and those who have not.
# Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.radcr.2019.03.033 .
## R e f e r e n c e s
[fig] Figure 1 -: CT-coronal view and axial views demonstrating SVC compression by the surrounding mediastinal tumour. [/fig]
[fig] Figure 2 -: A (left) and B (right)-venography demonstrating flow limiting high grade stenosis in the upper and mid SVC with reflex and flow into collateral veins.Fig. 3 -A (left) and B (right). Immediate venogram postdeployment of the sinus SL stent. A balloon plasty is performed on the right, in order to widen the narrowest obstructing point. day, the patient was taken to the angiography suite with a view to placing an SVC stent across the obstruction. The stenosis was crossed with a 150 cm Bentson guidewire (Boston Scientific, Marlborough, MA) and a 5-French, 80 cm long, balloon dilatation catheter (Cook Medical, Bloomington, IN) via 10-French, 40 cm Introducer set (Cook Medical, Bloomington, IN) [/fig]
[fig] Figure 4 -: Postballoon dilatation, the stent remains with a waist, and good flow is demonstrated across the previously near-obstructed point. [/fig]
[fig] Figure 5 and 6 -: On the left is the chest radiograph at presentation, prior to SVC stent deployment. On the right in the study acquired around the time the patient became dyspnoeic and experienced new pain, postprocedure. The stent can be seen in place and has not migrated, The right hemidiaphragm is now elevated. [/fig]
|
Pylephlebitis Caused by Actinomyces Bacteremia
Pylephlebitis is a thrombophlebitis of the portal vein or its branches, which usually occurs as a complication of intra-abdominal infections that are drained by the portal vein, most commonly as a result of diverticulitis or appendicitis. Diagnosis of pylephlebitis is achieved by visualizing a portal vein thrombosis in a patient with bacteremia or a recent intra-abdominal infection. Many microorganisms have been reported to cause this infection. However, Actinomyces has never been reported before as a cause of pylephlebitis. Here, we describe a case of a 56-yearold female who had pylephlebitis that was caused by Actinomyces bacteremia and was treated with intravenous antibiotics.
# Introduction
Pylephlebitis is a rare condition, which is also known as thrombophlebitis of the portal venous system [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. It is considered as a complication of intra-abdominal infection of areas that are drained by the portal venous system, most commonly caused by diverticulitis or appendicitis [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref]. The infection is usually polymicrobial, with Escherichia coli, Bacteroides, and Streptococci being the most commonly isolated organisms [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. The presentation of pylephlebitis is nonspecific and usually consists of fever, abdominal pain, and fatigue [bib_ref] Abdominal pain secondary to pylephlebitis: an uncommon disease of the portal venous..., Sherigar [/bib_ref] [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref]. Diagnosis is usually done by either Doppler ultrasonography or computed tomography (CT) that demonstrates a portal vein thrombus in the setting of bacteremia or a recent intraabdominal infection [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref]. Antibiotics are the cornerstone of treatment for pylephlebitis [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref]. However, the use of anticoagulation is not well defined in the treatment of this disease [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. Treatment is crucial to prevent complications or death. Despite all the advances in diagnostic techniques and treatment, pylephlebitis still has a high mortality rate of 11%-32% [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref].
## Case presentation
A 56-year-old female, with a history of a repaired Tetralogy of Fallot and pulmonary embolism while on warfarin, presented with epigastric pain and melena. The patient was febrile (101.2℉) but hemodynamically stable and did not appear to be septic. Labs on admission are shown in [fig_ref] TABLE 1: Vital signs and labs on presentation [/fig_ref] The patient was given 10 mg of vitamin K intravenously and six units of fresh frozen plasma. The esophagogastroduodenoscopy (EGD) showed two nonbleeding duodenal arteriovenous malformations (AVMs). Her total bilirubin level increased to 3.0 mg/dL on day three of her hospital stay. An abdominal ultrasound (US) scan and a CT scan with/without contrast [fig_ref] FIGURE 1: An abdominal computed tomography scan with contrast showing an acute portal vein... [/fig_ref] showed acute portal vein thrombosis extending into the splenic vein and segmental branches of the right and left hepatic lobes. No abscesses or other sources of infection were noted. The patient was started on 1 mg/kg enoxaparin daily (INR 1.5 on day three). The patient's initial fever and leukocytosis were attributed to portal vein thrombosis; thus, no antibiotics were given, pending blood cultures. The next day, the blood cultures grew Gram-positive cocci and rods (Micromonas miros and Actinomyces turicensis, respectively). She was started on IV vancomycin. However, she continued to spike fevers with worsening leukocytosis [fig_ref] FIGURE 2: The patient's white blood cell count [/fig_ref]. An echocardiogram did not show any valve vegetation. A tagged WBC scan showed no evidence of infection, making infective endocarditis unlikely. Her dental evaluation showed poor oral hygiene, multiple retained roots, pulpal necrosis, and mobile teeth. Repeated blood cultures grew Actinomyces meyeri. Both the hepatology and infectious diseases teams agreed this was likely a septic pylephlebitis secondary to Actinomyces bacteremia (likely stemming from the oral cavity). She was switched to IV penicillin G, after which her WBC count improved [fig_ref] FIGURE 2: The patient's white blood cell count [/fig_ref] and repeated blood cultures came back negative. She was discharged on IV ertapenem for six weeks followed by six weeks of oral amoxicillin and a follow-up appointment for oral surgery.
# Discussion
Pylephlebitis is a rare condition caused by an infected thrombosis of the portal venous system, and hence it is called suppurative thrombophlebitis [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. It is considered as a complication of an intra-abdominal infection that is drained by the portal venous system, mostly as a result of diverticulitis or appendicitis [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref]. It was first described in 1846 by Waller during an autopsy on a patient that had a case of hepatic abscess [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. Pylethrombosis is different from pylephlebitis in that it is a thrombosis in the portal venous system without infection [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref]. The incidence of pylephlebitis is unknown but thought to be close to 2.7 per 100,000 persons per year [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref].
Pylephlebitis is commonly caused by infections in the regions that are drained by the portal system. The infection starts in small mesenteric veins, then extends to larger veins of the portal system and the liver [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. Another way for pylephlebitis to develop is through bacterial seeding of a pre-existing portal vein thrombosis [bib_ref] Pylephlebitis after CT-guided percutaneous liver biopsy, Tandon [/bib_ref]. Diverticulitis and appendicitis are the most common causes, with other causes including pancreatitis, inflammatory bowel disease, or other abdominal infections [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. Pylephlebitis has also been reported to occur after some procedures such as CT, guided liver biopsy, hemorrhoidal banding, and gastric banding [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref] [bib_ref] Pylephlebitis after CT-guided percutaneous liver biopsy, Tandon [/bib_ref] [bib_ref] Pylephlebitis of the portal vein complicating intragastric migration of an adjustable gastric..., De Roover [/bib_ref]. Hypercoagulable states or clotting factor deficiencies are considered as a risk factor for pylephlebitis; other risk factors are cirrhosis, recent abdominal surgery, malignancy, smoking, steroid use, and immobility [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref].
The infection in pylephlebitis is usually polymicrobial; Bacteroides, E. coli, and Streptococci are the most common pathogens [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. Plemmons et al. [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] reviewed 19 cases from 1979 to 1993 and found that diverticulitis was the most common cause, being responsible for 13 cases (68%). The same study showed that 88% of the patients had bacteremia and that Bacteroides, Gramnegative bacilli, and Streptococci were the most commonly isolated organisms. Kanellopoulou et al. [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref] performed a retrospective literature review of all reported pylephlebitis cases in the English language literature from 1971 to 2009, and found 100 reported cases. Upon analysis of the etiology, diverticulitis was found to be the most common cause (30%), followed by appendicitis (19%), inflammatory bowel disease (6%), and pancreatitis (5%). Bacteremia was evident in 60% of the cases; a single microorganism was isolated in 47% of the cases, while two or more bacterial organisms were isolated in the rest. The most common pathogens were Bacteroides, E. coli, and Streptococci; none of these cases had Actinomyces. Choudhry et al. [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] performed a more recent retrospective study that included a chart review from 2002 to 2012, during which time they found 95 cases of pylephlebitis. While analyzing the causes, they found that pancreatitis was the most common cause, accounting for 31% of the cases, followed by diverticulitis (19%), peritonitis (15%), and intra-abdominal abscesses (13%). It is worth mentioning that this study was conducted in a tertiary center that had many referrals for pancreatic and hepatobiliary conditions, which may not be reflective of the general population.
The same study showed that bacteremia was present in 34 patients (44%). The most common organism cultured was Streptococcus viridans, followed by E. coli and Bacteroides fragilis. Again, Actinomyces was not isolated in any of the cases.
Pylephlebitis is difficult to diagnose given its nonspecific presentation; usually patients will have fever, abdominal pain, nausea, jaundice, and hepatomegaly [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Abdominal pain secondary to pylephlebitis: an uncommon disease of the portal venous..., Sherigar [/bib_ref] [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref]. Plemmons et al. [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] found that fever was the most common presenting symptom and was present in all their patients (19 cases), followed by abdominal pain which was found in 74% of the patients. Kanellopoulou et al. [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref] found that the most common presenting symptom was fatigue (95%), followed by fever (86%) and abdominal pain (82%). The same study analyzed laboratory tests and found that leukocytosis was the most common finding (80%), followed by elevation of liver enzymes (69%), hyperbilirubinemia (55%), and anemia (55%).
The diagnosis of pylephlebitis is often made using Doppler ultrasonography or CT that shows a portal vein thrombosis in a patient with bacteremia or intra-abdominal infection [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] [bib_ref] Pylephlebitis and pyogenic liver abscesses: a complication of hemorrhoidal banding, Chau [/bib_ref].
Treatment of pylephlebitis is important to prevent complications; antibiotics form the cornerstone of treatment. The choice of antibiotic should be based on the culture results (either from blood or a surgical sample). It is recommended to prescribe at least four weeks of antibiotic therapy in a patient who does not have a visualized abscess, and six weeks for a patient who has liver abscess with considerations of drainage [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref]. Another accepted regimen is to treat with two weeks of parenteral antibiotics followed by three to four weeks of oral treatment [bib_ref] Pylephlebitis: through these portals pass bad bugs, Adnan [/bib_ref].
The use of anticoagulation in pylephlebitis is a controversial topic [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref]. The purpose of anticoagulation is thought to be the prevention of the progression of thrombosis and the treatment of complications of portal vein thrombosis [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. Kanellopoulou et al. [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref] noted that early use of anticoagulation was associated with a decreased mortality rate and better recanalization. However, Plemmons et al. [bib_ref] Septic thrombophlebitis of the portal vein (pylephlebitis): diagnosis and management in the..., Plemmons [/bib_ref] did not find a statistically significant effect on mortality. Baril et al. [bib_ref] The role of anticoagulation in pylephlebitis, Baril [/bib_ref] performed a retrospective study on 44 patients with pylephlebitis to assess the use of anticoagulation; they came to a conclusion that anticoagulation should be considered in patients with a hypercoagulable state due to a deficiency of clotting factors, cancer, or in the case of mesenteric vein involvement as the risk of infarction will be higher.
The use of invasive methods such as thrombectomy, catheter-directed thrombolysis, and systemic thrombolysis has been reported [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. For example, Sherigar et al. [bib_ref] Abdominal pain secondary to pylephlebitis: an uncommon disease of the portal venous..., Sherigar [/bib_ref] described a case of a 59-year-old male who received parenteral antibiotics for nine days without improvement in symptoms or via imaging to CT scan; he was subsequently treated with a 10-mg bolus of alteplase followed by a tapered infusion over 24 hours, which resulted in resolution of symptoms thrombus itself, based on imaging, within 24 hours. In refractory cases, surgical thrombectomy or placement of a percutaneous drain into the portal vein might be necessary [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Management of suppurative pylephlebitis by percutaneous drainage: placing a drainage catheter into..., Pelsang [/bib_ref]. However, it is worth mentioning that surgical thrombectomy has been reported to have higher recurrence rates [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref].
As mentioned earlier, treatment is crucial to prevent complications or death. Pylethrombosis can lead to mesenteric ischemia, infarction, and can extend to other veins including superior mesenteric vein, splenic vein, or other nearby veins [bib_ref] Pylephlebitis: a review of 95 cases, Choudhry [/bib_ref] [bib_ref] Pylephlebitis: an overview of non-cirrhotic cases and factors related to outcome, Kanellopoulou [/bib_ref]. Other known complications include the formation of hepatic abscesses, sepsis, and the development of portal hypertension [bib_ref] Abdominal pain secondary to pylephlebitis: an uncommon disease of the portal venous..., Sherigar [/bib_ref] [bib_ref] The role of anticoagulation in pylephlebitis, Baril [/bib_ref]. Even with the use of antibiotics, pylephlebitis continues to have high mortality rates 11%-32% [1-2, 5].
# Conclusions
Pylephlebitis is a rare condition, but it has been reported. It needs high clinical suspicion given its nonspecific presentation. It should be considered in cases of portal mesenteric venous thrombosis and fever. Early identification and initiation of antibiotics is crucial in decreasing mortality rate significantly. The role of anticoagulation in pylephlebitis is a controversial topic; further studies are needed to assess its benefits and risks in this disease.
# Additional information disclosures
Human subjects: Consent was obtained by all participants in this study.
## Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 1: An abdominal computed tomography scan with contrast showing an acute portal vein thrombosis (A) extending into the splenic vein (B) and segmental branches of the right and left hepatic lobes. [/fig]
[fig] FIGURE 2: The patient's white blood cell count (A) and temperature (B) trends during the hospital course. [/fig]
[table] TABLE 1: Vital signs and labs on presentation. [/table]
|
Discrimination in the workplace, reported by people with major depressive disorder: a cross-sectional study in 35 countries
Objective: Whereas employment has been shown to be beneficial for people with Major Depressive Disorder (MDD) across different cultures, employers' attitudes have been shown to be negative towards workers with MDD. This may form an important barrier to work participation. Today, little is known about how stigma and discrimination affect work participation of workers with MDD, especially from their own perspective. We aimed to assess, in a working age population including respondents with MDD from 35 countries: (1) if people with MDD anticipate and experience discrimination when trying to find or keep paid employment; (2) if participants in high, middle and lower developed countries differ in these respects; and (3) if discrimination experiences are related to actual employment status (ie, having a paid job or not).Method: Participants in this cross-sectional study (N=834) had a diagnosis of MDD in the previous 12 months. They were interviewed using the Discrimination and Stigma Scale (DISC-12). Analysis of variance and generalised linear mixed models were used to analyse the data.Results: Overall, 62.5% had anticipated and/or experienced discrimination in the work setting. In very high developed countries, almost 60% of respondents had stopped themselves from applying for work, education or training because of anticipated discrimination. Having experienced workplace discrimination was independently related to unemployment.Conclusions: Across different countries and cultures, people with MDD very frequently reported discrimination in the work setting. Effective interventions are needed to enhance work participation in people with MDD, focusing simultaneously on decreasing stigma in the work environment and on decreasing self-discrimination by empowering workers with MDD.Brouwers EPM, et al. BMJ Open 2016;6:e009961.
# Introduction
Employment has many benefits that can contribute to the recovery of people with mental health problems. 1 2 However, in many countries, participation and reintegration of people with mental health problems in the workforce is problematic. Several factors cause this. Some are related to the individual, and some to the environment. An important barrier for full occupational participation and successful vocational integration is the stigma that is associated with mental health problems. [bib_ref] Important to investigate the dynamics of the stigma process, Angermeyer [/bib_ref] Stigma is a mark or sign of disgrace usually eliciting negative attitudes to its bearer and can be seen as a problem associated with knowledge (ignorance), attitudes ( prejudice) and behaviour (discrimination). [bib_ref] Stigma: ignorance, prejudice or discrimination?, Thornicroft [/bib_ref] Several studies have shown that although some cultural differences may exist, [bib_ref] A cross-cultural study of employers' concerns about hiring people with psychotic disorders:..., Tsang [/bib_ref] overall employers in many countries commonly express a range of concerns about hiring a potential employee with mental health problems. [bib_ref] Anticipated discrimination among people with schizophrenia, Uçok [/bib_ref] [bib_ref] Attitudes toward disabilitues and mental illness in work settings: a review, Mangili [/bib_ref] [bib_ref] Disability and work-related attitudes in employers from Beijing, Chicago and Hong Kong, Corrigan [/bib_ref] Concerns reported include the belief that people with mental health problems have limited productivity and job performance, especially in tasks requiring cognitive skills, that they are unreliable and might pose threats to the safety of other Strengths and limitations of this study ▪ Depression is a leading cause of disability worldwide, and for this study respondents with major depressive disorder (MDD) from as many as 35 countries were interviewed. ▪ This study examines the under-researched yet substantial problem of discrimination as a barrier to work participation of people with MDD. ▪ Interviews were used to gather direct self-reports rather than hypothetical scenarios or vignettes, which are often carried out in research on stigma and discrimination ▪ Limitations are the cross-sectional design of the study, and the fact that purposive sampling was used to recruit participants, which limits the generalisability of the results.
employees, customers or themselves, [bib_ref] Stigma as a barrier to employment: mental disability and the Americans with..., Scheid [/bib_ref] or behave in a strange and unpredictable manner, and that there is potential for symptom relapse. [bib_ref] Anticipated discrimination among people with schizophrenia, Uçok [/bib_ref] In addition, the anticipation of discrimination by people with MDD may lead them not to apply for a job, in the expectation of failure or rejection. Whereas most studies on mental health problems and discrimination in the workplace have focused on severe mental disorders such as schizophrenia, relatively few have focused on major depressive disorder (MDD). [bib_ref] Important to investigate the dynamics of the stigma process, Angermeyer [/bib_ref] This is remarkable, as MDD is one of the leading causes of the global burden of disease. [bib_ref] Depression, chronic diseases, and decrements in health: results from the World Health..., Moussavi [/bib_ref] It is one of the most prevalent of all causes of disability and therefore an important public health problem. Across different countries and cultures, stigma and discrimination form an important barrier to work reintegration, although this topic has hardly been studied. In this context, the aim of this study was to assess: (1) if people with MDD of working age anticipate and experience discrimination because of their mental health problems when trying to find or keep paid employment; (2) if people with MDD of working age from high, middle and lower developed countries differ in this respect; and (3) if discrimination experiences when trying to find or keep paid employment are related to present work status (ie, having a paid job) in working aged people with MDD. The design of this study was intentionally pragmatic so that as many as possible low income and middle income countries could participate using only locally available resources, because no external funding was available. Participants were recruited through local research staff who were asked to identify people attending specialist mental health services (either outpatient or day care in the public and private sectors) in the local area with a clinical diagnosis of MDD in the previous 12 months. All were asked to apply the Diagnostic and Statistical Manual Fourth Edition (DSM-IV) criteria in the same traditional way. Within centres, site directors were asked to identify a minimum of 25 participants who were, in their judgement, reasonably representative (as a group) of all people with a diagnosis of MDD attending specialist mental health services (either outpatient or day care in the public and private sectors in the local area). The minimum number of 25 for each site was defined for feasibility issues, particularly for non-European sites with no grant support. This method was intended to allow local staff to take into account the specific local service configuration and to draw participants from the whole range of appropriate local services. Staff at each site ensured that the sample had a spread across the adult age range (young people [bib_ref] Cross-national variations in reported discrimination among people with major depression worldwide: the..., Lasalvia [/bib_ref] [bib_ref] Internalized stigma of mental illness: psychometric properties of a new measure, Boyd Ritsher [/bib_ref] [bib_ref] Systematic review of beliefs, behaviours and influencing factors associated with disclosure of..., Brohan [/bib_ref] [bib_ref] Exploring the perspectives of people whose lives have been affected by depression, Mcnair [/bib_ref] [bib_ref] The stigma of mental illness: patients' anticipations and experiences, Angermeyer [/bib_ref] [bib_ref] Self-Stigma and the "why-try" effect: impact on life goals and evidence-based practices, Corrigan [/bib_ref] [bib_ref] Worker attitudes towards mental health problems and disclosure, Dewa [/bib_ref] , working years (25-65), older adults (≥65)) and a clear representation of female participants as MDD is twice as prevalent in women as men. Response rates were unknown. Since the present study focused on the working age population, students (N=72) and retired respondents (N=168) were excluded from the analyses. Full details of the method have been published previously. [bib_ref] Global pattern of experienced and anticipated discrimination reported by people with depressive..., Lasalvia [/bib_ref] Procedure Data were gathered during face-to-face interviews in 2010, between 1 January and 31 December. Inclusion criteria were (1) a clinical diagnosis of major depressive disorder during the previous 12 months (single episode or recurrent), as based on the DSM-IV criteria; (2) ability to speak and understand the main local language; and (3) aged 18 years or older. Individuals who were receiving psychiatric inpatient care during recruitment were excluded. The study was approved by the appropriate ethical review board at each study site. After complete description of the study to the subjects, written informed consent was obtained.
# Methods
## Study design
## Measures
Participants were assessed face to face by independent researchers not involved in the care process using the standardised Discrimination and Stigma scale (V.12), a structured interview for recording the discrimination experienced by an individual with a mental health problem. The DISC-12 interview starts with the statement 'Discrimination and stigma occur when people are treated unfairly because they are seen as being different from others. This interview asks about how participants have been affected by discrimination and stigma because of mental health problems'. The instrument consists of 32 questions, assessing discrimination in several life domains, such as marriage, parenting, housing and leisure. For the present paper, only the items that referred to discrimination in the work environment are reported on. For anticipated discrimination, the items used in this study were: 'Because of how others might respond to your mental health problem, have you stopped yourself from applying for work?' and 'Because of how others might respond to your mental health problem, have you stopped yourself from applying for education and training?' For experienced discrimination, the items used were 'Because of how others might respond to your mental health problem, have you been treated unfairly in finding a job?' and 'Because of how others might respond to your mental health problem, have you been treated unfairly in keeping a job?' All questions were answered on a fourpoint Likert scale (0=not at all, 1=a little, 2=moderately and 3=a lot).
For the second research question, consistent with the methodology of a previous ASPEN/INDIGO paper, [bib_ref] Cross-national variations in reported discrimination among people with major depression worldwide: the..., Lasalvia [/bib_ref] countries were divided into groups according to the Human Development Index (HDI). The HDI is a summary measure of human development established by the United Nations,which measures the average achievements of a country in three basic dimensions of human development: (1) long and healthy life (operationalised as life expectancy at birth), (2) access to knowledge, (ie, the mean number of years of schooling) and (3) standard of living, (ie, gross national income per capita). Since data were gathered in 2010, the HDI statistic of that year was used. Countries with a very high HDI score were England, Australia, Finland, Germany, Canada, Italy, Portugal, Belgium, France, Japan, Greece, the Netherlands, Scotland, Slovakia, Slovenia, Spain, the Czech Republic, Taiwan and Hungary. Countries with a high HDI score were Turkey, Malaysia, Brazil, Serbia, Bulgaria, Venezuela, Tunisia, Lithuania, Romania and Croatia. Since few countries had a low HDI, the medium low and low HDI group were taken together as one group for the analyses. This medium/low HDI group included Egypt, India, Morocco, Nigeria, Pakistan and Sri Lanka.
Internalised stigma, one of the independent variables included in the analyses for the third research question, was measured with the Internalized Stigma of Mental Illness Scale (ISMI). [bib_ref] Internalized stigma of mental illness: psychometric properties of a new measure, Boyd Ritsher [/bib_ref] Internalised stigma refers to the inner subjective experience of stigma and its psychological effects resulting from applying negative stereotypes and stigmatising attitudes to oneself. The ISMI is a 29-item instrument for self-rated assessment of the subjective experience of stigma, with higher scores indicating higher internalised stigma. Here, the total score on the ISMI was used.
## Statistical analyses
All analyses were performed using SPSS V.19. All p values were two-tailed with an accepted significance level of 0.05. For the first research question, percentages of anticipated and experienced workplace discrimination were reported per country. For the second research question, two separate ANOVAS were conducted, the first of which was with anticipated workplace discrimination as the dependent variable and HDI level as the independent variable. A second ANOVA analysis was conducted with experienced workplace discrimination as the dependent variable and HDI level as the independent variable. For the first and second research questions, answers to the questions on anticipated and experienced workplace discrimination were dichotomised into 'No' ('not at all') and 'Yes' ('a little', 'moderately', 'a lot'). For the third research question, multivariate logistic regression analysis was performed, using work status as the dependent variable (defined as 0=no paid employment and 1=employed), and 10 independent variables, including experienced workplace discrimination. First, univariate analyses were conducted including the following independent variables that were expected to be related to job outcome: experienced workplace discrimination, gender, age, ethnicity (ie, belonging to an ethnic minority), level of education, marital status, previous psychiatric treatment, age of first contact with mental health services, internalised stigma (ISMI total score) and HDI. Second, all variables that showed a significant relationship with the dependent variable on a univariate level ( p<0.05) were included in the multivariable logistic regression analysis.
# Results
A total of 834 people with MDD across 35 different countries were individually interviewed for this study. About half of all participants were married or cohabiting, and two-thirds of the participants were women. Characteristics of the sample are shown in table 1. Although there were differences in employment rate across sites, the employment rates per HDI group did not differ significantly.
As shown in table 2, for each separate question, about 40-50% of the participants indicated that discrimination was not a problem for them. However, when looking at the four items combined, about two-thirds (62.5%) of the total sample reported anticipated and/or experienced discrimination in the work setting due to their mental health problem. Almost one-third of participants indicated to have stopped themselves from applying for work because of anticipated discrimination.
Regarding the second research question, significant differences were found between the groups with different HDI levels. Specifically, participants in countries with a very high HDI reported significantly more often anticipated (χ 2 =26.01 (df=2), p<0.01) and also experienced (χ 2 =7.25 (df=2), p<0.05) discrimination than participants in countries with moderate/low HDI (see [fig_ref] Figure 1: Percentages and 95% CIs of respondents who reported having anticipated and experienced... [/fig_ref]. As can also be seen from figure 1, in all three groups the anticipated workplace discrimination scores were higher than the experienced workplace discrimination scores.
Concerning the third research question, as can be seen in table 3, several variables were not related to work status on a univariate level (ie, 'belonging to an ethnic minority', 'marital status', 'age of first contact with mental health services' and 'HDI'), for which reason they were not included in the multivariable model. Results from the multilevel logistic regression analysis showed that experienced workplace discrimination was independently and positively related to unemployment (0.61, 95% CI 0.43 to 0.86). Other variables that were significantly related to unemployment were 'low educational level' (0.48, 95% CI 0.34 to 0.69) and 'having ever been admitted to psychiatric treatment' (0.55, 95% CI 0.38 to 0.79).
# Discussion
The results of this study show that as many as 62.5% of participants reported having anticipated and/or experienced discrimination in the work setting. Anticipated workplace discrimination was reported more often than experienced workplace discrimination. Participants from countries with a very high HDI reported significantly more often anticipated and experienced workplace discrimination, although even in the medium/low HDI group about one-third of participants reported discrimination in the work setting. Regarding the third research question, it was found that experienced workplace discrimination was indeed independently related to unemployment.
These findings show that discrimination in the workplace is a common problem in many countries worldwide. Considering that inpatients were excluded from the study, for the total group of people with MDD these percentages may even be much higher. These findings are consistent with those of a recent review [bib_ref] Systematic review of beliefs, behaviours and influencing factors associated with disclosure of..., Brohan [/bib_ref] and†Total score on the Internalised Stigma of Mental Illness scale. [bib_ref] Internalized stigma of mental illness: psychometric properties of a new measure, Boyd Ritsher [/bib_ref] Scale ranges from 1 to 4, with higher scores indicating higher internalised stigma. ‡Combination of 'Would like to work but afraid to loose benefits', 'unable to work', 'choose not to work'. that of a large Australian study on the experiences and perspectives of people with MDD. [bib_ref] Exploring the perspectives of people whose lives have been affected by depression, Mcnair [/bib_ref] Here, participants indicated that stigma was a considerable problem, particularly regarding employment. In a similar German study, 81.5% of the 55 participants who had experienced a depressive episode anticipated stigmatisation in the occupational setting. [bib_ref] The stigma of mental illness: patients' anticipations and experiences, Angermeyer [/bib_ref] These studies from the depressed individual's perspective are in line with results of studies from employers' perspectives. Such studies have shown that employers tend to have negative attitudes towards people with mental health problems. [bib_ref] Important to investigate the dynamics of the stigma process, Angermeyer [/bib_ref] [bib_ref] Stigma: ignorance, prejudice or discrimination?, Thornicroft [/bib_ref] [bib_ref] A cross-cultural study of employers' concerns about hiring people with psychotic disorders:..., Tsang [/bib_ref] An important finding of this study was that participants anticipated workplace discrimination more often than they had actually experienced it. In another study, Uçok et al 8 found that anticipated discrimination was not necessarily associated with experienced discrimination. Similar to our results, Angermeyer et al 23 also found anticipated discrimination to be higher than experienced discrimination, and suggested that it could result in a tendency to avoid situations with a high risk of stigma. Corrigan et al 24 described this 'why try' effect as an overarching phenomenon encompassing self-stigma, followed by low self-esteem and self-efficacy, and a diminished behaviour to pursue life goals. However, not only people with mental ill health themselves anticipate to be discriminated in the workplace. A recent population-based survey of working adults in Canada showed that a third of workers would not tell their managers if they experienced mental health problems, mostly out of fear of damaging their careers. [bib_ref] Worker attitudes towards mental health problems and disclosure, Dewa [/bib_ref] Hence, findings from these studies and this study underline the clear need for interventions focusing on the empowerment of people with MDD in the work environment. Peer support plays an important role in enhancing empowerment and decreasing self-stigma 20 and may be useful in such programmes.
Since mental health problems are highly prevalent, but people with these disorders are often reluctant to disclose their condition, 21 22 27 28 employers usually are not aware of the fact that many of their employees have mental health problems. Although this is a major impediment for work adaptations, authors of a recent vignette study concluded that concealment of mental health problems may actually be wise, as employers tended to think more negatively about a worker with depression than with a physical disorder under the exact same circumstances. [bib_ref] Managers' reactions towards employees' disclosure of psychiatric or somatic diagnoses, Mendel [/bib_ref] Recently, several studies have been conducted on the topic of disclosure of mental illness in the workplace. [bib_ref] Systematic review of beliefs, behaviours and influencing factors associated with disclosure of..., Brohan [/bib_ref] For instance, a decision aid for employees on whether or not to disclose their mental health problems to an employer has been developed, which has been shown to effectively reduce decisional conflict in employees with mental health problems. [bib_ref] Decision aid on disclosure of mental health status to an employer: feasibility..., Henderson [/bib_ref] The findings of the present and other studies suggest that future programmes aimed at reducing stigma and discrimination should also involve stakeholders from the environment such as employers and occupational health professionals as they play a major role in, for instance, whether or not temporary workplace reasonable adjustments or accommodations are made. Boot et al 33 showed that workplace adjustments are associated with a reduction in sick leave duration and that 43% of workers with mental health problems reported a need for work adjustments.
The results of this study indicated that in very highly developed countries, significantly higher percentages of workplace discrimination were reported as compared to countries with a low/medium developmental score (research question 2). These findings differ from those of an intercultural study on employers' attitudes towards hiring and accommodating a person with disabilities at work. [bib_ref] Disability and work-related attitudes in employers from Beijing, Chicago and Hong Kong, Corrigan [/bib_ref] Here, it was found that Chinese employers were less likely to endorse hiring people with psychiatric disabilities than employers from the USA or Hong Kong. However, it should be noticed that within one HDI group, many different countries and cultures are represented which limits generalisability.
Whereas the size of this study, including 35 countries, is a considerable strength, the number of people interviewed per country was too small to draw any conclusions at country level. Nevertheless, the results indicated that even in countries with a medium to low developmental score, about one-third of participants reported discrimination in the work setting. Future research should focus on differences between countries and study, for instance, the effects of legislation. However, legislation will not entirely solve the problem, as legislation does not address self-stigma and also in countries with more advanced equality legislation experienced workplace discrimination rates were still high.
We also found that experienced workplace discrimination was significantly related to unemployment (research question 3). These findings are similar to those of a large household interview survey in six European countries. Specifically, they found that in participants with a mental health problem, perceived stigma was significantly associated with being unemployed, as well as with a decreased quality of life, higher work and role limitations and higher social limitations. [bib_ref] Perceived stigma among individuals with common mental disorders, Alonso [/bib_ref] An explanation for the finding that experienced workplace discrimination was independently related to unemployment is that the social stigma attached to mental health problems among employers may hinder them from hiring an employee with MDD. Alternatively this finding may be explained by the fact that during job interviews, applicants with MDD may not get the position because MDD is characterised by a variety of symptoms that may be disadvantageous during job interviews, such as markedly diminished interest in activities, impaired ability to think, concentrate or make decisions, fatigue, increased irritability and low self-worth. [bib_ref] Internalized stigma of mental illness: psychometric properties of a new measure, Boyd Ritsher [/bib_ref] These symptoms may influence both applicants' verbal and non-verbal behaviour, thereby diminishing their chances of being appointed.
When considering the results of this study, several limitations need to be taken into account. First, apart from the four items on the DISC questionnaire that measured anticipated and experienced workplace discrimination, little additional information was available on how participants perceived their work setting and why they felt discriminated. Future qualitative and longitudinal studies are needed to address this in more detail, focusing on the role of stakeholders such as supervisors, employers, colleagues and occupational health professionals. A second limitation is that the design of the study was crosssectional, for which reason no causality can be assumed. Hence, workplace discrimination may lead to unemployment, but unemployment may also lead to feelings of being discriminated against. Third, purposive sampling was used to recruit participants. This limits the generalisability of the results, as participants do not necessarily represent true prevalent cases in the community.
In conclusion, the results suggest that anticipated and experienced discrimination in the workplace is a highly common phenomenon in higher as well as in lower developed countries across the world. The topic of overcoming stigma and discrimination has been underresearched so far 35 but may offer new ways to improve work participation of people with MDD. In many countries, mental health problems such as MDD are associated with high costs for society, due to unemployment, absences and at-work performance deficits. [bib_ref] What does research tell us about depression, job performance, and work productivity?, Lerner [/bib_ref] [bib_ref] Costs of nine common mental disorders: implications for curative and preventive psychiatry, Smit [/bib_ref] [bib_ref] Factors associated with work participation and work functioning in depressed workers: a..., Lagerveld [/bib_ref] Previous studies have called for research addressing workplace environment issues to improve work participation of people with MDD. Stigma and workplace discrimination are such issues and there is a clear need for effective interventions.
[fig] Figure 1: Percentages and 95% CIs of respondents who reported having anticipated and experienced discrimination in the work setting in very high, high, moderately and lower developed countries. HDI, Human Development Index. [/fig]
[table] Table 2: Responses to the DISC-12* questions related to employment (N=834) [/table]
[table] Table 3: Multivariable logistic regression analysis work status [/table]
|
A rare presentation of isolated right-sided pleural effusion in the context of ovarian hyperstimulation syndrome: A case report
An isolated pleural effusion as the sole manifestation of early ovarian hyperstimulation syndrome (OHSS) is rare. A 38-year-old woman who had undergone in vitro fertilization presented with OHSS. Six days after transvaginal oocyte pickup, she presented with only an isolated right-sided pleural effusion and restricted respiratory capacity. A thoracentesis was successfull. Clinicians must be aware of unilateral pleural effusion, with a higher incidence on the right side, as a single-symptom presentation of OHSS. The case reported here illustrates the diversity and severity of OHSS.
# Introduction
Ovarian hyperstimulation syndrome (OHSS) is usually an iatrogenic disorder. The incidence of moderate to severe OHSS is 1-5% of all in vitro fertilization (IVF) cycles, with a mortality rate of 1:45000-1:50000 [bib_ref] The ovarian hyperstimulation syndrome, Blumenfeld [/bib_ref]. Most cases of OHSS arise only 2-7 days after oocyte pickup (OPU) due to the administration of exogenous human chorionic gonadotropin (hCG) on the day of ovulation induction, a so-called "early-onset OHSS." A decline in serum hCG concentration 7-10 days after hCG-induced ovulation leads to improved OHSS symptoms. In contrast, "late-onset OHSS" occurs 12-17 days after hCG administration almost exclusively due to endogenous hCG production resulting from pregnancy [bib_ref] Gonadotropin-releasing hormone agonist versus HCG for oocyte triggering in antagonist-assisted reproductive technology, Youssef [/bib_ref].
Knowledge of the risk factors enables preventive management of OHSS. Young patients with PCOS or high antral follicle count (AFC) and those who have received a high dose of gonadotropins are at an increased risk of OHSS. An additional risk factor is low BMI, related to high hormone responsiveness. The administration of hCG to induce ovulation considerably increases the risk of OHSS. An individually selected patient-specific dosage of gonadotrophins and careful monitoring during stimulation are of utmost importance in preventing OHSS. The use of an antagonist protocol for ovarian stimulation halves the risk of OHSS. In addition, patients at increased risk of OHSS should cryopreserve all the embryos that are acquired. To reduce the risk of OHSS, a gonadotropin-releasing hormone (GnRH) agonist should be used to trigger ovulation. We present a rare case of OHSS with isolated pleural effusion following controlled ovarian hyperstimulation.
## Case presentation
A 38-year-old woman (gravida I, para I) presented with secondary infertility for about 2 years. Her medical history included endometriosis stage I according to the revised American Society for Reproductive Medicine classification (rASRM) without deep endometriosis or adenomyosis and the endometriosis fertility index (EFI) was 6. Due to a unilateral tubal occlusion (re+ / li-) the previous pregnancy resulted from insemination therapy. The sperm quality of the partner was normal. Her ovarian reserve showed no evidence for PCO-like ovaries during transvaginal sonography, and her AMH level was 18 pmol/L. Her BMI was 21 kg/m 2 .
For ART, stimulation of the ovaries was performed using an antagonist protocol which included 225 IU hMG/day for a total of 12 days and ganirelix 0.25 mg/day for eight days, starting on day 6. For seven follicles >17 mm, ovulation induction was performed with rHCG (s.c. 250 μg of r-hCG [Ovitrelle®]) 36 h before OPU. Progesterone on the trigger day was 2.0 nmol/L. A total of 16 oocytes were retrieved, 13 of which were mature, and nine of which were suitable for in vitro fertilization. After five days, embryo transfer (one blastocyst 4AB) was performed. Four blastocysts were subsequently cryopreserved. One day after embryo transfer, the patient presented with a new onset of dyspnea in the right lateral position and nausea without vomiting. The patient's weight was 48.8 kg (pre-treatment weight 48 kg), had an abdominal circumference of 72 cm, was afebrile, and had a stable circulation. At the time of presentation after embryo transfer, the patient had a weight increase of 0.8 kg, a pulse rate of 76 beats/min, and a respiratory rate of 18 Transvaginal and transabdominal ultrasound showed enlarged ovaries: right ovary 80x51x30 mm, left ovary 45x31x65 mm, without intraabdominal ascites, but the patient presented with a unilateral pleural effusion on the right side [fig_ref] Figure 1: Sonographic presentation of enlarged ovaries without presence of ascites intraabdominally [/fig_ref]. Laboratory tests showed hematocrit, electrolytes, and renal function values within the normal range. The patient was admitted to hospital for monitoring due to the diagnosis of OHSS. The management of OHSS was commenced with 1.5 L of intravenous fluids, and thromboembolic prophylaxis with heparin (prophylactic dosage) was started.
During the patient's hospital stay, her dyspnea worsened, and her weight increased to 82 kg. Laboratory tests revealed a hematocrit value of 0.47, mild hyponatremia, and hyperkalemia. The patient had an oxygen saturation level of 94%. Chest X-rays showed a large right pleural effusion with almost complete atelectasis of the right lung [fig_ref] Figure 2: Chest X-ray reveals a severe pleural effusion on the right side [/fig_ref]. The left lung was regularly ventilated. Cardiac decompensation was ruled out by the massive pleural effusion evident on echocardiography, and thoracic drainage led to a total of 2 L of fluid being removed. hCG blood tests confirmed pregnancy 14 days after OPU.
The chest X-ray showed an apical pneumothorax 10 mm wide on the right side and a regressive pleural effusion. The infusion therapy was stopped after laboratory values were normalized. Due to an inadequate increase of hCG and the sonographic detection of a disturbed early pregnancy, drug therapy with misoprostol was begun. Subsequently, rapid improvement of symptoms followed a drop in hCG. Fortunately, after three months, an embryo transfer during a thawing cycle resulted in a further pregnancy and delivery without any complications.
# Discussion
OHSS is a serious complication of IVF that presents at different times of onset and with various degrees of severity. The most frequent symptoms are abdominal discomfort, nausea, and vomiting, which are exographically related to ascites and ovarian enlargement [bib_ref] New algorithm for OHSS prevention, Papanikolaou [/bib_ref]. OHSS is associated with increased vascular permeability, which is influenced by the renin-angiotensin-aldosterone system (RAAS) and the release of vasoactive substances, such as vascular endothelial growth factor (VEGF) and interleukins (IL) -1, − 2, and − 6, which alter the permeability of the vascular bed and, as a consequence, this typical symptomatology appears. [bib_ref] Ovarian hyperstimulation syndrome: review and new classification criteria for reporting in clinical..., Humaidan [/bib_ref] [bib_ref] Unilateral pleural effusion as the sole clinical presentation of severe ovarian hyperstimulation..., Irani [/bib_ref].
We present here a case of isolated pleural effusion in a patient with severe OHSS. In 2017, a systematic review was published which included ~30 patients with an atypical pleural effusion but only one patient presented with an ovarian manifestation of OHSS [bib_ref] Unilateral pleural effusion as the sole clinical presentation of severe ovarian hyperstimulation..., Irani [/bib_ref] [bib_ref] Unilateral pleural effusion as the main presentation of "early onset" severe ovarian..., Murray [/bib_ref]. Most patients with atypical isolated pleural effusion suffer from shortness of breath (oxygen saturation up to ~87%) and an exudative component. The findings in our case of isolated right-sided pleural effusion are correlated with the reports of the above-mentioned systematic review.
The pathophysiology of OHSS is not fully understood. After the release of vasoactive substances (especially VEGF, IL-1, IL-2, and IL-6), 10 or more days after HCG administration, mostly endogenous HCG when pregnancy has occurred vascular permeability is increased, which can lead to fluid displacement with a consecutive accumulation of fluids intraperitoneally and in the pleural space. The mechanism for the development of pleural effusion is still unclear, but it has been hypothesized that a pressure gradient is responsible for effusion. Fluid buildup in the peritoneum can lead to excess pressure, which can impair the normal function of the diaphragm. Negative intrathoracic pressure then draws fluid from the peritoneum into the pleura, which may explain findings of minimal to absent ascites, as observed in our case. These defects usually occur on the right side [fig_ref] Figure 3: Pathophysiology of right-sided pleural effusions during ovarian hyperstimulation [/fig_ref]. Prevention and early diagnosis of OHSS driven pleural effusion are very important; time of diagnosis determines whether a patient receives ambulatory or intensive care treatment. In our case, we found the appearance of pleural effusion after day 6 of OPU. Unfortunately, an isolated finding of pleural effusion as a presentation of severe OHSS is not recognized under the OHSS classification. There is currently no consensus for the classification of OHSS, although in 2016 the Royal College of Obstetricians and Gynaecologists (RCOG) proposed a classification system based on symptoms and severity [fig_ref] Table 1: Classification based on symptoms and severity according to the Royal College of... [/fig_ref] ,.
We propose here the following strategies that should be considered to prevent OHSS [bib_ref] New algorithm for OHSS prevention, Papanikolaou [/bib_ref]. As primary prevention, individualized stimulation with gonadotropin should take into account risk factors. In addition, before stimulation, potential complications such as OHSS should be clarified (for informed consent). The dose of gonadotropins in ovarian stimulation should be adapted to AMH and AFC, and where there is the possibility of a good response to ovarian stimulation, a GnRH antagonist should be administered. In secondary prevention, GnRH agonist administration should be given instead of hCG administration; all blastocysts should be frozen and a fresh transfer avoided, thus preventing endogenous hCG production according to the clinical guidelines from the ASRM and ESHRE. OHSS is an iatrogenic complication, and despite close monitoring during ovarian stimulation, it can have significant morbidity and mortality. Meticulous fluid management, physical examinations, and blood tests are key to patient management. Outpatient management is possible only in mild to moderate OHSS. If symptoms worsen, hospital admission is advised for rehydration, electrolyte correction, and thrombosis prophylaxis. In our case, a thoracentesis was performed due to the large right-sided effusion causing pulmonary compromise. The result was a significant reduction of dyspnea, improved respiratory function, and reduced further complications such as pulmonary embolism, infections, and abdominal ascites. The possibility of a patient undergoing hormonal treatment being at risk of OHSS or endometriosis presenting with unilateral pleural effusion should be kept in mind when assessing these patients.
# Conclusion
In conclusion, the very early appearance of a unilateral right-sided pleural effusion without ascites can be an atypical course of OHSS. Such a diagnosis necessitates prompt hospital management for any associated complications. Before ovarian stimulation treatment is begun, primary prevention of OHSS should be carried out if risk factors are present.
## Contributors
Angela Vidal was involved in patient care, conception of the case report, wrote the initial manuscript draft, revised it critically for important intellectual content and approved the final submission.
Christiane Wachter revised the manuscript critically for important intellectual content and approved the final submission.
Alexandra Kohl Schwartz revised the manuscript critically for important intellectual content and approved the final submission.
Carolin Dhakal was involved in patient care, assisted in the literature review, revised the manuscript critically for important intellectual content and approved the final submission.
[fig] Figure 1: Sonographic presentation of enlarged ovaries without presence of ascites intraabdominally. [/fig]
[fig] Figure 2: Chest X-ray reveals a severe pleural effusion on the right side. breaths/min. [/fig]
[fig] Figure 3: Pathophysiology of right-sided pleural effusions during ovarian hyperstimulation. [/fig]
[table] Table 1: Classification based on symptoms and severity according to the Royal College of Obstetricians and Gynaecologists (RCOG). [/table]
|
The relationship between food quality score with inflammatory biomarkers, and antioxidant capacity in young women
Diet has the potential to decrease oxidative stress and inflammation and this may be beneficial in several diseases. This study investigated the association between food quality score (FQS) with antioxidant and inflammatory properties in 171 apparently healthy young women. This cross-sectional study was conducted using a validated food frequency questionnaire to determine the dietary intake of participants. FQS was calculated by summing all the scores obtained from healthy and unhealthy food groups. The total antioxidant capacity and free radical scavenging activity of serum and urine were quantified using the ferric reducing/antioxidant power (FRAP) and α, αdiphenyl-βpicrylhydrazyl (DPPH) methods, respectively. Malondialdehyde (MDA) was measured using the formation of thiobarbituric acid reactive substances (TBARS). White blood cell (WBC) and neutrophil counts, mean platelet volume (MPV) and red blood cell distribution width (RDW), were measured. Neutrophil: lymphocyte ratio (NLR), platelet: lymphocyte ratio (PLR), and RDW: platelet ratio (RPR) were also calculated. A high food quality (rich in fruit and vegetables, nuts, whole grain, and low intake of sweetened beverage, potato chips and fried food from outside the home) was related to lower hematological inflammatory biomarkers including WBC count, RDW, NLR, and PLR. Multivariable-adjusted odds ratios (95% CIs) demonstrated that higher FQS group (third tertile vs. first tertile) was associated with a significant lower levels of urinary FRAP (OR adj = 0.82; 95%CI: 0.70 to 0.97), and DPPH.High food quality was associated with reduced of markers of inflammation and oxidative stress in Iranian young girl.K E Y W O R D Sinflammation, MDA, neutrophil, oxidative stress
# | introduction
It has been well established that diet quality is closely related to mortality and morbidity particularly from chronic diseases . An adequate intake of macro and micronutrients is required for rapid physical growth, maturity, and cognitive development in childhood and adolescence [bib_ref] Lifestyle correlates of dietary patterns among young adults: Evidence from an Australian..., Ushula [/bib_ref]. First and foremost, to improve dietary patterns (DPs), it is necessary to investigate the factors that influence dietary behaviors and how these factors influence diet quality. Dietary guidelines place a strong emphasis on the promotion of good nutrition and health [bib_ref] Diet quality in young adults and its association with food-related behaviours, Thorpe [/bib_ref].
The assessment of single nutrient intakes does not accurately reflect the overall diet quality, and hence some dietary scores, like the Food-Based Diet Quality Score (FQS), have been developed. Nutrient intake assessments do not require a database or software, and foodbased scores are easily adjusted for clinical use [bib_ref] A critical review of predefined diet quality scores, Waijers [/bib_ref]. FQS is usually determined by summing up different food groups that fall into two categories: healthy and unhealthy [bib_ref] The association of food quality score and cardiovascular diseases risk factors among..., Darooghegi Mofrad [/bib_ref]. FQS has previously been assessed in a limited number of studies where its association with the risk of metabolic syndrome [bib_ref] Diet quality indices in relation to metabolic syndrome in an Indigenous Cree..., Lavigne-Robichaud [/bib_ref] , coronary heart disease [bib_ref] Food quality score and the risk of coronary artery disease: A prospective..., Fung [/bib_ref] , cardiovascular disease (CVD; Darooghegi [bib_ref] The association of food quality score and cardiovascular diseases risk factors among..., Darooghegi Mofrad [/bib_ref] , and breast cancer [bib_ref] Food quality score and risk of breast cancer among Iranian women: Findings..., Hosseini [/bib_ref] has been evaluated.
Oxidative stress and inflammation are believed to be involved in several chronic diseases. Oxidative stress is defined as the homeostatic imbalance between oxygenderived metabolites, predominantly reactive oxygen species (ROS), and the potential of cellular antioxidant protection systems. Higher levels of ROS formation is closely related to oxidative stress. Oxidative damage of biomolecules is contributed to the etiology, development, and pathogenesis of a wide spectrum of chronic disorders, such as neurodegenerative, cardiovascular, autoimmune, tumors, lung, digestive, and inflammatory, among others, and plays a considerable role in the aging of cells [bib_ref] Oxidative stress in biological systems and its relation with pathophysiological functions: The..., Kruk [/bib_ref] [bib_ref] Reactive oxygen species: Oxidative damage and pathogenesis, Bandyopadhyay [/bib_ref].
There is growing evidence that systemic inflammatory responses can be evaluated by hematological parameters that are easily measured in the clinic; for example white blood cells (WBC), neutrophils, red blood cell distribution width (RDW), mean platelet volume (MPV), neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and RDW/platelet ratio (RPR; [bib_ref] Neutrophil-and platelet-tolymphocyte ratios are correlated with disease activity in rheumatoid arthritis, Fu [/bib_ref] [bib_ref] Red blood cell distribution width is a potential index to assess the..., Hu [/bib_ref] [bib_ref] Neutrophil-lymphocyte ratio and plateletlymphocyte ratio are 2 new inflammatory markers associated with..., Yang [/bib_ref]. The predictive or prognostic value of these parameters has been studied in a variety of conditions such as cancers [bib_ref] Preoperative hematologic inflammatory markers as prognostic factors in patients with glioma, Bao [/bib_ref] , CVDs [bib_ref] The utility of inflammation and platelet biomarkers in patients with acute coronary..., Kamińska [/bib_ref] , autoimmune , and parasitic diseases [bib_ref] Correlation between hematological parameters and ancylostomiasis: A retrospective study, Hu [/bib_ref].
Diet quality has the potential to decrease oxidative stress and inflammation that may be beneficial [bib_ref] Diet quality scores and oxidative stress in Korean adults, Kim [/bib_ref] [bib_ref] Poor dietary quality is associated with increased inflammation in Swedish patients with..., Bärebring [/bib_ref]. For instance, the consumption of various fruits and vegetables elevated the antioxidant capacity (TAC) of serum, saliva, and urine [bib_ref] Antioxidant status in humans after consumption of blackberry (Rubus fruticosus L.) juices..., Hassimotto [/bib_ref] [bib_ref] Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine..., Cao [/bib_ref] [bib_ref] Influence of lycopene and vitamin C from tomato juice on biomarkers of..., Jacob [/bib_ref] [bib_ref] Antioxidant status of young children: Response to an antioxidant supplement, Stewart [/bib_ref]. However, another study reported that there was no significant effect of following a specific dietary pattern on oxidative stress markers in healthy individuals [bib_ref] A dietary pattern that lowers oxidative stress increases antibodies to oxidized Ldl:..., Miller Iii [/bib_ref]. [bib_ref] Poor dietary quality is associated with increased inflammation in Swedish patients with..., Bärebring [/bib_ref] have reported that high dietary quality is inversely associated with inflammatory markers such as high sensitivity C-reactive protein (hs-CRP) and erythrocyte sedimentation rate (ESR). A recent systematic review analysis found that plant-based DPs are linked to reduced levels of inflammation and oxidative stress biomarkers, suggesting preventive approaches to chronic disease [bib_ref] Dietary patterns and biomarkers of oxidative stress and inflammation: A systematic review..., Aleksandrova [/bib_ref].
To our knowledge, no comprehensive evidence currently found to inspect the relationship between FQS and inflammatory status, and antioxidant capacity. With respect to the great importance of DPs in well-being and health, we performed a cross-sectional survey to investigate the association between FQS with antioxidant and inflammatory properties of the serum and urine of apparently healthy young women.
## | materials and methods
## | study design
The study was undertaken in Birjand, in northwestern of Iran, in January 2020 among female university student aged between 18 and 24 years old . A total of 330 females were initially screened for inclusion. We excluded those having any acute or chronic complications or who were taking medication or nutritional supplements. The final population sample comprised 171 healthy young women who were recruited from five different universities in Birjand using a multistage cluster sampling method.
Since we aimed to perform our investigation on a homogeneous population, in order to control for potential confounders, only single, apparently healthy women were included. The Ethical Committee of our university approved the study, and informed written consent was obtained from all participants.
## | adherence to fqs
A validated food frequency questionnaire (FFQ) was employed to estimate the food intake of individuals [bib_ref] Validation of a short semi-quantitative food frequency questionnaire for adults: A pilot..., Ahmadnezhad [/bib_ref]. A proficient dietitian instructed study volunteers to report their food intake frequency for each item within the last year on per day, week, month, rarely or never basis. Food analysis was performed using Diet Plan 6 software (forest field Software Ltd). Food quality scoring was performed by the scale developed by [bib_ref] Food quality score and the risk of coronary artery disease: A prospective..., Fung [/bib_ref]. FQS ingredients include vegetables, fruits, whole grains, nuts and legumes, yogurt, coffee as healthy foods as well as refined grains, sugar-sweetened beverages, desserts and ice cream, red and processed meats, potato and potato chips, and fried food consumed outside the home as unhealthy foods. We then classified the participants' intake into decile. The value between 1 and 10 was assigned to each healthy component. For unhealthy components, a reverse scoring process (values between 10 and 1) was assigned. Finally, the total FQS (in the range of 14-140) was calculated by summing all the scores obtained for each participant, so that a higher score indicates a healthier diet.
## | sampling
The blood and urine samples and FFQ data were collected on the same day. Blood and urine samples were collected after a 12 h fasting. The volunteers were instructed to avoid intense physical activity 24-h before the sampling. Blood samples were collected into both serum separator tubes and EDTA tubes. Sterile disposable container was used for collecting urine specimens from the first-morning section of urine from the middle stream according to a standard protocol. Serum and urine specimens were stored at −80°C until analysis.
## | biochemical measurements
The serum concentrations of, urea, creatinine, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin, direct bilirubin, total protein, albumin, calcium, phosphate, magnesium and uric acid, fasting blood glucose (FBG) and hs-CRP were quantified using commercial kits (Pars Azmun) and an auto-analyzer (Prestige 24i, Tokyo Boeki Ltd.).
## | complete blood count
Blood cell counts, hemoglobin levels, dimensional variables (MPV and RDW), and some combined parameters, such as NLR, PLR and RPR, were assessed by means of an automated commercial cell counter (Sysmex K-800).
2.6 | Serum and urine oxidative stress parameters 2.6.1 | Total antioxidant capacity (TAC) TAC was quantified using the ferric reducing/antioxidant power (FRAP) method [bib_ref] The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant..., Benzie [/bib_ref]. This procedure is based on the reduction of a ferric-tripyridyl triazine (Fe 3+ -TPTZ) complex to its ferrous (Fe 2+ ) colored appearance in the presence of antioxidant compounds.
The tests were carried out in 260 μl reaction mixtures including 250 μl of FRAP solution plus 10 μl serum, standard (FeSO 4 ) and blank (for each sample, a blank sample was used to remove turbidity). The absorbance was measured colorimetrically at 593 nm. All tests were undertaken in duplicate and TAC measure of samples was described in μmol TAC/L. For urine samples, the samples diluted 1:10 and the findings are demonstrated in μmol TAC/mg creatinine.
## | free radical scavenging activity
The free radical scavenging activity in samples was assessed using the A-Diphenyl-B-Picrylhydrazyl (DPPH) method [bib_ref] Assay of total antioxidant capacity: Comparison of four methods as applied to..., Janaszewska [/bib_ref]. Tests were conducted in reaction mixtures including 1 ml of 100 mM DPPH solution and 40 μl of each serum and blank samples. After incubation at room temperature for 10 min, each sample was centrifuged at 4000 g for 5 min at 37°C to remove cells. Absorbance was read using a microplate reader (Epoch-Biotek®) at 517 nm and compared with that of blank samples, including only DPPH and methanol solution. The results are reported in mmol Trolox equivalent/L. The urine tests were performed in reaction mixes including 250 μl of DPPH reagent and 10 μl urine (the samples diluted 1:10) and blank samples. The results are shown in mmol Trolox equivalent/mg creatinine.
## | malondialdehyde (mda) assay
The thiobarbituric acid-reactive-substances (TBARS), an index of lipid peroxidation, was measured using the method described by [bib_ref] Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method, Kei [/bib_ref]. The end product of fatty acid peroxidation, malonyldialdehyde (MDA), interacts with TBA to generate a colored complex. TBARS reagent (1 ml) was mixed with samples (100 μl), and the admixture was heated in a boiling water bath for 20 min. Next, 1 ml of N-butanol was used to extract TBARS adducts and solution centrifuged at 1500 g (10 min at 4°C). The supernatant was collected and record the fluorescence intensity at excitation and, emission wavelengths of 515 and 553 nm.
Findings were quantified by comparing them to the standard curve obtained from standard solutions under the same conditions. The TBARS concentration of samples is expressed in μmol TBARs/L. For urine samples the results are presented in μmol TBARs/mg creatinine.
## | other variables
Anthropometric parameters as well as blood pressure were measured according to standard instruction [bib_ref] Association between simple anthropometric indices and cardiovascular risk factors, Ho [/bib_ref].
# | statistical analysis
Kolmogorov-Smirnov tests were recruited to evaluate the normality of the distribution of variables. Subjects were sub-grouped into three categories according to tertiles of their FQS. One-way analysis of the variance (ANOVA) test (normal distribution parameters) or Kruskal-Wallis test (non-normal distribution parameters) were employed for comparison of quantitative variables between tertiles of FQS. Moreover we performed a multivariable logistic regression to explore the relationship between FQS with inflammatory and oxidative stress parameters using the FQS tertile groups as dependent variable (reference category: first tertile), after correction for energy intake, WHR, total protein and phosphate levels. All statistical analyses were undertaken using the SPSS version 16.0 and p values ≤0.05 were set as significant.
# | results
The FQS were used for categorizing of the participants into tertiles, with T1 set as the bottom tertile (lower score; range: 19-26; n = 55), T2 (middle score; range: 27-29; n = 57), and T3 as top tertile (higher score; range: 30-35; n = 59). Kolmogorov-Smirnov test indicated a normal distribution of all variables (p > 0.05), except serum hs-CRP and triglycerides.
No significant difference was found between the participants across tertiles of FQS regarding to demographic and anthropometric parameters, lipid profile, liver function enzyme tests, FBG, and albumin (p > 0.05; . However, WHR were lower in the third tertile than in the second and first tertile (p = 0.007). Individuals in the highest tertile of the FQS tended to have lower serum levels of total protein and phosphate compared with those in the lowest tertiles (p < 0.05; .
The dietary intake of subjects across tertiles of FQS assessments was shown in . The subjects with a higher tertile of FQS had a greater consuming of dietary fiber, MUFA, vitamin B6, vitamin C, magnesium, Fe, vegetables, fruits, legumes and nuts, and whole grains compared to lowest tertile. Furthermore, eating of fat, sugar sweetened beverage, potato chips, and fried food from outside the home higher more in participants who were placed in the third tertile of FQS versus to the first tertile (p < 0.05; .
Additionally, individuals in higher group of FQS, were more likely to have lower blood WBC, RDW, NLR, PLR as well as urinary FRAP and DPPH levels (p < 0.05). Serum FRAP was found to be lower in the bottom than in the top tertile of the FQS (p = 0.030; .
Multivariable-adjusted ORs (95% CIs) after controlling of energy intake, WHR, total protein and phosphate levels that higher FQS group (third tertile vs. first tertile) was associated with a significant lower levels of blood WBC .
# | discussion
Findings from this cross-sectional survey provide evidence that a high FQS (with a high intake of fruit and vegetables, nuts, whole grain, and low intake of sweetened beverage, potato chips and fried food from outside the home) was related to lower levels of inflammatory biomarkers including WBC count, RDW, NLR, and PLR. Higher FQS also associated to reduction of urinary FRAP, DPPH levels. To our knowledge, this is the first attempt investigating the association between FQS and biomarkers of inflammation and anti-oxidant capacity in healthy young women.
We also found that a high FQS was associated with a high intake of antioxidant-rich foods, which might reduce oxidative stress. We found that young healthy women with a high FQS have significantly lower urinary concentrations of FRAP, and DPPH. In Korean population, two food quality scales, Recommended Food Score (RFS) and the alternate Mediterranean Diet Index (aMED), were negatively associated with urinary MDA concentrations [bib_ref] Diet quality scores and oxidative stress in Korean adults, Kim [/bib_ref]. The eating of vegetables, particularly green leaves, and fruits connected with lower oxidative stress [bib_ref] Effect of diets, familial history, and alternative therapies on genomic instability of..., Paz [/bib_ref]. Interestingly, a Western dietary style identified by the intake of components with a high glycemic index may causes to impaired blood glucose homeostasis, and elevate oxidative stress [bib_ref] Relation between a diet with a high glycemic load and plasma concentrations..., Liu [/bib_ref]. There is inconsistent evidence on the association between dietary fruit and vegetables and urinary markers of oxidative stress. [bib_ref] Antioxidant status in humans after consumption of blackberry (Rubus fruticosus L.) juices..., Hassimotto [/bib_ref] found reduced urinary TAC concentrations after a single ingestion of blackberry juices. [bib_ref] Intake of polyphenol-rich pomegranate pure juice influences urinary glucocorticoids, blood pressure and..., Tsang [/bib_ref] announced the elevation in plasma, but not urinary FRAP levels following fruit juice intake. Urinary TAC was also unaltered post 3 weeks consuming of a antioxidants fortified dried fruit and vegetable [bib_ref] Antioxidant status of young children: Response to an antioxidant supplement, Stewart [/bib_ref]. Vitamin C and magnesium reduce NADPH oxidase as a superoxideproducing enzyme, which supports that increasing antioxidant capacity can lead to the attenuation of ROS [bib_ref] Dash diet lowers blood pressure and lipid-induced oxidative stress in obesity, Lopes [/bib_ref]. It has been reported that FRAP levels were proportional to the mitigating power of the major non-enzymatic antioxidants in serum [bib_ref] The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant..., Benzie [/bib_ref] ; so, this assay was chosen to evaluate the antioxidant status of young women in the present study.
We found serum FRAP was lower in the bottom than in the top tertile of the FQS; this finding indicates that the better diet quality causes to increased non-enzymatic antioxidant defenses.
Oxidative stress is related with an imbalance between free radical generation and antioxidant defenses throughout the body, it is usually necessary to assess the counterpart of oxidation, the total antioxidant status. Additional indices suggest an association between antioxidant status/ capacity status, and oxidative stress. As antioxidants can act additively or synergistically, and absorbed and used in the human body in different ways, so the judgment based on total antioxidant activity provides more credible data rather than the quantification of one antioxidant Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; WHR, waist to hip ratio. a Significant between tertile 1 and 3. b Significant between tertile 2 and 3. c Significant between tertile 1 and 3. T A B L E 1 Baseline clinical characteristics of study participants individually. These include indices that focus the total scavenging abilities of serum sample, following, for example, addition of a radical forming compound. So, we used the most common tests for evaluation including FRAP and DPPH assays.
Our findings demonstrated that the subjects with a higher FQS (greater consuming of dietary fiber, MUFA, vitamin B6, vitamin C, magnesium, Fe, vegetables, fruits, legumes and nuts, and whole grains) were more likely to have lower blood levels of WBC, RDW, NLR, PLR. A long with us, [bib_ref] Diet-quality scores and plasma concentrations of markers of inflammation and endothelial dysfunction, Fung [/bib_ref] reported that Alternate Healthy Eating Index (AHEI), and aMED which more emphasize on DPs rich in fruit, vegetables, nuts, whole grains, and sea foods are significantly correlated with lower indices of inflammation including serum CRP and IL-6 in middle aged women. In consistent, a DP high in sugar and fat has been connected with low-grade inflammation identified by high concentrations of CRP [bib_ref] Relation between a diet with a high glycemic load and plasma concentrations..., Liu [/bib_ref]. There is increasing evidence that a healthier diet or health-protecting foods with anti-inflammatory contents, such as high intakes of grains and fiber, vegetables, fruit magnesium [bib_ref] Iron and its relation to immunity and infectious disease, Oppenheimer [/bib_ref] , and vitamin C [bib_ref] Vitamin C in the treatment and/or prevention of obesity, Garcia-Diaz [/bib_ref] , are inversely associated with serum inflammatory mediators. It has been shown that intake of nuts causes to decreased levels of inflammation and cholesterol; therefore cardioprotective effects throughout the body [bib_ref] Nuts and CVD, Ros [/bib_ref]. Although, no associations was observed between diet quality indices with inflammatory biomarkers, such as hs-CRP and IL-6, in youth diabetic patients [bib_ref] Dietary quality and markers of inflammation: No association in youth with type..., Liese [/bib_ref]. Food quality score is a validated tool to capture the overall diet quality according to food intake which provides valuable estimation about the DPs of individuals or populations and health status. This approach focuses on total diet rather than using a single food-intake determination approach. We controlled for important potential confounder variables that could affect the relationships. Moreover, energy-adjusted intakes of FQS items were used, which can decrease the risk of misclassification of study subjects. But, present study has several limitations. In epidemiological studies, evaluation of diet depends on self-reported information which is susceptible to inevitable recall bias. Also, even though urinary FRAP, DPPH and MDA levels were adjusted by urinary creatinine, these amounts were measured by spot urine specimens. Finally, the cross-sectional nature of this study does not make it conceivable to disclose a causality effect.
# | conclusion
Overall, our data suggest that consuming a good quality diet characteristically rich in fruits, vegetables, nuts, and whole grain and low in discretionary foods is associated with lower values of inflammatory markers and higher anti-oxidant capacity biomarkers in young women. Future studies will attempt on underlying mechanisms. Moreover, future dietary modification studies may provide a public health knowledge on whether diet modification interventions may be beneficial in combating pathologies through targeting oxidative stress and inflammation.
# Author contributions
AB was involved in conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, and writing-original draft preparation. FN was involved in investigation and methodology. ZK was involved in resources and software. MM was involved in validation and visualization. GF was involved in conceptualization (supporting), data curation (supporting), and writing-review and editing. All of the authors have read and confirmed the final manuscript. |
Positive detection of SARS‐CoV‐2 combined HSV1 and HHV6B virus nucleic acid in tear and conjunctival secretions of a non‐conjunctivitis COVID‐19 patient with obstruction of common lacrimal duct
Background: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. Methods: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. Results: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1 + CD95 + cells was accounting for 94.8% in CD3 + CD8 + T subset and for 94.8% in CD3 + TCRcd + T subset. Conclusions: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases.
# Abstract.
Background: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. Methods: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. Results: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1 + CD95 + cells was accounting for 94.8% in CD3 + CD8 + T subset and for 94.8% in CD3 + TCRcd + T subset. Conclusions: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases. The current outbreak of Coronavirus Disease 2019 (COVID-19) has spread rapidly all over the world. More than 80 000 cases in China and 300 000 cases in the other countries have been reported until March 24th. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We reported a case that positive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) combined herpes simplex virus type 1 (HSV1) and human herpesvirus type 6B (HHV6B) virus nucleic acid in tear and conjunctival secretions of a nonconjunctivitis COVID-19 patient with obstruction of common lacrimal ducts in order to understand the ocular change of COVID-19 and to be a basis for clinical strategy.
# Methods
The patient's agreement was obtained for each sample collection. Tear and conjunctival secretion samples were collected by eye swab. Eyelids were everted, and samples were obtained by sweeping the inferior fornices of both eyes with sterile cotton swabs with topical anaesthesia. The swabs were placed into Hank's balanced salt solution. Samples were transported in ice to the laboratory in our hospital and centre for disease control (CDC). The samples were tested for SARS-CoV-2 in our hospital by use of polymerase chain reaction (PCR) with the CDC recommended Kit. Routine blood test and flow cytometry are operated according to routine methods.
## Case report
A 70-year-old, native resident of Xi'an city (Shaanxi Province, China) was admitted to the First Affiliated Hospital of Xi'an Jiaotong University complicated with a fever, fatigue, cough and sputum for one day. Just one week ago, he has contacted with some relatives that came from Wuhan Province (China).
After being treated in quarantine ward, a series of laboratory examinations including nasopharyngeal swab were detected for SARS-CoV-2. According to the records, the initial physical examination revelled T 37.7°C, and SpO2 92% without oxygen supplement. The blood routine examination showed white blood cells (6.92 9 10 9 /l), lymphocyte per cent (5.8%), neutrophil per cent (88.7%) and C-reactive protein (15.4 mg/l). IL-6 in serum was 40.90 pg/ml. The changes of granulocyte (A) and IL-6 (B) in the whole course were shown in [fig_ref] Figure 1: The change of granulocyte [/fig_ref]. The test for influenza A and B showed negative. Computerized tomography (CT) of lung showed significant ground-glass opacities. The CDC confirmed that both the twice nasopharyngeal swab detections were all positive for SARS-CoV-2. Sure enough, the man suffered COVID-19 (mild type).
The whole clinical course was shown in. After combined treatment, (lopinavir and ritonavir tablets per os for 6 days, moxifloxacin per os for 7 days, interferon nebulization for 16 days, traditional Chinese medicine per os for 7 days), the man's body temperature returned to normal after onset 12 days. After 23 days, his chief complaint recovered, and the groundglass opacities decreased in CT of lung were detected with the negative nasopharyngeal swabs for several times continuously (24 hr apart). Before discharged, nasopharyngeal swab and eye swab were collected again at the same time. Surprisingly, the nasopharyngeal swab was negative and the eye swab was positive on Day 25 of the course. Reexamination was the same results and next-generation sequencing (NGS) confirmed PCR results. Besides, HSV1 and HHV6B virus nucleic acid were also detected by NGS. However, any symptoms and signs of conjunctivitis were not found in his eyes. Only the patient had a history of obstruction of common lacrimal duct in his left eye with mild tearing and without any secretion. Through lacrimal duct irrigation, obstruction of common lacrimal duct in his left eye was diagnosed, and levofloxacin with Arbidol eye drops was taken. The left eye swab quickly turned undetectable. The whole changes of C t value in swabs were shown in.
The gene detection of lopinavir and ritonavir for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC, and it means that the efficiency of lopinavir and ritonavir tablets in this patient would be significantly decreased. The flow cytometry results of lymphocyte subsets showed PD-1 + CD95 + cells was accounting for 94.8% in CD3 + CD8 + T subset, and for 94.8% in CD3 + TCRcd + T subset [fig_ref] Figure 3: Flow cytometry results of lymphocyte subsets [/fig_ref].
To sum up, his nasopharyngeal swabs were positive for 22 days, but the eye swabs were still continuously positive for 2 weeks. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks and then became near normal.
# Discussion
According to recent news reports, several ophthalmologists in Wuhan (China) were sacrificed due to COVID-19. Although COVID-19 indeed transmitted mainly through respiratory droplets and contact with infected patients, some studies suspected that it might be related with the contaction of body fluid of patients, including tear. Xia and colleagues suggested that SARS-Cov2 transmission through the ocular surface should not be ignored [bib_ref] 2019-nCoV transmission through the ocular surface must not be ignored, Lu [/bib_ref]. In this case, we collected the eye swab from a clinical cured COVID-19 patient and detected the positive expression of SARS-CoV-2.
Respiratory-related public health events, such as SARS, were reported to be associated with ophthalmology [bib_ref] The severe acute respiratory syndrome coronavirus in tears, Loon [/bib_ref] [bib_ref] Ocular symptoms and treatment of Ebola virus disease, Vegh [/bib_ref]. No matter SARS or Ebola, the patients' tears were detected the virus nucleic of coronavirus or Ebola virus [bib_ref] Assessment of the risk of Ebola virus transmission from bodily fluids and..., Bausch [/bib_ref]. The novel virus SARS-CoV-2, which caused COVID-19, is similar to SARS virus, which it shares more than 79% of its sequence. Our case positively detected SARS-CoV-2 virus in tears and conjunctival secretions. Similarly, some published paper has the same results of SARS-CoV-2 virus in tears [bib_ref] Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2..., Xia [/bib_ref]. However, our case has many features differently with other reported cases. There were 5 points:
1 Virus was detected for continuously until turned negative, not only one time-point. 2 The case was not conjunctivitis patient and positively detection when recovered.
3 Different results between two eyes of the patient were related with obstruction of common lacrimal duct on left eye. 4 Positive eye sample virus lasted for 2 weeks more after nasopharyngeal swab became negative. 5 System immune states were evaluated including blood routine, other virus co-infection and Lymphocyte subsets.
Continuously detection may comprehensive analysis the dynamic change of this case. The occurrence of asymptomatic patients with SARS-CoV-2 may pose a significant public health issue and infection control challenge. China's National Centre for Disease Control analysed 72 314 COVID-19 cases and found out 889 (1.2%) cases were asymptomatic (Novel Coronavirus Pneumonia Emergency Response Epidemiology 2020). One report indicated that an asymptomatic person was able to transmit SARS-CoV-2 to another patient in Germany [bib_ref] Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany, Rothe [/bib_ref]. These results reminded us that asymptomatic cases might play a role in the transmission. Similarly, the patient in our case did not suffer conjunctival congestion or conjunctivitis, but positive virus in eye sample may cause infection through eye tissues/tears or when doing ophthalmic examination. It reminded ophthalmologists should take more carefully protection for themselves and ophthalmic patients.
Coincidentally, we positively detected SARS-CoV-2 in his left eye sample repeatedly, of which common lacrimal duct was obstructed. Some study revealed that lacrimal duct obstruction and acute dacryocystitis were associated with Epstein-Barr virus infection [bib_ref] Nasolacrimal duct obstruction and acute dacryocystitis associated with infectious mononucleosis (Epstein-Barr virus), Steele [/bib_ref]. Therefore, we speculated that this case might concern with obstruction of common lacrimal duct. To our knowledge, obstruction of common lacrimal duct may decrease the virus clear through lacrimal drainage system. This point may be the reason why the different results between two eyes. Further works are needed to demonstrate this speculation. \In this case, the man entered into convalescence as his body temperature returned to normal and the clinical symptoms like cough were all disappeared. Positive eye sample virus lasted for 2 weeks more after nasopharyngeal swab became negative. However, the SARS-CoV-2 could still be detected, which mean that the virus was shedding persistently. In 2005, a retrospective study depicted the dynamic profiles of viral persistence in SARS patients. Cultivable SARS-CoV-2 was detected in stool or urine specimens for longer than 4 weeks in three convalescent patients [bib_ref] Persistent shedding of viable SARS-CoV in urine and stool of SARS patients..., Xu [/bib_ref]. In 2018, a study revealed that the prolonged shedding of Avian Influenza A (H7N9) RNA in the respiratory tract was associated with delayed initiation of neuraminidase inhibitor (NAI) treatment and with use of corticosteroids [bib_ref] Factors associated with prolonged viral shedding in patients with avian influenza A(H7N9)..., Wang [/bib_ref]. Recently, an article showed the median duration of viral shedding of SARS-CoV-2 was 20 days from illness onset. The shortest was 8 days, and the longest among survivors was 37 days [bib_ref] Clinical course and risk factors for mortality of adult inpatients with COVID-19..., Zhou [/bib_ref]. In our case, the nasopharyngeal swabs kept positive for 22 days and the eye swabs kept positive for nearly 2 weeks more after the nasopharyngeal swabs became negative. After lacrimal duct irrigation and topical eye drops treatments, the SARS-Cov-2 in his eye samples turned undetectable. Thus, the duration of viral shedding was 36 days, might even be 38 days, as we did not collect eye swabs on Day 37 and 38.
The reasons of the prolonged shedding of SARS-CoV-2 in eye sample might be related with these factors as follows.
(1) The patients had low efficiency for lopinavir and ritonavir because of type CC of ABCB1 (3435T>C) rs1045642. Although recognized specific drug for COVID is not found, lopinavir and ritonavir were recognized that they might inhibit SARS-CoV-2. Therefore, lopinavir and ritonavir are our routine treatment in our hospital. The patients' gene determined his low effective for the two drugs and slowly virus shedding. Recently, a study revealed that in 199 hospitalized adult patients with severe COVID-19, no benefit was observed with lopinavir-ritonavir treatment beyond standard care [bib_ref] A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19, Cao [/bib_ref]. Future trials are needed in the further.
(2) Relative immunosuppressive state may also influence virus clear from his body. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, high PD-1 + CD95 + cells proportion in CD3 + CD8 + T subset and combined herpes simplex virus type 1 (HSV1) and HHV6B virus latent infection may be reflect that his immune was suppressed. These were no enough functional lymphocyte to kill the virus, and PD-1 + CD95 + cells can inhibition of T-cell activation and induction of immunocytes apoptosis [bib_ref] Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic..., Brezar [/bib_ref]. In the NGS testing report, HSV1 and HHV6B nucleic acid were also detected. HSV1 and HHV-6B are also responsible for lifelong chronic infections, most often asymptomatic, in the vast majority of the general adult population. In ophthalmology, HSV1 and HHV6 could remain latent in the human anterior segment and aqueous humour. When immune was suppressed, the two viruses can cause ocular diseases and be detected. Therefore, we speculated that the latent infection of HSV1 and HHV6B might be activated as immunocompromised during the course of COVID-19.
Our case reminds that more eye swabs should be collected from confirmed and suspected COVID-19 patients, especially from those immunocompromised, such elder patients or had a history of immunodeficiency diseases, those with eye symptoms, such as conjunctival congestion or conjunctivitis and those had a history of ocular diseases, such as obstruction of common lacrimal duct. Furthermore, when nasopharyngeal swab is negative and the eye swab is positive, it is a question that whether this man is a convalescent patient or asymptomatic infected patient. Deeply studies should be researched. It should be emphasized that PCR assay only detect the fragments of viral nucleic acid, so it represents differences from isolation and identification of the virus. And positive virus nucleic detection in tears does not absolutely mean that the virus definitely transmits from eyes.
[fig] Figure 1: The change of granulocyte (A) and IL-6 (B) in the whole course. Each datum was relative to the normal value, respectively. [/fig]
[fig] Figure 2: (A) The whole clinical course and results of swabs and treatments of this patient. (B) The NGS detection of SARS-CoV-2 (n = 6), HSV1 (n = 36) and HHV6B (n = 4). (C) The results of gene detection of lopinavir-ritonavir for drug use guidance. (D) The whole changes of C t value in swabs. [/fig]
[fig] Figure 3: Flow cytometry results of lymphocyte subsets: (A) PD-1 + CD95 + cells were accounting for 94.8% in CD3 + CD8 + T subset and (B) for 94.8% in CD3 + TCRcd + T subset. [/fig]
|
Factors Associated with Halitosis in White-Collar Employees in Shanghai, China
ObjectiveTo investigate the factors associated with halitosis in Chinese white-collar employees.Materials and MethodsSubjects in three randomly selected office buildings in Shanghai, China, were enrolled in this cross-sectional study using cluster random sampling. Oral malodor was assessed by measuring volatile sulfur compounds (VSCs) with a portable sulfide monitor. Subjects' oral health, including dental caries, periodontal status, and tongue coating, was evaluated clinically. A questionnaire was used to obtain information about participants' demographic characteristics, oral hygiene habits, and health behaviors.ResultsOf the 805 subjects invited to participate in this study, 720 were enrolled (89.4% response rate). Data from these subjects were used for statistical analyses. The prevalence of halitosis was 33.2%. In the final regression model, halitosis was significantly related to tongue coating thickness, periodontal pocket depth, no food consumption within 2 hours prior to oral examination, and less intake frequency of sweet foods.ConclusionsIn this Chinese white-collar population, tongue coating and periodontal disease were associated with halitosis. Oral hygiene education should be provided at the population level to encourage the maintenance of oral health and fresh breath. Consumption of sweet foods may reduce VSC production, although this finding requires further investigation.
# Introduction
The term "halitosis" refers to an unpleasant or offensive odor emanating from the mouth, regardless of whether its source is oral or non-oral [bib_ref] Production and origin of oral malodor: a review of mechanisms and methods..., Tonzetich [/bib_ref] [bib_ref] Prevalence of halitosis in the population of the city of Bern, Switzerland:..., Bornstein [/bib_ref] [bib_ref] Influence of oral health and lifestyle on oral malodour, Eldarrat [/bib_ref]. Other terms used to describe this
## Study population
This cross-sectional study was conducted in three randomly selected districts among the six districts with concentrations of office buildings in Shanghai, China. One office building in each district and two departments in each building were selected using cluster random sampling. All staff members in these departments were verbally invited to participate in the study by department directors who had been informed of the purpose and structure of the study. The sample size was calculated on the basis of a prevalence of halitosis of 20% in the general Chinese population, as determined by Halimter [bib_ref] Oral malodor-related parameters in the Chinese general population, Liu [/bib_ref]. The minimum sample size for the study was determined to be 512 participants; allowing for a 15% non-response rate, a sample of at least 590 participants was sought. The Ethics Committee of the Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University approved this study. All participants provided written informed consent prior to examination and questionnaire administration. The study was performed in accordance with the principles of the Helsinki Declaration.
## Examination procedure
In each office building, we rented a room for several days for examination purposes. Subjects were asked to complete the questionnaire, and then oral malodor was assessed. Clinical oral examination was conducted with each subject seated in a portable dental chair. Three dentists were involved in the investigation, each of whom was responsible for one procedure. The examination procedure, which required about 2 hours for each subject, was conducted during the working hours of each office building. Subjects were not given specific instructions (e.g., to brush) or prohibitions (e.g., to not eat).
## Oral malodor assessment
Oral malodor was assessed with a Halimeter (Interscan, Chatsworth, CA, USA) by measuring VSCs originating from the oral cavity. Each subject was asked to close his/her mouth for at least 1 minute. He/she was then instructed to hold a disposable tube above the posterior tongue dorsum, with the mouth slightly open while breathing through the nose. The peak value [in parts per billion (ppb)] displayed by the Halimeter was recorded. A VSC value of 110 ppb or greater was considered to indicate the presence of halitosis, according to the manufacturer's instructions .
## Clinical oral examination
A trained and licensed dentist assessed participants' dental caries status, gingival inflammation, periodontal status, plaque accumulation, and tongue coating. Oral examinations were conducted after oral malodor assessment to avoid the potential effect of possible gingival bleeding after probing on the accuracy of the latter. Decayed, missing, and filled tooth (DMFT) and decayed, missing, and filled surface (DMFS) indices were calculated. Dental caries status was assessed using the visual-tactile criteria proposed by the World Health Organizationfor the diagnosis of pitand-fissure and smooth-surface lesions. Gingival inflammation and plaque accumulation were examined using the gingival index (GI) and plaque index (PLI) at four sites (mesial, distal, buccal, and lingual) on six index teeth (#3, #8, #11, #19, #25, and #28) [bib_ref] Indices for prevalence and incidence of periodontal disease, Ramfjord [/bib_ref] [bib_ref] Periodontal disease in pregnancy. I. Prevalence and severity, Loe [/bib_ref] [bib_ref] Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition, Silness [/bib_ref]. Periodontal probing depth (PPD) was measured at four sites on each tooth using a scaled periodontal probe. Tongue coating was evaluated according to the area covered (0 = none, 1 = <1/3 of the tongue, 2 = 1/3-2/3 of the tongue, 3 = >2/3 of the tongue) and thickness (0 = none; 1 = thin, tongue papillae visible; 2 = thick, tongue papillae invisible) [bib_ref] Characteristics of patients complaining of halitosis and the usefulness of gas chromatography..., Oho [/bib_ref].
To assess intraexaminer reproducibility, 10% of subjects were reexamined. Cohen's κ values for all clinical measurements (including dental caries status, gingival inflammation, periodontal status, plaque accumulation, and tongue coating evaluation) ranged from 0.87 to 0.98.
## Questionnaire
To supplement the oral examinations and obtain information about factors associated with halitosis, participants were asked to complete a 52-item self-administered questionnaire. This instrument has been used in halitosis clinics [bib_ref] Characteristics of patients complaining of halitosis and factors associated with halitosis, Lu [/bib_ref] and was modified and updated for the whitecollar employee population. It was used to collect information about subjects' sociodemographic characteristics (gender, age, education level, income), dietary habits (e.g., frequency of fruit, sweets, meat, and tea consumption; smoking and alcohol consumption habits), oral hygiene habits (toothbrushing frequency, use of dental floss and mouthwash, tongue scraping), and oral discomfort (e.g., self-reported gingival inflammation and bleeding, oral ulcers, tongue coating, dry mouth, food impaction). Most questionnaire items concerned respondents' habits in the previous month. Subjects were also asked to provide medical history data, including information about gastrointestinal diseases and discomfort.
# Statistical analysis
Descriptive statistics (means, standard deviations, and percentages) were calculated for subjects' sociodemographic characteristics and VSC measurements. Chi-squared tests were performed to examine differences in VSC values [dichotomized as 0 (VSC < 110 ppb) and 1 (VSC 110 ppb)] according to clinical and lifestyle factors. Student's t-test and one-way analysis of variance were used to examine differences in VSC values. Multiple factor analyses were performed to explore factors associated with oral malodor. VSC values (dichotomized as 0 and 1) were examined using multiple logistic regression, and continuous VSC values were examined using analysis of covariance. All variables with P values < 0.2 in bivariate analyses were entered into these two regression models. All statistical analyses were conducted using SPSS software (ver. 20.0; IBM Corporation, Armonk, NY, USA), with the significance level set to P < 0.05.
# Results
## Study participants
Of the 805 white-collar workers invited to participate in the study, 720 [347 men, 373 women; mean age, 30.4 (range, 22-70) years] were enrolled (89.4% response rate). Subjects who were absent on the examination days due to business trips or illness and those unwilling to undergo dental examination were excluded from the study. Clinical and survey data from the 720 subjects were included in the final analyses. No difference in age or gender distribution was noted between subjects who did and did not participate in the study.
## Oral malodor and general characteristics
The mean VSC value was 117 ± 103 ppb. VSC values exceeded 110 ppb, the cutoff value for halitosis, in 33.2% of subjects. Halitosis was more prevalent in men than in women (P < 0.01). VSC values were higher in participants with higher body mass indices (BMIs) (P < 0.05). No significant difference in the prevalence of halitosis according to participants' place of birth, education level, or monthly income was observed [fig_ref] Table 1: Relationships between oral malodor measurements and sociodemographic and background characteristics of white-collar... [/fig_ref].
## Relationships between oral malodor and clinical and lifestyle factors
Relationships between oral malodor measures and clinical factors are shown in [fig_ref] Table 2: Relationships between oral malodor measurements and clinical factors # [/fig_ref]. Tongue coating area and thickness, as well as mean PPD, were associated positively with halitosis (P < 0.01). No significant association was found between oral malodor measures and DMFT or DMFS indices or GIs.
No oral hygiene habit was related to oral malodor [fig_ref] Table 3: Relationships between oral malodor measurements and oral hygiene habits # [/fig_ref]. The percentage of subjects with VSCs 110 ppb was associated with eating within 2 hours prior to oral malodor measurement (P < 0.01), fruit intake frequency (P = 0.03), and work stress (P = 0.043; [fig_ref] Table 4: Relationships between oral malodor measurements and health behaviors # [/fig_ref]. VSC values were lower in subjects who had eaten within 2 hours prior to assessment (P < 0.01) and those with more frequent intakes of fruits, sweet foods, and tea (P < 0.05; [fig_ref] Table 4: Relationships between oral malodor measurements and health behaviors # [/fig_ref]. No significant association between oral malodor measures and self-reported oral or systemic diseases or oral discomfort was observed.
Regression models indicated that the percentage of subjects with VSCs 110 ppb was associated significantly with tongue coating thickness, PPD, and no food consumption within 2 hours prior to assessment . VSC values were associated significantly with tongue coating thickness, PPD, no food consumption within 2 hours prior to assessment, and less frequent intake of sweet foods .
# Discussion
This is the first study to evaluate the prevalence and factors related to halitosis in the specific population of white-collar employees in Shanghai, China. More than one-third (33.2%) of VSC values in the study population exceeded 110 ppb, similar to the results of previous studies [bib_ref] Factors associated with self-reported halitosis (SRH) and perceived taste disturbance (PTD) in..., Nalcaci [/bib_ref] [bib_ref] Prevalence of halitosis in young male adults: a study in Swiss army..., Bornstein [/bib_ref] [bib_ref] Oral malodor and related factors in Japanese senior high school students, Yokoyama [/bib_ref]. Although organoleptic methods are the gold standard for oral malodor assessment, we only measured VSC levels with the Halimeter monitor. As a portable sulfur detector, the Halimeter provides useful data for clinical studies of halitosis [bib_ref] Comparison of volatile sulfur compound concentrations measured with a sulfide detector vs...., Furne [/bib_ref]. Omitting organoleptic methods also simplified the examination procedure and saved time. However, this omission was a definite limitation of this study, and future studies should include both measurements.
In the present study, more men than women had VSCs 110 ppb and higher VSC values in bivariate analysis, consistent with the results of a study conducted in Brazil [bib_ref] Oral malodour and its association with age and sex in a general..., Nadanovsky [/bib_ref]. This finding may be explained by better oral knowledge, attitude, and oral hygiene among women [bib_ref] Gender differences in knowledge, attitude, behavior and perceived oral health among adolescents, Ostberg [/bib_ref] [bib_ref] Level of education and gender-specific selfreported oral health behavior among dental students, Al-Omiri [/bib_ref]. In the present study, gender was not associated with halitosis in regression analyses adjusted for confounding factors. In contrast, halitosis was found to be more prevalent among women than men in a study conducted in Kuwait. As a self-reported questionnaire was used to identify halitosis in that study, the results may reflect women's overestimation of their oral malodor status [bib_ref] Factors associated with self-reported halitosis in Kuwaiti patients, Al-Ansari [/bib_ref]. Most epidemiological studies of halitosis, including a large-scale study conducted in China in 2002, have found no association between oral malodor and gender [bib_ref] Oral malodor-related parameters in the Chinese general population, Liu [/bib_ref] [bib_ref] Association among bad breath, body mass index, and alcohol intake, Rosenberg [/bib_ref] [bib_ref] Factors associated with self-reported halitosis (SRH) and perceived taste disturbance (PTD) in..., Nalcaci [/bib_ref] [bib_ref] Oral malodor and related factors in Japanese senior high school students, Yokoyama [/bib_ref] [bib_ref] Exploring pathways for socio-economic inequalities in self-reported oral symptoms among Korean adolescents, Jung [/bib_ref]. These discrepancies in study findings may due to differences in diagnostic methods and study populations. The association between gender and halitosis should be investigated further. The finding that VSC values were higher in subjects with BMIs > 24 in bivariate analysis is similar to the results of a study conducted in Israel [bib_ref] Association among bad breath, body mass index, and alcohol intake, Rosenberg [/bib_ref]. High BMI has been found to be associated with many diseases, such as diabetes, hypertension, sleep apnea, and periodontitis [bib_ref] Relationship between upper body obesity and periodontitis, Saito [/bib_ref] [bib_ref] ABC of obesity. Obesity-time to wake up, Haslam [/bib_ref]. Sleep apnea may cause dry mouth, a risk factor for oral malodor [bib_ref] Clinical assessment of bad breath: current concepts, Rosenberg [/bib_ref].
The dorsum of the tongue has long been considered the most important site for the development of halitosis, as abundant desquamated cells and leukocytes are retained and anaerobic bacteria grow on its irregular surface [bib_ref] A review of the current literature on aetiology and measurement methods of..., Van Den Broek [/bib_ref] [bib_ref] Volatile sulfur compounds in mouth air from clinically healthy subjects and patients..., Yaegaki [/bib_ref] [bib_ref] Factors associated with self-reported halitosis (SRH) and perceived taste disturbance (PTD) in..., Nalcaci [/bib_ref] [bib_ref] Identification of oral bacterial species associated with halitosis, Haraszthy [/bib_ref] [bib_ref] Halitosis: a review of associated factors and therapeutic approach, Cortelli [/bib_ref] [bib_ref] Oral malodour-a review, Hughes [/bib_ref] [bib_ref] Halitosis (breath odor), Scully [/bib_ref]. Although tongue coating thickness and area were associated with halitosis in bivariate analysis, only thickness remained significantly associated with halitosis after adjusting for confounding factors in the present study. Thus, the content, rather than extent, of tongue coating may contribute to malodor production. We also found that eating within 2 hours prior to examination dramatically reduced the percentage of subjects with VSCs 110 ppb and VSC values, perhaps because of the mechanical cleansing ("scraping") of the tongue surface by food [bib_ref] Morning breath odor: influence of treatments on sulfur gases, Suarez [/bib_ref].
Several studies have indicated that periodontal disease is also a source of halitosis [bib_ref] Oral malodor-related parameters in the Chinese general population, Liu [/bib_ref] [bib_ref] Association between oral malodor and adult periodontitis: a review, Morita [/bib_ref] [bib_ref] The relationship between volatile sulfur compounds and major halitosis-inducing factors, Lee [/bib_ref] [bib_ref] Characteristics of 2000 patients who visited a halitosis clinic, Quirynen [/bib_ref] [bib_ref] Association between volatile sulfur compounds and periodontal disease progression in elderly non-smokers, Makino [/bib_ref] [bib_ref] Association between oral malodour and periodontal disease-related parameters in the general population, Apatzidou [/bib_ref]. In adjusted analyses, we found that PPD was significantly associated with halitosis. In a recent study, halitosis was more likely to be detected in subjects with periodontitis (odds ratio = 9.2) [bib_ref] Association between oral malodour and periodontal disease-related parameters in the general population, Apatzidou [/bib_ref]. However, some researchers did not find association between halitosis and periodontitis and periodontal pocket is considered a "closed" environment that only a small fraction of sulfur compounds escape into the oral cavity [bib_ref] Relationship of oral malodor to periodontitis: evidence of independence in discrete subpopulations, Bosy [/bib_ref] [bib_ref] Bad breath and periodontal disease: how related are they?, Rosenberg [/bib_ref]. Periodontal pathogenic bacteria, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, have been shown to contribute to VSC production [bib_ref] The formation of hydrogen sulfide and methyl mercaptan by oral bacteria, Persson [/bib_ref] [bib_ref] The relationship between the presence of periodontopathogenic bacteria in saliva and halitosis, Awano [/bib_ref] [bib_ref] Methyl mercaptan production by periodontal bacteria, Nakano [/bib_ref]. A recent study demonstrated that the amount of P.gingivalis residing on the tongue dorsum may play a key role in oral malodor production in periodontitis patients [bib_ref] Association between oral malodour and periodontal disease-related parameters in the general population, Apatzidou [/bib_ref]. So the reason for the significant relationship between PPD and oral malodor may not contribute to the periodontal pocket, but to the periodontal pathogenic bacteria especially the bacteria residing on tongue dorsum. In the present study, no radiographic assessment of bone loss was performed and the increased pocket levels may be attributed to gingival hyperplasia and pseudo-pocketing in absence of clinical attachment level measurements, the collected data may not reflect the true periodontal situation. This limitation should be avoided in future studies. In analyses controlling for confounding factors, such as age and gender, we found that more frequent intake of sweet foods reduced VSC concentrations. The presence of carbohydrates, such as glucose and sucrose, has been reported to inhibit the expression and activity of trypsinlike enzyme, which is capable of degrading peptides that may produce malodorous compounds by producing an acidic environment [bib_ref] Importance of nutrition in gingival crevice microbial ecology, Loesche [/bib_ref] [bib_ref] Role of Streptococcus mutans in human dental decay, Loesche [/bib_ref] [bib_ref] Microbiology and treatment of halitosis, Loesche [/bib_ref]. Another study showed that 10 children with moderate to high caries activity who were more likely to consume sugar-containing snacks habitually were free from halitosis [bib_ref] Oral malodor in children and volatile sulfur compound-producing bacteria in saliva: preliminary..., Paryavi-Gholami [/bib_ref]. The present study may be the first epidemiological investigation to reveal that the consumption of sweet foods can inhibit VSC production. Fruit consumption was also associated with halitosis in bivariate analysis. As halitosis originates in the oral cavity as a result of proteolytic degradation by anaerobic Gram-negative oral bacteria of various sulfur-containing substrates [bib_ref] Production and origin of oral malodor: a review of mechanisms and methods..., Tonzetich [/bib_ref] , we assume that a greater frequency of fruit intake was accompanied by reduced protein consumption, resulting in less VSC production. Furthermore, as most fruits are sweet, the mechanism of halitosis inhibition may be similar to that of sweet food. Further large-scale epidemiological studies are needed to explore associations between diet and halitosis. Tea is a very popular beverage in China. Green tea has been reported to reduce VSC levels immediately after consumption in vivo and in vitro [bib_ref] Effect of green tea on volatile sulfur compounds in mouth air, Lodhia [/bib_ref]. The inhibition of halitosis may due to the antibacterial effect of green tea and suppression of the mgl gene, which encodes L-methionine-α-deamino-γ-mercaptomethane-lyase (METase), an enzyme producing methyl mercaptan [bib_ref] Tea catechin EGCg suppresses the mgl gene associated with halitosis, Xu [/bib_ref] [bib_ref] Evaluation of the effect of green tea extract on mouth bacterial activity..., Moghbel [/bib_ref]. In the present study, more frequent tea intake was related to lower VSC levels in bivariate analysis, but not in the regression analysis adjusted for confounding factors. We did not ask participants about the type and timing of tea consumption, which limited our ability to explore this association. The relationship between tea consumption and halitosis should be investigated further.
More than 90% of the subjects in this study complained of moderate to severe work stress. A greater proportion of participants with heavy work stress had VSCs 110 ppb in the bivariate analysis. One animal experiment showed that rats under stress produced more VSCs [bib_ref] Oral concentration of volatile sulphur compounds in stressed rats, Kurihara [/bib_ref]. In a recent study, stress was shown to increase the production of VSCs, especially hydrogen sulfide, in men and women [bib_ref] Influence of gender and stress on the volatile sulfur compounds and stress..., Lima [/bib_ref]. However, work stress was not included in the final regression model in the present study. Thus, the relationship between work stress and halitosis requires further evaluation.
This study was an epidemiological study of halitosis in a specific population rather than the general population. Thus, the results may only be applicable to individuals with similar backgrounds and working conditions. Caution should be taken when seeking to apply the results to other populations.
# Conclusion
In the present study, tongue coating thickness and PPD were significantly related to measures of oral malodor. As most cases of halitosis originate from the oral cavity, tongue cleaning and periodontal health maintenance may help to reduce oral malodor. The results of our study suggest that appropriate intake of sweet foods, while controlling for the risk of caries development, can reduce VSC production. However, the relationship between halitosis and sweet food consumption, as well as the appropriate amount of such foods to control VSC production without causing caries, requires further investigation.
Supporting Information S1 STROBE Checklist. STROBE Checklist cross-sectional. (DOC)
[table] Table 1: Relationships between oral malodor measurements and sociodemographic and background characteristics of white-collar employees in China # . [/table]
[table] Table 2: Relationships between oral malodor measurements and clinical factors # .Obtained by Chi-squared test, Student's t-test (two groups), and one-way ANOVA.VSC: volatile sulfur compound; SD: standard deviation. [/table]
[table] Table 3: Relationships between oral malodor measurements and oral hygiene habits # . [/table]
[table] Table 4: Relationships between oral malodor measurements and health behaviors # . PLOS ONE | DOI:10.1371/journal.pone.0155592 May 17, 2016 [/table]
|
Autophagy Modulators in Cancer: Focus on Cancer Treatment
# Introduction
Autophagy (self-eating) is defined as a homeostatic process that enables the lysosomal degradation of unnecessary or dysfunctional organelles or proteins. There are three types of autophagy: microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy. Microautophagy is involved with the direct uptake of cargo by the lysosome, and little is known about its mechanism. CMA is a selective process and involves substrates with specific motifs (KFERQ or similar KFERQ generated by modification). Heat-shock cognate chaperone of 70 kDa (Hsc70) recognizes the KFERQ motif of the substrate and binds and moves together to the lysosomal membrane. Chaperone selectively binds to LAMP2A on the lysosomal membrane and internalizes the substrate into the lysosome through LAMP2A. Thus, CMA has not only selective substrates but also specific machinery components.
Macroautophagy, usually considered as "autophagy" and thereafter referred to as autophagy, is primarily induced by nutrient starvation (e.g., glucose starvation or amino acid starvation). Cells deal with starvation by recycling cellular organelles and proteins and then generating energy. Autophagy is initiated with the formation of a phagophore, which is also known as an isolation membrane. The membrane source of phagophore has been proposed to originate from the smooth endoplasmic reticulum (ER), mitochondria or plasma membrane. The conjugation of LC3 to phosphatidylethanolamine (PE) yields recruitment to the nascent phagophore structure. The precursor proLC3 is cleaved by Atg4 protease into LC3-I with a C-terminal glycine residue, and the C-terminus is then conjugated to the polar head of PE by Atg complex to form LC3-II. The lipidated LC3 (LC3-II) is localized on the membrane's inner and outer sides and contributes to the expansion and closure of the phagophore. As LC3-II migrates faster than LC3-I in gel electrophoresis experiments, it is widely used as a marker for assessing autophagy induction. Phagophore grows, engulfs cytosolic components and closes to become an autophagosome. Then, the autophagosome fuses with a lysosome and degrades sequestered contents. Cellular response to nutrient deprivation is thought to be non-selective autophagy and usually involves random uptake of cytoplasm. Conversely, selective autophagy selectively eliminates specific cellular components, such as damaged organelles, or protein aggregates. Moreover, pathogens are targeted for degradation by selective autophagy. Cargo-specific or protein aggregates. Moreover, pathogens are targeted for degradation by selective autophagy. Cargo-specific names have been used to classify the various types of selective autophagy (e.g., pexophagy, mitophagy, aggrephagy, glycophagy, lipophagy ribophagy and xenophagy). In particular, mitophagy is strongly associated with tumorigenesis.. Schematic diagram of autophagic progression. There are three types of autophagy in mammalian cells: macroautophagy, microautophagy and chaperone-mediated autophagy. When macroautophagy is induced, cytoplasmic materials are engulfed by double membranes (phagophore), closed (autophagosome), fused with the lysosome (autolysosome) and degraded. The KFERQ motif is recognized by Hsc70 and binds with LAMP2A in CMA. LAMP2A mediates the translocation of the substrate into lysosomes. Microautophagy directly uptakes the cytoplasmic cargo.
Autophagy-modulating drugs have been increasingly used in clinical trials; simultaneously, many phenotypic screens are being conducted for new drug discovery, which can modulate autophagy for therapeutic purposes. However, it is not easy to screen and identify new autophagy-modulating chemicals. The brief analysis of the role of autophagy modulators through LC3-II or autophagosome accumulation during drug screening may be misleading. An increase in LC3-II or autophagosomes can be interpreted as autophagy induction and blockade: an increase in the rate of autophagosome formation or a decrease in the rate of autophagosome clearance following fusion with lysosomes. To solve this problem, autophagic flux probes, such as GFP-LC3-RFP-LC3ΔG or mRFP-GFP-tagged LC3, were developed and used for high-throughput screening to identify autophagy modulators. Autophagy flux was quantitatively monitored by calculating the GFP/RFP ratio or LC3 puncta: the decreased GFP/RFP ratio is detected by the autophagy inducer, whereas the autophagy inhibitor detects the increased GFP/RFP ratio. However, an assay using an autophagic flux probe has a limitation, as it does not distinguish whether the autophagy modulators inhibit the initiation step or lysosomal fusion step. To apply autophagy modulators as therapeutics, more research beyond screening is required to determine which steps of autophagy are regulated by these chemicals. Through various autophagy screening methods, researchers are discovering novel autophagy modulators and/or adding new functions as autophagy modulators to existing drugs. Autophagy-modulating drugs have been increasingly used in clinical trials; simultaneously, many phenotypic screens are being conducted for new drug discovery, which can modulate autophagy for therapeutic purposes. However, it is not easy to screen and identify new autophagy-modulating chemicals. The brief analysis of the role of autophagy modulators through LC3-II or autophagosome accumulation during drug screening may be misleading. An increase in LC3-II or autophagosomes can be interpreted as autophagy induction and blockade: an increase in the rate of autophagosome formation or a decrease in the rate of autophagosome clearance following fusion with lysosomes. To solve this problem, autophagic flux probes, such as GFP-LC3-RFP-LC3∆G or mRFP-GFP-tagged LC3, were developed and used for high-throughput screening to identify autophagy modulators. Autophagy flux was quantitatively monitored by calculating the GFP/RFP ratio or LC3 puncta: the decreased GFP/RFP ratio is detected by the autophagy inducer, whereas the autophagy inhibitor detects the increased GFP/RFP ratio. However, an assay using an autophagic flux probe has a limitation, as it does not distinguish whether the autophagy modulators inhibit the initiation step or lysosomal fusion step. To apply autophagy modulators as therapeutics, more research beyond screening is required to determine which steps of autophagy are regulated by these chemicals. Through various autophagy screening methods, researchers are discovering novel autophagy modulators and/or adding new functions as autophagy modulators to existing drugs.
## Autophagy: tumor suppressor or promoter?
In normal cells, autophagy contributes to the maintenance of homeostasis. Autophagy is a mechanism to deal with starvation, but it also plays an essential role in removing substances that can be toxic to cells. For instance, autophagy is induced in response to reactive oxygen species (ROS) to remove them and protect the cells from apoptosis, whereas autophagy impairment causes the accumulation of oxidative stress. Moreover, since ROS is involved in DNA damage and genetic instability, the removal of ROS by autophagy may be critical for blocking the transformation of normal cells.
However, in cancer cells, the role of autophagy has remained controversial. Autophagy has been reported to have both antitumor and tumor-promoting effects in cancers. Since the role of autophagy varies according to the cancer stage and tumor type, it is necessary first to check how autophagy is dysregulated in target cancers. Due to the opposite effects of autophagy, either inhibitors or inducers of autophagy could be exploited for cancer therapy depending on cancer context, respectively.
In the early stages of cancer development, autophagy is believed to play a protective role against cancer initiation. Inhibition of autophagy or defects of autophagy can lead to impaired removal of toxic materials such as damaged organelles, unfolded proteins or ROS. Here, inducing autophagy might serve as a cancer prevention or treatment. Additionally, Beclin1, which is the core subunit of the PI3K complex and is involved in initiation of autophagosome, heterozygote mice exhibited spontaneous development of malignant tumors, indicating that autophagy inhibition can induce tumorigenesis. Furthermore, decreased expression of autophagic genes (Atg5, Beclin1, Atg7) and autophagic activity was observed in hepatocellular carcinoma (HCC). In glioblastoma (GBM), low levels of ULK2 transcripts by DNA methylation were reported. ULK2 overexpression induced autophagy and inhibited astrocyte transformation and tumor growth in glioblastoma. Altogether, these studies indicate that autophagy genes function as tumor suppressors in several tumors.
However, tumor-promoting effects of autophagy are more prominent in various cancers. Once the carcinogenic phenotype is established, cancer cells exploit autophagy mechanisms to satisfy their energy requirements. Particularly, autophagy contributes to the adaptation and survival of cancer cells in unfavorable conditions such as hypoxia or nutrient-deficient conditions. Therefore, inducing autophagy in this situation may rather promote tumor progression. Notably, enhanced autophagy flux was observed in various tumors.
Additionally, autophagy has also been determined to promote the invasion of hepatocellular carcinoma cells through the activation of epithelial mesenchymal transition (EMT). Autophagy is also associated with the metastatic ability of pancreatic cancer. These studies suggest that autophagy is critical for cancer metastasis. However, it was reported that autophagy induction in glioblastoma cells rather impairs migration and invasion. Interestingly, SNAIL, which is responsible for glioblastoma cell movement, was degraded upon autophagy induction. This is another example showing that autophagy does not have the same effect on all cancer types.
Autophagy is also related to cancer stem cells (CSCs) in various cancers such as breast, ovarian, liver and brain cancer. Basal levels of autophagy are required to maintain the balance between pluripotency and differentiation in CSCs. Intriguingly, changes in the basal levels of autophagy by either autophagy inducer or inhibitor can result in a decrease in pluripotency or an increase in differentiation and senescence in CSCs.
Several studies have already reported that autophagy flux is enhanced in various CSCs, and, in this case, suppressing autophagy has a positive effect in terms of cancer treatment. In mammosphere conditions, a culture system in which mammary CSCs/progenitor cells can be propagated, a greater autophagy flux was displayed than in normal adherent culture conditions. The knockdown of Beclin1 impaired CSC maintenance and proliferations. Additionally, ovarian CSCs have enhanced autophagic flux, and the inhibition of autophagy decreased the self-renewal ability and chemoresistance of ovarian CSCs. It has been reported that the enhanced autophagy flux in liver CSCs contributes to the adaptation of liver CSCs to the tumor microenvironment, such as hypoxia and nutrient-deficient conditions. Therefore, autophagy inhibitors may make liver CSCs CSC resistance to chemotherapy is also associated with autophagy. Many studies have shown that combining cytotoxic drugs and autophagy inducers or inhibitors increases the sensitivity of CSCs. For instance, autophagy induction by rapamycin promoted cell differentiation and made GSCs sensitive to radiation therapy. Azathioprine, an immunosuppressant for rheumatoid arthritis or Crohn's disease, induces autophagy through mTORC1and also sensitizes GBM to chemotherapy or radiotherapy. In terms of autophagy inhibition, chloroquine was able to increase the chemosensitivity of glioma cells to temozolomide. Altogether, autophagy modulators can be used for cancer treatment because the imbalance in autophagy leads to cancer cell death.
## Modulators of initial autophagy and cancer treatment
The initiation stage of autophagy is controlled by a series of autophagy-related genes (ATGs). Specifically, AMPK, mTOR, and unc-51-like kinase 1 (ULK1, also known as Atg1) play a central role in autophagy initiation. Under conditions of nutrient sufficiency, active mTOR inhibits AMPK activation and phosphorylates ULK1 at the S758 site in humans (Ser757 in mouse). Phosphorylation of ULK S758 was also found to inhibit the interaction between ULK1 and AMPK. Upon nutrient starvation conditions, activated AMPK phosphorylates ULK1 at various sites and initiates autophagy. Additionally, autophagy is accelerated by AMPK-induced phosphorylation of Raptor and TSC1, which is associated with mTOR inhibition. Firstly, an autophagy activator can regulate autophagy initiation and serve as a treatment for cancer. This is described inand. contributes to the adaptation of liver CSCs to the tumor microenvironment, such as hypoxia and nutrient-deficient conditions. Therefore, autophagy inhibitors may make liver CSCs difficult to survive in an unfavorable tumor microenvironment, which may help improve anticancer therapeutic effects. CSC resistance to chemotherapy is also associated with autophagy. Many studies have shown that combining cytotoxic drugs and autophagy inducers or inhibitors increases the sensitivity of CSCs. For instance, autophagy induction by rapamycin promoted cell differentiation and made GSCs sensitive to radiation therapy. Azathioprine, an immunosuppressant for rheumatoid arthritis or Crohn's disease, induces autophagy through mTORC1and also sensitizes GBM to chemotherapy or radiotherapy. In terms of autophagy inhibition, chloroquine was able to increase the chemosensitivity of glioma cells to temozolomide. Altogether, autophagy modulators can be used for cancer treatment because the imbalance in autophagy leads to cancer cell death.
## Modulators of initial autophagy and cancer treatment
The initiation stage of autophagy is controlled by a series of autophagy-related genes (ATGs). Specifically, AMPK, mTOR, and unc-51-like kinase 1 (ULK1, also known as Atg1) play a central role in autophagy initiation. Under conditions of nutrient sufficiency, active mTOR inhibits AMPK activation and phosphorylates ULK1 at the S758 site in humans (Ser757 in mouse). Phosphorylation of ULK S758 was also found to inhibit the interaction between ULK1 and AMPK. Upon nutrient starvation conditions, activated AMPK phosphorylates ULK1 at various sites and initiates autophagy. Additionally, autophagy is accelerated by AMPK-induced phosphorylation of Raptor and TSC1, which is associated with mTOR inhibition. Firstly, an autophagy activator can regulate autophagy initiation and serve as a treatment for cancer. This is described inand.
## Ampk activator
Activating AMPK is believed to be a promising cancer therapeutic because AMPK is involved in regulating cell growth-related pathways such as cell proliferation, protein synthesis and lipid biosynthesis. AMPK acts as a sensor of AMP/ATP or ADP/ATP ratio and contributes to maintaining homeostasis when cellular energy is low.
Metformin, which has already been used for a long time as a drug against type 2 diabetes, induces autophagy by activating AMPK indirectly. Inhibition of mitochondria respiratory chain complex I by metformin increases the AMP/ATP ratio, which then induces AMPK activation. Several studies have suggested that metformin suppresses cell proliferation and induces apoptosis in several cancer cells, including renal, colorectal liver and pancreatic cancer cells. An in vivo study demonstrated tha administration of metformin to hamsters with high-fat diet has diminished the occurrence of pancreatic cancer following exposure to pancreatic carcinogens. Additionally patients with type 2 diabetes who were given metformin had a significantly lower risk o developing pancreatic and hepatocellular cancer than patients who received other medications. These studies suggest that AMPK activation and subsequent autophagy induction may have a preventive role in cancer incidence. Based on these studies metformin is currently undergoing various clinical trials for cancer patients withou diabetes. Interestingly, the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improved progression-free survival in phase 2 randomized clinical trials. Additionally, various phase 1 clinica trials were conducted for patients with glioblastoma or advanced/refractory cancer, and the stability of metformin was confirmed.
5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activator of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to
## Ampk activator
Activating AMPK is believed to be a promising cancer therapeutic because AMPK is involved in regulating cell growth-related pathways such as cell proliferation, protein synthesis and lipid biosynthesis. AMPK acts as a sensor of AMP/ATP or ADP/ATP ratio and contributes to maintaining homeostasis when cellular energy is low.
Metformin, which has already been used for a long time as a drug against type 2 diabetes, induces autophagy by activating AMPK indirectly. Inhibition of mitochondria respiratory chain complex I by metformin increases the AMP/ATP ratio, which then induces AMPK activation. Several studies have suggested that metformin suppresses cell proliferation and induces apoptosis in several cancer cells, including renal, colorectal liver and pancreatic cancer cells. An in vivo study demonstrated that administration of metformin to hamsters with high-fat diet has diminished the occurrence of pancreatic cancer following exposure to pancreatic carcinogens. Additionally patients with type 2 diabetes who were given metformin had a significantly lower risk of developing pancreatic and hepatocellular cancer than patients who received other medications. These studies suggest that AMPK activation and subsequent autophagy induction may have a preventive role in cancer incidence. Based on these studies metformin is currently undergoing various clinical trials for cancer patients without diabetes. Interestingly, the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improved progression-free survival in phase 2 randomized clinical trials. Additionally, various phase 1 clinica trials were conducted for patients with glioblastoma or advanced/refractory cancer, and the stability of metformin was confirmed.
5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activator of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to
## Ampk activator
Activating AMPK is believed to be a promising cancer therapeutic because AMPK i involved in regulating cell growth-related pathways such as cell proliferation, protein synthesis and lipid biosynthesis. AMPK acts as a sensor of AMP/ATP or ADP/ATP ratio and contributes to maintaining homeostasis when cellular energy is low.
Metformin, which has already been used for a long time as a drug against type 2 diabetes, induces autophagy by activating AMPK indirectly. Inhibition of mitochondria respiratory chain complex I by metformin increases the AMP/ATP ratio, which then induces AMPK activation. Several studies have suggested that metformin suppresse cell proliferation and induces apoptosis in several cancer cells, including renal, colorectal liver and pancreatic cancer cells. An in vivo study demonstrated tha administration of metformin to hamsters with high-fat diet has diminished the occurrence of pancreatic cancer following exposure to pancreatic carcinogens. Additionally patients with type 2 diabetes who were given metformin had a significantly lower risk o developing pancreatic and hepatocellular cancer than patients who received othe medications. These studies suggest that AMPK activation and subsequent autophagy induction may have a preventive role in cancer incidence. Based on these studies metformin is currently undergoing various clinical trials for cancer patients withou diabetes. Interestingly, the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improved progression-fre survival in phase 2 randomized clinical trials. Additionally, various phase 1 clinica trials were conducted for patients with glioblastoma or advanced/refractory cancer, and the stability of metformin was confirmed.
5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activato of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to
## Ampk activator
Activating AMPK is believed to be a promising cancer therapeutic because AMPK is involved in regulating cell growth-related pathways such as cell proliferation, protein synthesis and lipid biosynthesis. AMPK acts as a sensor of AMP/ATP or ADP/ATP ratio and contributes to maintaining homeostasis when cellular energy is low.
Metformin, which has already been used for a long time as a drug against type 2 diabetes, induces autophagy by activating AMPK indirectly. Inhibition of mitochondria respiratory chain complex I by metformin increases the AMP/ATP ratio, which then induces AMPK activation. Several studies have suggested that metformin suppresses cell proliferation and induces apoptosis in several cancer cells, including renal, colorectal liver and pancreatic cancer cells. An in vivo study demonstrated tha administration of metformin to hamsters with high-fat diet has diminished the occurrence of pancreatic cancer following exposure to pancreatic carcinogens. Additionally patients with type 2 diabetes who were given metformin had a significantly lower risk o developing pancreatic and hepatocellular cancer than patients who received other medications. These studies suggest that AMPK activation and subsequent autophagy induction may have a preventive role in cancer incidence. Based on these studies metformin is currently undergoing various clinical trials for cancer patients withou diabetes. Interestingly, the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improved progression-free survival in phase 2 randomized clinical trials. Additionally, various phase 1 clinica trials were conducted for patients with glioblastoma or advanced/refractory cancer, and the stability of metformin was confirmed.
5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activator of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to
## Ampk activator
Activating AMPK is believed to be a promising cancer therapeutic because AMPK is involved in regulating cell growth-related pathways such as cell proliferation, protein synthesis and lipid biosynthesis. AMPK acts as a sensor of AMP/ATP or ADP/ATP ratio and contributes to maintaining homeostasis when cellular energy is low.
Metformin, which has already been used for a long time as a drug against type 2 diabetes, induces autophagy by activating AMPK indirectly. Inhibition of mitochondrial respiratory chain complex I by metformin increases the AMP/ATP ratio, which then induces AMPK activation. Several studies have suggested that metformin suppresses cell proliferation and induces apoptosis in several cancer cells, including renal, colorectal, liver and pancreatic cancer cells. An in vivo study demonstrated that administration of metformin to hamsters with high-fat diet has diminished the occurrence of pancreatic cancer following exposure to pancreatic carcinogens. Additionally, patients with type 2 diabetes who were given metformin had a significantly lower risk of developing pancreatic and hepatocellular cancer than patients who received other medications. These studies suggest that AMPK activation and subsequent autophagy induction may have a preventive role in cancer incidence. Based on these studies, metformin is currently undergoing various clinical trials for cancer patients without diabetes. Interestingly, the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improved progression-free survival in phase 2 randomized clinical trials. Additionally, various phase 1 clinical trials were conducted for patients with glioblastoma or advanced/refractory cancer, and the stability of metformin was confirmed. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activator of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to inhibit autophagy by a pathway independent of AMPK. Similar to other AMPK activators such as metformin, phenformin, salicylate and 2-deoxy-D-glucose (2-DG), AICAR inhibits cell growth; however, it also inhibits cell growth in AMPK-deficient cells, suggesting that AMPK-independent regulation exists. In other words, AICAR may have nonspecific effects on AMPK or control independent of autophagy. Recently developed AMPK activators are more specific. A-769662, an AMPK activator, showed specific AMPK-dependent retardation of cell growth and metabolism. GSK621, a direct and specific activator of AMPK, induces cytotoxicity and autophagy in acute myeloid leukemia (AML). Continuous efforts are made to develop specific and potent AMPK activators and use them for cancer treatment.
## Mtor inhibitor
Rapamycin/sirolimus, a representative mTOR inhibitor and autophagy inducer, was initially approved as an immunosuppressant to prevent allograft rejection. Due to its poor solubility and pharmacokinetics of rapamycin, several rapamycin analogs (rapalogs) were developed. Autophagy is induced in cancer cells not only by rapamycin but also by rapalogs. Temsirolimus, a rapalog, was approved by the Food and Drug Administration (FDA) in 2007 for treating advanced renal cancer carcinoma (RCC). Thereafter, everolimus was approved for RCC treatment by FDA in 2009. Additionally, everolimus was later approved as a treatment for various cancers, including astrocytoma, breast cancer, angiomyolipoma and neuroendocrine cancer. However, rapalogs have achieved only modest effects in clinical practice. The reasons for clinical limit have not been fully established, but it may involve multiple mTORC1 regulatory negative feedback loops in cancer cells. To overcome this limitation, multiple clinical trials are currently evaluating the efficacy of rapalogs plus chemotherapy combination therapy. AZD-8055 is a potent bioavailable ATP-competitive mTORC1 and mTORC2 inhibitor and is under phase I clinical trial in patients with various tumors. In preclinical data, AZD8055 exhibited a more potent anticancer effect than rapamycin in the brain tumor xenograft model. Efforts focused on developing novel mTOR inhibitors or optimal combinations with rapalogs will have great potential to yield an improved efficacy.
Nutraceutical products obtained from normal food can control autophagy. Epigallocatechin gallate (EGCG), a polyphenol compound obtained from green tea, has been reported to modulate autophagy by affecting the balance of the mTOR-AMPK pathway. Fisetin, a member of the flavonoid group of polyphenols, inhibits mTOR activity and induces autophagic-programmed cell death in several cancer cells, including prostate carcinoma and oral squamous cell carcinoma.
Various inhibitors of autophagy initiation (autophagy blockers) were developed and actively used in biological experiments to determine the role of autophagy in cancer cells. However, autophagy blockers have not entered clinical trials yet. Since various preclinical experiments are in progress, it is expected that autophagy blockers that enter clinical trials will emerge when numerous data are accumulated. Autophagy blockers are discussed below in terms of cancer treatmentand.
## Ulk1/2 inhibitors
Several ULK1 or 2 activators, such as BL-918, typically target Parkinson's disease but not cancers. Rather, ULK1/2 inhibitors have been developed as oncolytic drugs in certain cancers. In preclinical data, SBI-0206965, ULK1 inhibitor, reduced cell growth and promoted apoptosis in neuroblastoma. SBI-0206965 and MRT-68921, another ULK1 inhibitor, was able to induce apoptosis in AML with FLT3-ITD mutation. However, ULK1 inhibitors alone are not sufficient for tumor suppression in certain cancers. Recently, a paper was published explaining the reason why the anticancer effect of ULK inhibition monotherapy was ineffective in pancreatic ductal adenocarcinoma (PDAC). Intriguingly, autophagy inhibition induced micropinocytosis, which is the uptake of extracellular fluid droplets containing proteins and other macromolecules. As micropinocytosis provides energy to autophagy-compromised cells, ULK inhibition alone could not induce apoptosis in PDAC. Indeed, combination treatment of MRT-68921 and macropinosome formation inhibitor synergistically induced tumor regression in an in vivo model.
## Pi3k inhibitors
Although the PI3K-AKT-mTOR axis is also critical for autophagy regulation, modulators of upstream kinases are not specific in autophagy regulation and show mixed autophagy readouts depending on cell types or treatment concentration and time. For example, 3-methyladenine (3-MA) is used as an autophagy inhibitor, but it also inhibits PI3K class I and class III. PI3K class III stimulates autophagic sequestering, whereas PI3K class I inhibits it. Although the overall effect of 3-MA is considered an autophagy inhibitor, PI3K inhibitors are not suitable for clinical setting due to the complexity of the results.
## Modulators of lysosomal activity
Autophagosome fuses with a lysosome and eventually degrades the contents, including cargo proteins or organelles. Chloroquine (CQ) and a derivative of CQ, that is, hydroxychloroquine (HCQ), are the most well-known and clinically used chemical blockers. CQ and HCQ have been used as antimalarial drugs. CQ and HCQ inhibit autophagy by increasing intralysosomal pH, but the exact mechanism is not well understood. Autophagy inhibition limits the energy supply for cancer growth. Additionally, several damaged proteins or mitochondria can be accumulated in CQ-or HCQ-treated cells, resulting in the ER stress. Triggering excessive ER stress is an excellent strategy to kill cancer cells. Apoptosis is eventually induced because cancer cells cannot tolerate excessive ER stress. In this context, CQ and HCQ show synergistic enhancement when combined with various therapeutic agents in cancer treatment. Treatment with CQ or HCQ along with radiation therapy or standard therapy is already in clinical trials. There are multiple reports that CQ sensitizes glioblastoma to radiation or chemotherapy. Favorable toxicity of CQ in patients was determined, and phase III clinical trials are ongoing (NCT00224978). According to the results of a recently published phase II clinical trial, HCQ along with gemcitabine and nab-paclitaxel resulted in a greater pathological tumor response in pancreatic cancer.
## Pi3k inhibitors
Although the PI3K-AKT-mTOR axis is also critical for autophagy regulation, modulators of upstream kinases are not specific in autophagy regulation and show mixed autophagy readouts depending on cell types or treatment concentration and time. For example, 3-methyladenine (3-MA) is used as an autophagy inhibitor, but it also inhibits PI3K class I and class III. PI3K class III stimulates autophagic sequestering, whereas PI3K class I inhibits it. Although the overall effect of 3-MA is considered an autophagy inhibitor, PI3K inhibitors are not suitable for clinical setting due to the complexity of the results.
## Modulators of lysosomal activity
Autophagosome fuses with a lysosome and eventually degrades the contents, including cargo proteins or organelles. Chloroquine (CQ) and a derivative of CQ, that is, hydroxychloroquine (HCQ), are the most well-known and clinically used chemical blockers. CQ and HCQ have been used as antimalarial drugs. CQ and HCQ inhibit autophagy by increasing intralysosomal pH, but the exact mechanism is not well understood. Autophagy inhibition limits the energy supply for cancer growth. Additionally, several damaged proteins or mitochondria can be accumulated in CQ-or HCQ-treated cells, resulting in the ER stress. Triggering excessive ER stress is an excellent strategy to kill cancer cells. Apoptosis is eventually induced because cancer cells cannot tolerate excessive ER stress. In this context, CQ and HCQ show synergistic enhancement when combined with various therapeutic agents in cancer treatment. Treatment with CQ or HCQ along with radiation therapy or standard therapy is already in clinical trials. There are multiple reports that CQ sensitizes glioblastoma to radiation or chemotherapy. Favorable toxicity of CQ in patients was determined, and phase III clinical trials are ongoing (NCT00224978). According to the results of a recently published phase II clinical trial, HCQ along with gemcitabine and nab-paclitaxel resulted in a greater pathological tumor response in pancreatic cancer. Vacuolar-type proton adenosine triphosphatase (v-ATPase) is an ATP-driven proton pump responsible for controlling the acidification of lysosomes. Bafilomycin A1 and concanamycin A are v-ATPase inhibitors known to prevent the acidification of lysosomes. Several in vivo xenograft mouse studies revealed that bafilomycin A1 can inhibit tumor growth, including bladder, breast and liver cancers. Although bafilomycin A1 is more famous and commonly used in many preclinical experiments to identify an inhibitory role in cancer growth, several more selective and potent v-ATPase concanamycin A are v-ATPase inhibitors known to prevent the acidification of lysosomes. Several in vivo xenograft mouse studies revealed that bafilomycin A1 can inhibit tumor growth, including bladder, breast and liver cancers. Although bafilomycin A1 is more famous and commonly used in many preclinical experiments to identify an inhibitory role in cancer growth, several more selective and potent v ATPase inhibitors exist (e.g., salicylihalamide A, lobatamides and oximidines). Unlike bafilomycin A1 and concanamycin A, these chemicals inhibit mammalian V-ATPases at low concentrations but do not affect fungi and yeast v-ATPases. However, there are few reports that these chemicals inhibit tumor growth and autophagy, but further studies are still needed to confirm these findings.
## Modulation of cma in cancer
The recognition of substrates initiates CMA with KFERQ-like motifs by cytosolic chaperone Hsc70, and the substrates are then delivered to the lysosomal surface. The targeted substrates bind to lysosomal-associated membrane protein 2A (LAMP2A) and are translocated inwardly across the lysosomal membrane and are eventually degraded. The role of CMA in oncology has not been fully understood as encompassing both pro-survival and pro-death parts in different contexts.
Similar to macroautophagy, CMA plays a critical supportive role in cancer cell proliferation because it satisfies the excessive energy needs of cancer cells by degradation of substrates. However, given CMA substrates, CMA degrades not only oncogenic proteins (e.g., PKM2, mutant p53, HK2, HIF-1α) but also tumor suppressors (unphosphorylated PED, Rnd3). These observations suggest that the role of CMA in oncology is more controversial than macroautophagy, especially regarding cancer treatment.
The distinct and notable characteristics of CMA are the selectivity of the target substrate and specificity of core components. CMA degrades only substrates with KFERQ-like motifs. Oncoproteins or oncoprotein activators with the KFERQ motif might be a promising therapeutic target through CMA modulation. Hsc70 and LAMP2A are identified as the core components required for CMA. Specifically, LAMP2A is the rate-limiting component of the CMA pathway. LAMP2A is under the negative regulation by nuclear retinoic acid receptor-α (RAR-α). Indeed, RAR-α inhibition by synthetic derivatives of all-trans-retinoic acid activated only CMA without regulating other RAR-α-dependent transcriptions. However, RAR-α derivatives have the disadvantage that they can be used only when cancer cells are under the regulation of RAR-α. The application of RAR-α derivatives to cancer cells has not been published. Development of more diverse CMA modulators is required for application to anticancer treatment.
## Autophagy modulators associated with epigenetics
Although autophagy is rapidly regulated and occurs in the cytoplasm by starvation signal, transcriptional and epigenetic regulation in the nucleus are also deemed essential for the overall autophagy process. Nutrient starvation regulates not only upstream kinase of autophagy, such as AMPK or mTOR, but also the translocation of transcription factors to the nucleus or histone modifications of target genes. Transcriptional regulation of autophagy is required to maintain prolonged autophagy. Furthermore, because autophagy receptors and LC-3 proteins are degraded with cargo, cells replenish autophagy proteins with transcriptional upregulation of autophagy-related genes.
## Mit/tfe family
The major transcription factor of autophagy regulation is the transcription factor EB (TFEB). TFEB belongs to the microphthalmia/transcription factor E (MiT/TFE) family of transcription factors that bind coordinated lysosomal expression and regulation element. Under starvation conditions, TFEB translocates to the nucleus to promote the transcription of lysosome biogenesis and autophagy-related genes. Its phosphorylation status at various sites tightly controls the nuclear import or export of TFEB and induced by mTORC1 or GSK3β. According to an interesting publication, increased mRNA and protein expression of MITF, TFE3 and TFEB was detected in pancreatic ducal adenocarcinoma (PDA) cell lines and patient tumors. PDA is one of the most lethal of cancers with a 5-year survival rate of only 6% after diagnosis; it is a cancer determined to be highly dependent on autophagy for the supply of essential nutrients. Overexpressed MiT/TFE family transcription factor activates autophagy and maintains intracellular amino acid pools. However, small molecules that can directly modulate the MiT/TFE family have not yet been developed. Since the MiT/TFE family is an attractive target for PDA treatment, it is expected to be an excellent therapeutic agent if potent and selective modulators are developed.
## Histone modifications
Autophagy regulates, directly or indirectly, histone modifications to control transcription. Negative regulation occurs through histone H3 K9 or K27 methylationand. Under nutrient-rich conditions, H3K9 methylation is maintained by G9a methyltransferase in autophagy-related genes. However, a decrease in G9a recruitment and subsequent H3K9 dimethylation is observed by starvation. EZH2, a methyltransferase of H3 K27, regulated the expression of mTOR pathway-related genes through H3K27 methylation and knockdown of EZH2-induced autophagy. In addition, EZH2 inhibitors such as GSK126 and EPZ-011989 induced autophagy. Interestingly, the epigenetic inhibitor induces autophagy by altering the transcription of the target genes. Moreover, in vivo studies revealed that these EZH2 inhibitors showed significant tumor growth inhibition in lymphoma and myeloma. However, studies on how autophagy induction by EZH2 inhibitors affected overall tumor growth inhibition remain lacking. enhancing α-tubulin acetylation and alleviated Cockayne syndrome (CS), a condition of premature aging mainly caused by mutations in the csb gene. Currently, SAHA is approved to treat T-cell lymphoma (CTCL), malignant mesothelioma and multiple myeloma. In the case of cancer in which reduced autophagy promotes tumorigenesis, treatment with SAHA can be effective. Another HDAC inhibitor, that is, MGCD0103/mocetinostat, has been reported to inhibit autophagy. MGCD0103 decreased the autophagic flux in primary lymphocytic leukemia cells by activating the mTOR pathway and downregulating autophagy gene transcription. This suggests that since the role of autophagy is different for each cancer type, it is crucial to identify the detailed function of autophagy according to cancer type and select an appropriate therapeutic agent. In addition, epigenetic regulators can control not only histone modification but also non-histone modification. If HDAC inhibitors modulate histone acetylation, overall transcription may be activated, but it remains difficult to predict how transcription will alter if they modulate an unknown non-histone target. Since target specificity can vary depending on the type of drug as well as the concentration and treatment time of the drug, a fundamental study on how and which target is controlled before application to cancer patients is required.
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as Histone H3R17 methylation is associated with autophagy induction. Under nutrient starvation conditions, stabilized CARM1 methylated H3R17 and activated autophagy and lysosomal genes. As CARM1 methyltransferase activity on H3R17 is inhibited by ellagic acid, which is a naturally occurring phenolic acid abundant in fruits and vegetables, treatment with ellagic acid was able to block autophagy induction. Furthermore, ellagic acid inhibited pancreatic cancer growth in the xenograft model.
There are many reports that histone deacetylase (HDAC) inhibitors regulate autophagy, but the effects are a little complex and controversial. It has been reported that SAHA/vorinostat, one of the HDAC inhibitors, inhibits mTOR and induces autophagy. Additionally, treatment with SAHA improved autophagy flux by enhancing α-tubulin acetylation and alleviated Cockayne syndrome (CS), a condition of premature aging mainly caused by mutations in the csb gene. Currently, SAHA is approved to treat T-cell lymphoma (CTCL), malignant mesothelioma and multiple myeloma. In the case of cancer in which reduced autophagy promotes tumorigenesis, treatment with SAHA can be effective. Another HDAC inhibitor, that is, MGCD0103/mocetinostat, has been reported to inhibit autophagy. MGCD0103 decreased the autophagic flux in primary lymphocytic leukemia cells by activating the mTOR pathway and downregulating autophagy gene transcription. This suggests that since the role of autophagy is different for each cancer type, it is crucial to identify the detailed function of autophagy according to cancer type and select an appropriate therapeutic agent. In addition, epigenetic regulators can control not only histone modification but also non-histone modification. If HDAC inhibitors modulate histone acetylation, overall transcription may be activated, but it remains difficult to predict how transcription will alter if they modulate an unknown non-histone target. Since target specificity can vary depending on the type of drug as well as the concentration and treatment time of the drug, a fundamental study on how and which target is controlled before application to cancer patients is required.
## Conclusions and future perspective
Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as tumors develop. Additionally, cancer cells use autophagy to meet their increased metabolic demands by recycling macromolecules and supplying building blocks. Accordingly, autophagy modulators should be applied to consider various cancer characteristics such as cancer development stage, cancer types, cancer microenvironments and oncogenic mutations. Of course, it is necessary to determine the state of autophagy of cancer before applying activators or inhibitors.
Many clinical trials using autophagy modulators are combinatorial therapy with standard chemotherapy or radiotherapy. Since enhanced or impaired autophagy in cancer is one of the various characteristics of cancers, it will be difficult to show dramatic anticancer effects with autophagy modulator alone. However, the effect of disappearing chemotherapy resistance caused by prolonged standard treatments is positive by inducing an imbalance of autophagy. These effects may be due to the induction of apoptosis, alterations in the tumor microenvironment or a still unknown mechanism. More detailed mechanistic studies of how autophagy modulators overcome chemotherapy resistance are needed for the stronger efficacy of autophagy modulators.
It is interesting that epigenetic drugs such as EZH2 inhibitors or HDAC inhibitors regulate autophagy. However, the specific mechanism by which epigenetic drugs affect autophagy remains largely unknown. Since the effect of autophagy in cancer depends on cancer type and tumor stage, it is challenging to determine whether autophagy regulation by epigenetic drugs will have a positive/negative effect on cancer treatment. Therefore, it is necessary to study the autophagy mechanisms and treatment outcomes of epigenetic drugs in a wider range of cancers.
Metformin has been used for a long time in patients with type 2 diabetes and has been clinically proven to be safe. Moreover, HCQ and CQ have been used in patients with malaria. Repurposing drugs with potential antitumor properties might have the advantage of improving survival while saving time and money. These drugs have successfully passed phase I clinical trials for cancer patients and are currently undergoing phases II/III. It is expected for use in the treatment of cancer patients after its effectiveness and safety have been confirmed.
Recently, immunotherapy is attracting attention as a new cancer treatment. So far, the synergistic effect of autophagy and antibody-targeted therapy has frequently been reported. However, autophagy also enhances or attenuates the effectiveness of immunotherapy, depending on the cancer type. Currently, phase I/II clinical trials on HCQ in combination with nivolumab/ipilimumab in advanced melanoma are recruiting patients (NCT04464759). If positive clinical results are obtained, it is expected that the effective range of autophagy modulators as cancer immunotherapeutics will be further expanded. |
Researchers’ perspective of real-world impact from UK public health research: A qualitative study
Research funded by the National Institute for Health Research Public Health Research Programme is being undertaken in a complex system which brings opportunities and challenges for researchers to maximise the impact of their research. This study seeks to better understand the facilitators, challenges and barriers to research impact and knowledge mobilisation from the perspective of UK public health researchers. A qualitative study using semistructured interviews, informed by the Payback Framework, with public health researchers who held a research award with the National Institute for Health Research Public Health Research programme up to March 2017 was conducted. Following a thematic analysis, three strongly interlinked themes were extracted from the data and three key factors were highlighted as important for facilitating knowledge mobilisation and impact in UK public health research: (1) Public health researcher's perception of the purpose of the research (2) Approaches to undertaking Knowledge mobilisation activities (3) The complex nature of public health research in the wider research context. These have been reflected onto the Payback framework. Public health researchers can maximise the likelihood for impact by being aware of the context in which they are undertaking research, using different methods, and employing several strategies to take advantage of opportunities. There is a need to support researchers with knowledge mobilisation activities and for funders to identify their expectations of the impact resulting from research. Our findings have relevance to public health researchers and funders interested in increasing the benefit that research brings to society.
# Introduction
The National Institute for Health Research (NIHR) is a major funder of health research in the UK and funds research to improve the health and the wealth of the nation. The NIHR is an important funder of public health research [bib_ref] Mapping public health research across the National Institute for Health Research, Guegan [/bib_ref] , and one route for this is via the Public Health Research (PHR) programme. Established in 2008, the PHR programme funds research to evaluate the effectiveness and cost effectiveness of interventions that take place outside of the National Health Service (NHS) to inform delivery and improve the health of the public and reduce health inequalities. Increased international interest of research funders to understand if and how impact of research takes place is particularly relevant to public health given the multi-sector reach of this research. This interest comes from a shrinkage of the research budget and a need to demonstrate what has happened as a result of public investment on research [bib_ref] Promoting an open research culture, Nosek [/bib_ref]. For example [bib_ref] Models and applications for measuring the impact of health research: update of..., Raftery [/bib_ref] , made recommendations that the NIHR routinely assess the impact of the trials it funds on subsequent systematic reviews and guidelines.
In this paper, research impact means any demonstrable change or effect that has happened in the real world as a result of the research (i.e. outside of the academic environment) [bib_ref] Chasing the 'impact unicorn'-myths and methods in demonstrating research benefit, Bayley [/bib_ref]. Research impact can be seen as a measure of the effectiveness of knowledge mobilisation (KMb) activities which connect knowledge produced to the 'real world' [bib_ref] Building the concept of research impact literacy, Bayley [/bib_ref]. Understanding the way in which knowledge is mobilised (i.e., how knowledge is produced, exchanged, disseminated, and translated) to inform policy and practice is important for research teams who are under increasing pressure to undertake activities which seek to maximise the impact of their research. In theoretical evaluations, logic models and frameworks represent, in a simple diagram, the underlying theory of how an intervention(s) leads to outcomes and wider impacts, as well as helping to organise what evidence is needed and what data needs to be collected. Logic models have been used by research funders to demonstrate the impact of the research they fund [bib_ref] The impact of the National Institute for Health Research Health Technology Assessment..., Guthrie [/bib_ref] and one of the most widely used models used in evaluating impact from health research is the Payback Framework [bib_ref] The impact of the National Institute for Health Research Health Technology Assessment..., Guthrie [/bib_ref] [bib_ref] How can payback from health services research be assessed?, Buxton [/bib_ref] [bib_ref] The 'Payback Framework' explained, Donovan [/bib_ref].
The interface between researchers and research users plays an important role in evaluating health research impact [bib_ref] The 'Payback Framework' explained, Donovan [/bib_ref]. Overall, models of how research is adopted into policy show that "adoption of knowledge is interpreted as something that requires partnership working between researchers and policy makers" [bib_ref] The 'policy-preferences model': a new perspective on how researchers can facilitate the..., Brown [/bib_ref]. However, often it is the researchers alone who shoulder the responsibility for reporting impact, which is not desirable [bib_ref] Knowledge translation of research findings, Grimshaw [/bib_ref]. For instance, it has been proposed that the assessment of research impact (such as the Research Excellence Framework (REF) in the UK [13]) often centralises the role of the academic and overlooks the role of evidence-users [bib_ref] Building the concept of research impact literacy, Bayley [/bib_ref]. However, the evidence-user perspective is often centralised in studies looking at KMb [bib_ref] Health policy-makers' perceptions of their use of evidence: a systematic review, Innvaer [/bib_ref] [bib_ref] Increasing the use of evidence in health policy: practice and views of..., Campbell [/bib_ref] [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref] specifically because evidence-users are responsible for implementing research findings into practice. Therefore, there appears to be an unbalanced commitment between those who are responsible for making evidence-informed decisions within the health care system (i.e., policy-makers, practitioners, commissioners) and those (i.e. researchers, evidence producers) who bear the responsibility to demonstrate that knowledge mobilisation of their research informed policy-making decisions and as a result generated impact. This is especially relevant in Public Health (PH) research. Within the UK, PH research is funded through diverse mechanisms and has many pathways to impact [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref]. The effectiveness of these pathways and the impact the research has have been found to depend on a number of factors. For example, contextual factors (e.g. working relationships, values, interpretations of the evidence), researcher roles (e.g. negotiators), collaboration, and KMb outcomes all play a role in the use of evidence within the decision-making process [bib_ref] A model for collaborative working to facilitate knowledge mobilisation in public health, Mccabe [/bib_ref]. Similarly, constructs relating to the researcher (expertise, motivation), support (support structure, collaboration, relationship building and understanding needs) and utilising wider dissemination all contribute to generating PH policy impact [bib_ref] How can we achieve impact from public health research? A meta-ethnography of..., Gentry [/bib_ref]. Similar approaches from influential Australian public health researchers have been reported, who were found to employ a range of tactics to bridge the gap between their research and policy including engaging, targeting, consulting, and forming relationships with key stakeholders throughout the research process. Their findings suggested that the underpinning values of the researcher played a role in what they considered most important for research impact/ KMb. In addition, with regards to KMb, Australian public health academics considered how they interact with (and value) policy makers and the skills they need to do this, the constraints of their own research environment (context), and the professional identity of the researcher [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref]. For curiosity-driven researchers, undertaking quality research and ensuring that the research was published in high impact journals was highly valued. Conversely, 'policy-driven' researchers valued activities which ensured research was influential to policy.
Given the importance of KMb and its role in helping to achieve research impact, it is vital that research funders within the UK context, such as NIHR PHR programme, better understand how they can support public health researchers to effectively mobilise research findings to inform public health decisions. This is particularly important because researchers are responsible for reporting their KMb activities and impact(s) achieved to funders and sponsors. Thus, the aim of this paper is to better understand the facilitators, challenges, and barriers to research impact and KMb from the perspective of public health researchers funded by the NIHR PHR programme informed by the Payback Framework.
# Methods
## Design
This study is a qualitative exploration of public health researcher perspectives. It was part of a wider exploratory impact assessment primarily to demonstrate accountability which was undertaken in 2017 on all research awards (n = 113) funded by the NIHR PHR programme between May 2009 and 14 th March 2017 [Lakin K, Baker G, Thomas S, Worswick L, Dorling H. NIHR Public Health Research Programme: Exploring the influence of research on policy & practice. Unpublished internal report; 2018]. The Payback framework underpinned the wider impact assessment. This framework uses a logic model of the research to create a multi-dimensional characterisation of paybacks resulting from research. This framework was selected as it has been used to evaluate the impact of other NIHR funding research programmes [bib_ref] The impact of the National Institute for Health Research Health Technology Assessment..., Guthrie [/bib_ref] , and the PHR Programme had a specific interest in exploring two of the Payback categories, namely Benefits to informing policy, and Benefits to [public] health sector. For this study, public health researchers' experience of the barriers, challenges and facilitators to impact was explored within this context to contribute to a collection of award level case studies building an understanding of the researcher-research user interface within the context of public health. A phenomenological qualitative approach underpinned the study as we were interested in describing and understanding the experiences of our participants. Data were then collected using semi-structured interviews that were informed from the Payback framework (see S1 File).
## Recruitment
A purposive and self-selecting sample was used to identify principal investigators from NIHR PHR programme funded research awards which 1) had demonstrated evidence to suggest that the research findings had been influential to policy and/or the [public] health care sector following a desk-analysis of the PHR portfolio based on the Payback framework [bib_ref] How can payback from health services research be assessed?, Buxton [/bib_ref] [bib_ref] The 'Payback Framework' explained, Donovan [/bib_ref] and 2) were completed and the findings were published within the NIHR PHR Journal. Research awards which had been influential to policy and/or the [public] health care sector were targeted because this allowed an exploration of proven facilitators and barriers to the potential impacts being realised. Award selection based on completed research was most appropriate given the time-lags that often exist between completing the research and realised influence [bib_ref] The answer is 17 years, what is the question: understanding time lags..., Morris [/bib_ref] [bib_ref] How long does biomedical research take? Studying the time taken between biomedical..., Hanney [/bib_ref].
From a total of 113 PHR awards available, nine principal investigators from different PHR funded projects were eligible and invited to participate in an interview or to nominate another member of their research team. Invitations were sent out by email by a member of the project team.
## Ethics approval and consent to participate
Although not standard process within research impact assessments, ethical approval from the Faculty of Medicine University of Southampton Ethics Committee was sought (ID 27460). Study participants were informed about the objectives of the study, any risks and benefits to them, the fact that participation was entirely voluntary and that they could withdraw from the study at any time. Verbal consent was obtained, recorded and stored separately before the start of each interview, as approved in the ethics application. Verbal consent was obtained as this was felt to be the most practical approach given that all interviews were conducted via telephone. Participants were sent a copy of the information sheet and consent form in advance of the arranged interview and were asked to verbally confirm consent. Participant names were anonymised in the interview transcripts. Records linking participant names, consent and transcripts were stored according to the approved protocol.
## Data collection
Semi-structured telephone interviews were undertaken (Aug-Sep 2017) with one interviewer and one note-taker and lasted up to one hour. The interviewer and note-taker undertook the interviews together in a private room at the NIHR Evaluation, Trials and Studies Coordinating Centre in Southampton. The interview framework was adapted from [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref] , which aligned to the Payback Framework, and sought to understand the nature of the influence or impact the research had realised. However, it also sought to understand how change came about or what stopped it, thereby investigating the way in which knowledge from research was mobilised. The initial interview framework was sent to participants in advance of the interviews, and a question was added to the framework following the first interview to reflect new areas for exploration in further interviews.
# Analysis
Interviews were transcribed by a transcription service and reviewed for accuracy. NVivo 11 qualitative data analysis software was used to support the thematic analysis. Data were analysed according to [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref]. Following familiarisation, transcripts were coded. A second coding exercise led to the identification of common themes and sub-themes. Through an iterative process, themes and interpretations were reviewed and discussed by the research team to refine themes through merging, overlapping and separating out key concepts. The coding process was inductive as no prior themes or framework was considered and the themes were data driven. During the analysis, authors were mindful that they work within the NIHR and have a range of expertise in public health, health services research, health evaluation and commissioning, which strengthened the analysis and minimised bias. No one from the project team had a relationship with the participants prior to study commencement. Quotes were reported verbatim. The COREQ guidelines (COnsolidated criteria for Reporting Qualitative research) [bib_ref] Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews..., Tong [/bib_ref] for reporting qualitative data were utilised throughout and used as an additional reporting quality check.
## Consent for publication
All participants consented for their interview data to be used as part of a thematic analysis which would be published. As good practice, a copy of this manuscript was sent to all study participants to inform them of our plan to publish via a journal article and ensure that the interpretation reflected the discussions during the interview.
# Results
Seven researchers representing six research awards were interviewed. This number of interviews was sufficient to provide illustrative examples based on our knowledge of the awards and our experience of qualitative interviews [bib_ref] Sample Size in Qualitative Interview Studies: Guided by Information Power, Malterud [/bib_ref]. The research awards covered a range of areas relevant to public health practice (community and education, physical activity/obesity, transport) and research designs (systematic review, randomised controlled trial, natural experiment).
Three over-arching themes, with sub-themes, were extracted from the data using thematic analysis: 1). Public health researcher's perception of the purpose of the research; 2). Approaches to undertaking KMb activities; and 3). The complex nature of public health research in the wider research context.
## Public health researcher's perception of the purpose of the research
This theme suggests that how public health researchers perceive impact and KMb is guided by their views of the purpose of research. There was a variety of views regarding what was considered impact, how to achieve impact and whose responsibility it was to demonstrate impact. Researchers discussed impact and KMb in terms of publications, engagement with the public, policy makers and the media, dissemination, patient and public involvement, changing practice, guidelines or policy, and involvement in the UK REF.
And we've tried to illustrate, you know, how big an impact we have had, not just in terms of, you know, the number of publications, which has been, yes, much bigger than I think we had anticipated, but also the wider, kind of, policy impact. Participant 4
These preferences to impact and KMb sat on a continuum that ranged from a fixed pipeline approach to a more integrated, collaborative perspective. Researchers at the pipeline end of the continuum preferred to produce knowledge and evidence via publication that was then available for others to use. This preference tended to give less emphasis to the researcher being involved in how research findings were used in the world after project completion.
## I think my job is to produce some knowledge, and then it's, kind of, not really my skillset to think: well. . . you know, it's also none of my business how that gets used in the world, it's just not what i think i my job is. participant 1
By contrast, at the opposite end of the continuum, researchers preferred to undertake their research in a more collaborative and engaging way to bring about change within communities or by influencing changes to policy or practice. These researchers felt that undertaking research was a collective responsibility.
Participatory research, characterise impact as change that happens during the process as much as what happens beyond it. And if you involve people, [. . .] the impact will go beyond, be more likely to happen because people own the research. So people who have been involved will own it and, therefore, more likely change what they do. Most researchers seemed aware of their preferences on this continuum. However, they also understood the importance of different activities in facilitating impact and KMb of their research. As such, despite their preferences, researchers still engaged in or employed strategies to ensure different types of activities were undertaken (see theme 2).
Preferences on the continuum were discussed within the context of objectivity and advocacy. It was suggested that the boundary between advocacy and objectivity was sometimes difficult to navigate, with researchers indicating that certain activities aligned them more to one or the other. This could act as a barrier to researchers discussing and planning impact as they sometimes felt uncomfortable attaching themselves to findings or speculating on potential impacts.
Some researchers, I think, tend to lean more on the advocacy side than the objectivity side, but we've been really careful to try and make sure we can provide evidence when people want it, but. . . we remain objective, not advocates. Participant 4
## Approaches to undertaking knowledge mobilisation activities
This theme encompassed the strategic approaches that researchers used to facilitate impact and KMb. There were three subthemes (2.1.) strategy and opportunism; (2.2.) enduring networks and partnerships; and (2.3.) role and skills of the team.
## Strategy and opportunism.
A few participants noted that although they did not have a formal strategy for impact at the start of the research, writing pathways to impact statements as part of funding applications, or the requirements for REF2014, were influential to their thinking about impact and how it can be evidenced. However, challenges were noted around the type of evidence needed.
## One of the major challenges in maintaining and recording, or collating the evidence of impact, is lack of resource. participant 3
Researchers reported different impact strategies. One was to give prominence to the research outputs, while another strategy was to plan in advance and to give prominence to having the right people involved.
I don't believe we can control things or control people, but if you set up practices that encourage people to increase impact. . .then that is where the individuals who are involved [will have an] impact. However, a few participants noted that the way research impacts the real world can be 'accidental' and therefore a challenge to plan for or predict. Taking advantage of 'windows of opportunity' was viewed positively (see also theme 3). This could involve undertaking planned activities which maximise the chance of encountering opportunities, such as networking, or taking advantage of chance encounters.
You just happen to meet somebody, a policy maker, at an event. Or, you get invited to a conference from somebody in the audience. And then. . . the actual process is by which it happens are very difficult to plan in advance. Another strategy was to disseminate and increase the reach of research using media.
I think, in terms of impact for practice and probably policy, the best way of dissemination was through media: television, radio. . . Yes. I think that's the best. . .. . . it's the high level, perhaps television, sort of, snapshots, that really, really help in terms of getting it out there and getting people to be interested in it. Participant 3 However, one participant noted that this also came with challenges because the way research findings are presented in the media can mean public reaction is difficult to predict.
There were some concerns about, kind of. . . ..how the message. . . how our findings might get spun, I guess, in the public domain. There was a kind of concern about what we found, and how that might get utilised. So, I guess it was. . . it's not so much impact, as, kind of, a concern that we'd have to be quite careful with the messaging, in terms of what we found. Participant 1
All of these challenges indicate that there is a need to approach impact strategies with flexibility.
2.2 Enduring networks and partnerships. All participants agreed that networks and collaborations played a role in achieving impact. Views ranged from "they were very good", to networks and relationships being key to the "success" of the research. Most participants noted that enduring relationships are most effective in facilitating impact. As one interviewee stated.
It's having the time to have those conversations to build those relationships that then means you have a mechanism, going forward, that allows ongoing implementation and ongoing impact to happen. Participant 3
One participant felt that building relationships was a way to show how they, as a researcher, could be useful to others. Another view was that networks provided a way to raise their profile and research in their field. Some participants felt that engaging with community stakeholders was nice to do but unnecessary. For other participants, local community involvement was a key part of the research process. Almost all participants stated that they engaged with stakeholders, but for some this was is not always easy.
Policymakers aren't used to really making links with public health researchers. And so they might have different views or opinions about what we should be evaluating. Participant 4 2.3 Role and skills of the team. Some of the challenges to generate impact within public health were expressed in terms of the skills required to undertake KMb activities, which were felt to be outside of the researcher's area of expertise. In order to overcome this, nearly all participants acknowledged that there is a need to work with people with a range of skills. A number of key skill areas were mentioned: networking, communicating, the right professional background, and the ability to be alert to opportunities.
What I don't do very well is public engagement or policy impact. And it is worth thinking about hiring somebody who does have those skills, as well. . . rather than just hiring somebody. . . who's really good at academic research. Participant 1 A couple of participants specifically mentioned the role of knowledge brokers or community members, which were either internal or external to the core research team.
[They] help us not just with writing press releases but also things like evidence submissions and evidence briefs that we give to policymakers and practitioners Participant 4
We wanted the actual end user, if you like, to be part of the process. So for me, impact is. . . it comes from involving people in the process of research. There was an acknowledgement that having people with a range of skills on a project team is beneficial, particularly when researchers have additional skills which can be useful for the team. However, that wasn't to say that all team members had to have a mixed skill set, and some participants expressed preference to conduct activities close to their research strengths.
I want the freedom to be able to focus on the academic outputs, and do what I do well. Participant 1
There's lots of people out there that are natural brokers, they link people up together Participant 7
Undertaking research with external stakeholders presented opportunities for skills building. However, a challenge was bringing together team members. Furthermore, some participants indicated that more support and training for researchers is needed to overcome a barrier of effective conversations with non-academic groups or for dissemination activities.
Anything that could be done to overcome. . . problems that solidify relationships with researchers, academics, policy makers, and practitioners, would be good. Participant 2 However, irrespective of the team skill-mix, a key barrier to impact was keeping the team together once the funding had finished, or finding capacity to undertake KMb activities during the research.
The challenge around doing that is that we don't have funding to take the time to write the articles before we finish the work. Participant 5
## The complex nature of public health research in the wider research context
The nature of public health research is complex. Under this theme there are two subthemes that highlight the facilitators and barriers to KMb and impact delivery within the wider research context: (3.1.) the role of the research funder and (3.2.) the role of wider environmental and societal context. These subthemes interact with the research activities outlined in theme two.
## The role of the research funder.
Understanding the funder's expectations for impact was discussed by some participants. However, there were concerns around how far researchers should go to plan for impact at application stage.
## I think researchers are a little anxious that if they kind of guess or put some hypothesis for that, they might not be seen very favourably by funders who like to see very prescriptive plans that are all sewn up. participant 7
There was a view that funders could do more to support planning for impact, particularly when they commission the research.
The funders could help by being very clear when they are putting out a request for proposals, that they're anticipating certain types of impact that might come out as a result of the research needs that they see. Linked to this, there were views on the help and training support that funders could provide to prepare researchers to engage in impact and KMb activities.
## You know, every case is going to be different, and it really is a part of building up your own skills, but at least if you have some sort of training in terms of what you might expect, it would really help going forward. participant 3.
Most participants felt that changes need to be made in the funding model to include time for "impact" or dissemination activities. One idea was a pot of funding, a "sprinkling that continues after the project" (Participant 1), that would provide continued capacity once the research has finished, for activities including "media, television, radio" (Participant 3). Some participants reported that they had to secure additional funding or time for these types of activities either as part of their research funding, from their collaborators, or their academic institutions.
There were views about research methodologies that may be more appropriate to public health and the need for funders to take more risks to make sure that the type of research being funded uses the most appropriate methodology. These may be different to the 'gold standard' of randomised control trials, and researchers and funders should be more flexible in their approach to generate evidence in public health spaces. Although it was acknowledged that trials are useful, they are not always feasible and where this is the case, other designs should be used.
## [funders] should focus on the, sort of, massive things in different centres, which can nudge people in the right direction of making healthier decisions. and often those kinds of interventions won't be trialable because there will be things that we can't control the allocation of them. participant 2
Others noted the limitations around systematic reviews in public health, which were described as good but "messy" (Participant 5). Some participants also questioned how useful they were to public health practitioners in order to inform practice.
## Health systems are not finding systematic reviews very helpful, to tell them what to do-the practice participant 6
In comparison, realist reviews were viewed as a more helpful methodology to address questions within complex systems.
3.2. The role of wider environmental and societal context. The wider environmental and societal context in which public health researchers operate was mentioned by some participants. For example, there was an observation around the way in which the health sector uses evidence, in that it is dismissive of or removes evidence which provides context. In this space, the political environment (agenda) was viewed as an uncontrollable influential factor to the impact of public health research.
## By the time it did get funded, it had become quite politically contentious [. . .] there was concern about how findings would be utilised. participant 1
However, participants discussed the value of the serendipitous opportunities that influence the impact of public health research. The fast-paced, changing nature of public health arena means that a constant search for windows of opportunities and investment in enduring networks is needed. This is linked to Theme 2.
The mechanism of how research brings about change was one area in which all participants were unified; that the mechanism of change is often through a "combination of things", or as a result of an increasing body of evidence that "chips away at the edges" (Participant 1). It was acknowledged that unlike other, more clinical research, there is not really the same type of pathway in public health research. Often, research impact in public health can be about changing perceptions, which in turn, adds to a developing "cultural discourse" and "social movement".
## Alongside that evidence, and alongside various other bits of evidence, and other little bits of research i was doing, i fed into, effectively [a] movement amongst academics, and policy makers. participant 2
There was an acknowledgement of changes in the way public health services are provided where social enterprises and complex organisations are more involved. The opportunities that this new configuration of public health services present, and the challenges that arise from these were recognised.
## Commissioners demonstrated a real willingness to have a different sort of relationship with community providers. participant 6
Participants identified barriers in the form of differences between practices in academia and the ways in which knowledge spreads in the community, which is on ongoing struggle that some researchers are familiar with. Participants also discussed how the temporary, fixedterm contracts in academia mean that research teams are in a constant state of flux. This runs counterintuitively to the need for long-term commitment required to produce impact in public health.
One thing that kind of undermines [. . .] impact is the constant organisational restructuring, and people. Participant 1
## Linking findings to theory
Our findings highlight areas for consideration for the Payback framework with regards to public health research. [fig_ref] Fig 1: Study findings mapped onto the Payback Framework Logic Model for assessing research... [/fig_ref] that for stages 0-3 of the framework (research topic identification and research activity processes) our findings emphasise the need for and role of the research support community, such as HEIs and researcher funders, to support researchers to plan and undertake KMb activities. Interface B (Dissemination) highlights the need to take advantage of windows of opportunity to inform stage 4 (policy developments), which in turn present opportunities for adoption (stage 5). Networks and partnerships play an important role throughout all stages of the framework and are particularly important for maximising the adoption of research findings within public health contexts. Finally, our findings reflect the researcher perspective in terms of preferences, role and skills and how they can influence if and how they plan for and undertake KMb activities.
# Discussion
The aim of this study was to better understand the facilitators, challenges and barriers to research impact and KMb from public health researchers within the UK context, working outside the NHS setting. Three main themes were extracted from the data. These themes were all interlinked, showing the intricate nature of public health. From these themes, three key factors were highlighted as important for facilitating KMb and impact in public health, which are reflected onto the Payback framework: 1) An awareness of the context is essential in UK public health research and researchers preferences to KMb activities help navigate this context to achieve impact; 2) seizing on and proactively creating windows of opportunity to engage stakeholders who may be in a position of power in the policy arena or embedded in the community are vital to impact and KMb in public health; 3) funders and research organisations have opportunities to specify their expectations and to take a flexible stance towards impact supporting research teams to navigate the public health context.
## Context is important in public health research
Researchers in public health operate within a complex system where health outcomes are related to a multitude of interdependent environmental, social and physical determinants [bib_ref] The need for a complex systems model of evidence for public health, Rutter [/bib_ref]. Factors such as changes in the political landscape, public opinion and organisational restructures present challenges to the research environment. This means that the context in which a public health researcher works (across third sector, multi-government agencies and private sector) is likely different to other types of health research. Context has been recently highlighted as an area that should receive greater consideration to aid understanding of population health interventions. The importance of context has been reiterated by public health researchers in Australia [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref] and UK [bib_ref] A model for collaborative working to facilitate knowledge mobilisation in public health, Mccabe [/bib_ref]. Our findings mirror this complexity when researchers discuss the need to navigate context and to ensure their research is relevant. For example, researchers reported that evidence did not always follow a linear evidence-based medicine model and there were concerns raised by participants about how to demonstrate and evidence impact, particularly when it is conceptual (i.e. adds to a body of knowledge) in nature.
Whilst [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref] found that researchers questioned the type of evidence needed from public health research to inform policy, our findings suggest that researchers were aware of the type of evidence needed and were using non-standard research designs to ensure that the research generated relevant evidence. Evidence derived from realist reviews, natural experiments, nonrandomised trials, novel statistical methods and complex systems approaches provide alternative ways to explore complex interventions [bib_ref] The need for a complex systems model of evidence for public health, Rutter [/bib_ref] [bib_ref] Alternatives to randomisation in the evaluation of public health interventions: design challenges..., Bonell [/bib_ref] and some of these approaches were used by the researchers. This is because in complex social interventions, isolating health outcomes in order to measure statistically observable effects is difficult and often not the main goal. Moreover, there are practical challenges in trying to randomise service evaluations and population-level interventions, where other forms of evidence maybe better suited [bib_ref] The need for a complex systems model of evidence for public health, Rutter [/bib_ref] [bib_ref] Applied public health research-falling through the cracks?, Simmons [/bib_ref] [bib_ref] Evidence, hierarchies, and typologies: horses for courses, Petticrew [/bib_ref] [bib_ref] Randomized Controlled Trials: How Can We Know "What Works, Cowen [/bib_ref]. Despite this, we found that researchers feel frustrated by the research funding process and the inability to undertake research that is fit for purpose. Indeed, as pointed out in [bib_ref] Applied public health research-falling through the cracks?, Simmons [/bib_ref] , there is a mismatch between the type of evidence required by public health organisations, those who develop evidence-based guidance, and the types of research that funders are willing to support. The findings in [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref] and in our paper seem to evidence this mismatch.
Researchers recognised the need to collaborate and/or engage to ensure that the research is positioned within the right context. However, in line with [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref] , a lack of capacity (skills and time) to do so was a barrier to effective KMb (and therefore impact). Public health researchers need to engage with a broad range of stakeholders who often have different needs, priorities and entrenched practices. Researchers undertook a range of activities to navigate this space, but our findings suggest that more support is needed to help public health researchers with these activities.
## Researcher preferences to knowledge mobilisation activities can facilitate navigation of the public health context
Our findings show that researcher preferences to impact sat on a continuum from a 'traditional academic' to a coproduced approach. This type of continuum has been reported by others [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref] [bib_ref] From 'Ivory Tower Traditionalists' to 'Entrepreneurial Scientists'? Academic Scientists in Fuzzy University-Industry..., Lam [/bib_ref]. Building on this, we found that the continuum for PH researchers is influenced by views of academic rigour, emphasising objective generation of evidence on one end and co-production and engagement activities on the other. Influential PH researchers in Australia rarely just let research speak for itself and instead are aware of the applied nature of PH research. Our findings lend weight to this, and even where a researcher's preference could be viewed as a more traditional academic (i.e. to produce good quality evidence), their research, by its very nature, had policy goals or was policy-driven i.e. "policy-makers identify and prioritise particular problems that then become the focus of research", ]. Similar to [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref] , public health researchers in this study had a strong awareness of professional identity around researcher role and skill-set, and how they viewed their contribution to society. By contrast, we found no evidence that they viewed undertaking KMb activities as a competitive advantage to their peers. In general, however, our findings suggest that researchers were aware of their preference, which often facilitated how they navigated the complexities of the context in which they work. Such awareness drove different approaches and strategies to impact and KMb. However, as concluded in [bib_ref] Academic perspectives and experiences of knowledge translation: a qualitative study of public..., Collie [/bib_ref] , while researchers in applied research are increasingly expected to engage in KMb activities, this has not been followed by funding or other incentives and rewards which help to overcome barriers. Similar findings have been reported for PH research funded in the UK [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref]. One way in which funders could support researchers is to allow for flexible impact plans and strategies in order to respond to the changing complex environment. Moreover, our findings suggest that researchers believe that funders could be clearer on the intended changes or impacts of the research they are funding, and what evidence, data or indicators they need from researchers to demonstrate this change.
## Public health researchers need to seek out windows of opportunity and engage with a wide range of stakeholders
One way in which almost all researchers navigated the public health context was to be aware of windows of opportunity to engage with policy and key stakeholders, and that this was a facilitator to impact. Changes in the policy and health landscape opened up opportunities for researchers to engage. Such opportunities also presented challenges. Indeed, partnerships can be easier to forge when the policy area is a 'hot topic' and therefore high on the political agenda but this makes sustaining partnerships in long term more challenging [bib_ref] Experiences of knowledge brokering for evidence-informed public health policy and practice: three..., Frost [/bib_ref]. Our findings suggest that researchers were aware of the need to expand the composition of the research team to take this into account including collaborating (and coproducing) with key stakeholders, building on skill sets to engage or building on existing networks and partnerships. As found in [bib_ref] A model for collaborative working to facilitate knowledge mobilisation in public health, Mccabe [/bib_ref] , researchers evaluating public health interventions often had to change/shift their role depending on the context. A systematic review of KMb in the third sector found that strong relationships with academia were important to the mobilisation of research evidence and that having the ability to coproduce research could help to overcome some of the barriers by ensuring that the research was applicable to a local context [bib_ref] How do third sector organisations use research and other knowledge? A systematic..., Hardwick [/bib_ref]. Other strategies included using local or national research brokers. Knowledge brokers are being increasingly used to facilitate KMb between the producers and users of evidence to overcome the struggles of mobilising research knowledge into policy and practice. A number of different aims for knowledge brokers have been identified to facilitate KMb [bib_ref] Knowledge Brokering: The missing link in the evidence to action chain?, Ward [/bib_ref]. Mobilising knowledge to inform decision-making takes time but an important aspect for a broker is the ability to respond to windows of opportunity (e.g. hot topics, capitalising on chance encounters and creating networking opportunities) while at the same time hold evidence to inform future policies [bib_ref] Experiences of knowledge brokering for evidence-informed public health policy and practice: three..., Frost [/bib_ref]. Despite such strategies, our findings are in line with others [bib_ref] Mechanisms and pathways to impact in public health research: a preliminary analysis..., Boulding [/bib_ref] and suggest that there is a need to provide more support for researchers such as the provision of training and building of skills to help with the range of stakeholders that relationships need to be forged with and the different ways in which the research is communicated to different audiences.
## What this study adds
The recent yet increasing interest around impact to measure research performance is presenting challenges to stakeholders involved in research. This study contributes to the field by shedding light on challenges PH researchers in the UK are struggling with in defining, generating and demonstrating impact, and more importantly, what actions could be taken to develop the skills needed to track and capture impact from research.
This work is presented from the point of view of the PH researchers and provides a snapshot of the landscape of how impact operates in this context. Recommendations arising from the researcher's perspective here suggest that funders and research organisations should consider how best to support research teams. Such support could include specifying expectations towards impact, being open to the different methodologies that researchers in this field can adopt, provision of funding to support and enable KMb activities, committing to develop the skills that researchers need to engage with a wide range of stakeholders, and to evaluate track and capture impact from their research. This study has been laid out as a platform to encourage further work in the field. To develop a full picture of the views and interactions of all stakeholders, next steps would be to compare the views here with those from evidence-users as well as those who receive the benefits of research, to investigate whether impact is perceived in the same way by all stakeholders. Future research could consider the role of non-academic public health professionals working as knowledge brokers or as part of research team in increasing the impact of research.
Our findings highlight areas for consideration on the Payback framework specifically in the context of researchers undertaking public health research. Our findings have been mapped onto the framework emphasising the additional elements researchers should consider when planning for impact. As reflected in the model, our findings suggest that while the interface between researchers and research users is important, this is more complex than a two-way interface. The role of the research funder and the research institution, is important in this space to ensure that the right risks are being taken to fund the right research, with a team with the right skills in KMb (and provide training and support to achieve this). This will be of interest to anyone who is undertaking a theory driven impact evaluation or assessment of public health research to support what factors could influence the resulting outcomes and impacts of the research.
Since the introduction of the PHR programme, responsibilities for commissioning and providing public health services in England were transferred from the NHS to local authorities. Although such transfers of responsibility may not have resulted in changes to the research environment for the researchers interviewed in this study, it is possible that it may open up new opportunities to engage in evidence-based policy-making, which is applicable to public health [bib_ref] Reversing the pipeline? Implementing public health evidence-based guidance in English local government, Atkins [/bib_ref]. The number of interviews was small, and it is possible that a larger sample size might yield additional findings. We only looked at research which had completed but we acknowledge that impact may have resulted from active research. The research was undertaken from the perspective of the NIHR; participants were chosen from studies funded by the NIHR only and other funders might have different support provision for researchers in public health programmes. In addition, the authors work within the NIHR and although our selection of quotes gave a broad range of opinions, we do not know whether this factor might have influenced the openness of the participant responses. Our findings relate to public health research, specifically research which is undertaken outside of the NHS, and may not be generalisable to public health research conducted in NHS settings.
# Conclusion
The findings in this study are largely consistent with the existing research examining the views of public health researchers on impact and KMb. However, its contribution provides better clarity on public health researchers within the UK context, and in particular, on research being undertaken outside the NHS setting and this has been reflected in the Payback framework. Our findings support evidence in the literature which increasingly shows that context is important for the intervention/policy being evaluated. We would add that context is also highly relevant for the research environment and that the skills and approaches of researchers to navigate this context are important. Researchers need to be aware of their preferences for engaging with stakeholders and KMb in order to devise impact strategies that are more likely to succeed. Our findings show that by being aware of the context in which they are undertaking the research, using different research methods, and employing the right strategies to take advantage of opportunities, the researchers were able to maximise the likelihood for change to occur, and any change could be reported back to the research team through the enduring relationship between the parties.
Supporting information S1 File. Interview framework. Semi-structures questions that were asked of participants during telephone interviews. (DOCX) draft manuscript. Thank you to all our peer reviewers for providing helpful feedback during the ethics approval process and on this paper, your contributions made this stronger piece of research.
# Author contributions
Conceptualization: Kay Lakin, Genevieve Baker, Sarah Thomas.
[fig] Fig 1: Study findings mapped onto the Payback Framework Logic Model for assessing research impact. The Payback Framework Logic Model with areas for consideration resulting from this research highlighted in bold. The framework was adapted from [10]. https://doi.org/10.1371/journal.pone.0268675.g001 [/fig]
[table] PLOS ONE: | https://doi.org/10.1371/journal.pone. [/table]
|
Comprehensive analysis of immunoglobulin and clinical variables identifies functional linkages and diagnostic indicators associated with Behcet’s disease patients receiving immunomodulatory treatment
Background: Behcet's disease (BD) is a relapsing systemic vascular autoimmune/inflammatory disease. Despite much effort to investigate BD, there are virtually no unique laboratory markers identified to help in the diagnosis of BD, and the pathogenesis is largely unknown. The aim of this work is to explore interactions between different clinical variables by correlation analysis to determine associations between the functional linkages of different paired variables and potential diagnostic biomarkers of BD.Methods: We measured the immunoglobulin proteome (IgG, IgG1-4, IgA, IgA1-2) and 29 clinical variables in 66 healthy controls and 63 patients with BD. We performed a comprehensive clinical variable linkage analysis and defined the physiological, pathological and pharmacological linkages based on the correlations of all variables in healthy controls and BD patients without and with immunomodulatory therapy. We further calculated relative changes between variables derived from comprehensive linkage analysis for better indications in the clinic. The potential indicators were validated in a validation set with 76 patients with BD, 30 healthy controls, 18 patients with Takayasu arteritis and 18 patients with ANCA-associated vasculitis.(Continued on next page) Results: In this study, the variables identified were found to act in synergy rather than alone in BD patients under physiological, pathological and pharmacological conditions. Immunity and inflammation can be suppressed by corticosteroids and immunosuppressants, and integrative analysis of granulocytes, platelets and related variables is likely to provide a more comprehensive understanding of disease activity, thrombotic potential and ultimately potential tissue damage. We determined that total protein/mean corpuscular hemoglobin and total protein/mean corpuscular hemoglobin levels, total protein/mean corpuscular volume, and plateletcrit/monocyte counts were significantly increased in BD compared with controls (P < 0.05, in both the discovery and validation sets), which helped in distinguishing BD patients from healthy and vasculitis controls. Chronic anemia in BD combined with increased total protein contributed to higher levels of these biomarkers, and the interactions between platelets and monocytes may be linked to vascular involvement.Conclusions: All these results demonstrate the utility of our approach in elucidating the pathogenesis and in identifying novel biomarkers for autoimmune diseases in the future.
# Introduction
Behcet's disease (BD) is a chronic and relapsing vascular autoimmune/autoinflammatory disease of unknown cause, displaying involvement of multiple organs. BD is highly prevalent in countries along the "Silk Road"; it can be induced by persistent and excessive immune reactions via autoantigen-activated dendritic cells and T or B cells and leads to endothelial cell damage and vasculitis. Increases in human IgM, IgG and IgA levels have been found in BD patients, and the production of immunoglobulin isotypes is associated with mucocutaneous, ocular and systemic involvement in naïve active BD patients.
Clinically, corticosteroids and immunosuppressants are employed in rheumatoid immune diseases to attenuate the inflammatory response and tissue damage and relieve clinical symptoms due to their anti-inflammatory and immunosuppressive effects on the immune system. However, their long-term use may lead to immune disordersand increased susceptibility to viral infection. For example, Djaballah-Ider et al. found that corticosteroid therapy significantly reduced serum immunoglobulin isotype markersand inflammatory mediators related to disease pathogenesis, including IL-18 and IFN-γ regardless of the clinical manifestations in BD. found that thalidomide has both anti-inflammatory and regulatory effects in BD, decreasing the levels of the TNF-α receptor, CD8/CD11b + T cells and natural killer cells in early treatment while increasing CD4 + CD45RO+ memory T and γδ + T cells in later treatment.
The systems in the human body (e.g., coagulation, inflammation, etc.) are known to execute their functions cooperatively instead of alone, which can be reflected by clinically measured variables. For example, in a study of 48 participants with cirrhosis and nonalcoholic fatty liver disease, Niu et al. revealed the association of five plasma proteins (DPP4, ANPEP, TGFBI, PIGR and APOE) with liver enzymes through a global correlation map of clinical and proteomic data, implying their associations with cirrhosis and nonalcoholic fatty liver disease. Nathan et al. investigated the personal, dense and dynamic data from 108 individuals during a 9month period and generated a correlation network between clinical variables, proteomes and genome sequences, which revealed communities of related analytes associated with physiology and disease. For example, the negative correlation between levels of cystine in plasma and polygenic risk scores for inflammatory bowel disease revealed that genetic predisposition of diseases may be manifested by analyte changes and suggested that supplementation with cystine in a healthy population at high risk may stop the transition to disease by preventing inflammation and oxidative damage. Nathan proposed that measurement of personal data clouds over time can improve understanding of health and disease and are the essence of precision medicine.
Evidence has suggested that multiple pathological pathways are involved in BD with no single common denominator related with BD. However, no common or dominant pathological factor for BD has been identified until now. Moreover, the correlations of clinical variables in BD and their associations with BD diagnosis, progression and therapy are largely unknown. The hypothesis of this work is to explore the interactions between different clinical variables by correlation analysis to determine the associations between the functional linkages of different paired variables and potential diagnostic biomarkers of BD. To address this issue, we first measured the immunoglobulin proteome (IgG, IgG1-4, IgA, IgA1-2) using a plasma microarray and performed a comprehensive correlation analysis of the immunoglobulin proteome and 29 clinical variables. We defined the physiological, pathological and pharmaceutical relationships based on the correlations of all variables in the healthy controls (HCs) and BD patients without and with immunomodulatory therapy. Furthermore, we calculated the ratio changes between clinical variables to identify the specific indicators for the diagnosis of BD and differential diagnosis from other types of vasculitis.
# Materials and methods
Demographic and clinical characteristics of subjects All plasma samples were obtained from the Peking Union Medical College Hospital, where BD patients were diagnosed according to the 1990 International Study Group (ISG) criteriaand the International Criteria for Behcet's Disease (ICBD), and patients with Takayasu arteritis (TA) and those with ANCA-associated vasculitis (AAV) were diagnosed respectively according to.Furthermore, all patients with BD were assigned to four groups according to medication use, which included BD patients without treatment (BD-N), treatment with corticosteroids (BD-C), treatment with immunosuppressants (BD-I) or treatment with both (BD-C&I). Patients using immunosuppressants are defined as those who are under drug treatments, including Azathioprine, Cyclosporine, Thalidomide, Cyclophosphamide, Leflunomide, Hydroxychloroquine and Tripterygium glycosides. Blood samples were anticoagulated with EDTA, centrifuged at 12,000 rpm for 10 min, and the upper plasma layer was collected and frozen at − 80°C until use. This study was approved by the Medical Ethics Committee of Peking Union Medical College Hospital (JS-2049), informed consent was obtained from all subjects. All research on humans was performed in accordance to the Declaration of Helsinki.
Quantification of the immunoglobulin proteome using plasma microarray All plasma samples were retrieved from the − 80°C freezer, thawed on ice and centrifuged at 12,000 rpm for 10 min at 4°C. . The unbound molecules were removed by washing the slide with 0.05% PBST three times and deionized water two times in the dark. Then, the resulting slide was air-dried and scanned by GenePix® 4300A (Molecular Devices, California, USA) at a wavelength of 488 nm (IgG1, IgG2 and IgA2), 532 nm (IgG) or 635 nm (IgA, IgA1, IgG3 and IgG4).
The quantification of immunoglobulin proteins in plasma was performed by using a standard curve fitted with a 4-or 5-parameter logistic model using the "nplr package" in R as previously described. The linkage analysis of the immunoglobulin proteome and all variables was performed by calculating the Pearson's or Spearman's correlation coefficient between two variables according to their normality. Pearson's correlation coefficient was performed when the data of both variables had a normal or log-normal distribution. Hierarchical clustering analysis of the correlation coefficient matrices was performed using Euclidean distance and the complete method in the pheatmap package in R, with which the positively and negatively correlated variables were clustered together in a heatmap. A P value of < 0.05 was considered to be statistically significant.
In addition, ratios between every two clinical variables were calculated. Ratios more than one with significant difference when comparing BD-N and HC were retained for further comparison between groups in the validation set. A P value of < 0.05 was considered to be statistically significant.
# Results
## Quantification of the immunoglobulin proteome using a plasma microarray
A schematic illustration of high-throughput immunoglobulin proteome detection in plasma and the following data analysis is shown in. Briefly, all 129 plasma samples were printed onto a microscope slide using a microarray together with a series of concentrations of immunoglobulin protein standards. The resulting array was then detected by a fluorescein-labeled anti-immunoglobulin secondary antibody within 30 min. Standard curves were constructed by using the signals from immunoglobulin protein standards, with which the concentrations of immunoglobulin protein in all plasma samples can be quantified. Statistical and correlation analyses were employed to identify the variable linkages that are related to BD and clinical treatment with corticosteroids and immunosuppressants, respectively.
A representative image of the fluorescence detection of the plasma microarray is shown in . The signal of the immunoglobulin IgA standard was increased with increasing concentrations of the protein standards. The IgA in plasma samples displayed different signals on the microarray. The r correlation within and between different arrays was 0.91 and 0.96, respectively . The r correlations within arrays are calculated between blocks for different samples in the same array. In addition, we printed identical proteins on the top and bottom of the slide to evaluate the effect of printing location on plasma protein detection. The results indicate that the r correlation between the two locations was 1.00 for the immunological proteome (IgA, IgA1-2, IgG, and IgG1-4) , D). All these results demonstrate the high reproducibility of immunoassays using plasma microarrays.
Differential expression analysis of the plasma immunoglobulin proteome in BD patients receiving immunomodulatory therapy
Using this platform, we quantified the immunological proteome (IgA, IgA1-2, IgG, and IgG1-4) in the plasma of 66 HCs and 63 BD patients without and with corticosteroid and immunosuppressant treatment. Prior to the statistical analysis, we analyzed the effect of age and sex on the expression of the immunoglobulin proteome. The results indicate that there was no correlation between immunoglobulin and age in either HCs or BD patients. The same results were obtained for sex and the immunoglobulin proteome in HCs. However, the expression of IgG4 in male BD patients was higher than that in female BD patients (P = 0.0326).
Compared to the HCs, no significant changes were observed in the expression levels of IgG, IgG1-4, IgA and IgA1-2 in patients with BD-N . The expression levels IgG1, IgG2 and IgG4 were suppressed by using combination treatment of corticosteroids and immunosuppressants , b) since statistical significance (P < 0.05) was achieved for IgG1 between BD patients without and with the combination of corticosteroids and immunosuppressant treatment, as well as for IgG2 and IgG4 between HCs and BD patients receiving the combination of corticosteroids and immunosuppressant treatment (P < 0.05, . The results demonstrate that the immune system could be suppressed by corticosteroids and immunosuppressants through the regulation of immunoglobulin proteome expression.
Furthermore, we performed global linkage analysis to elucidate the potential mechanism promoted by the correlations of these immunological proteins during the progression of BD and the patients receiving immunomodulatory medication . We defined correlations (r) of 0.20-0.39 as weak linkage, 0.4-0.59 as moderate linkage, 0.60-0.79 as strong linkage and 0.8-1.0 as very strong linkage. The red and blue colors of the circles represent positive and negative correlations, respectively. The size of the circle and intensity of the color are proportional to the correlation coefficients. We defined the physiological, pathological and pharmacological linkages according to the change of linkage under the disease and therapeutic situations. For example, the correlation of IgG-IgG1 (r = 0.62~0.89) and IgG-IgG2 (r = 0.39-0.67) remained constant in the HCs and BD patients without and with immunomodulatory therapy and were assigned as the physiological linkages . However, the correlations of IgA-IgG3 and IgA1-IgG3 were increased in BD patients compared with HCs and were assigned to the pharmacological linkage . Conversely, the correlation of pharmacological linkage, and IgA-IgG4 in the BD group was increased only in the patients who received immunomodulatory therapy (BD-C, BD-I and BD-C&I) .
## Analysis of clinical variables in bd patients received immunomodulatory therapy
We then analyzed the changes in 29 variables, which were associated with inflammation, coagulation and nutrition, from clinical tests in HCs and BD patients without and with therapeutic treatment. The abbreviations of their full names are shown in.
First, we found that the clinical variables associated with inflammation are highly regulated in BD patients compared to HCs, including WBC, NEUT, CRP. The WBC level was high in BD patients treated with corticosteroids either alone or in combination with immunosuppressants (P < 0.05 for BD-C vs. HC). The same results were obtained for NEUT, CRP and ESR, which is in accordance with the function of immunosuppressants in inhibiting immunity and inflammation.In addition, compared to ESR, CRP was significantly Second, in this study, we found no significant difference in nutrition variables between BD-N and HC (P < 0.05,. However, the use of corticosteroids alone or in combination with the suppressants reduced the expression of albumin (Alb, P < 0.01 for BD-C vs. HC and BD-C&I vs. HC) and therefore downregulated the total protein levels, especially in the BD-C&I group (TP, P < 0.01 for BD-C&I vs. HC/BD-N/BD-I). Moreover, we found that Alb and TP decreased in patients with gastrointestinal involvement compared with those without gastrointestinal involvement and healthy controls, which may be due to impaired digestive function and a poor nutrition condition.
Third, among these platelet variables associated with the coagulation process, MPV and PCT were observed to be significantly higher in the BD-N group than in the HC group (P < 0.01), indicating an increase in the volume of platelets and thus an enhanced ability of thrombosis. Moreover, the use of corticosteroids increased the number of platelets (PLT, P < 0.01 for BD-C vs. HC and BD-C vs. BD-C&I) and induced a corresponding increase in PCT while decreasing MPV in BD patients by reducing the destruction of platelets (P < 0.01 for BD-C vs. BD-N and BD-C vs. BD-I).
Fourth, we analyzed the changes in the ratio between these hematological indicatorsneutrophil-tolymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and lymphocyte-to-monocyte ratio (LMR) in the HC and BD groups. NLR and PLR were increased in the BD groups compared with the HC group (P < 0.01), while LMR was decreased (P < 0.01).
The heatmap analysis of these variables further demonstrated the increase in inflammation and coagulation as well as the decrease in nutritional status in BD patients without and with immunomodulatory treatment. All clinical variables with significant difference between BD-N and HC are listed in.
## Comprehensive linkage analysis of clinical variables in bd patients receiving immunomodulatory therapy
Based on the previous finding, we speculated that all variables associated with our physiological system perform their functions cooperatively. To address this question, we comprehensively analyzed the linkages of all 37 variables in five groups of 129 samples by nonhierarchical clustering analysis, which led to 6845 linkages. The results indicate that the linkages of all variables were changed in the HCs and BD patients without and with immunomodulatory therapy, in which the linkages of the positive (red color) or negative (blue color) correlations were clustered and displayed as modules. For example, the variables of liver and renal functions were clustered together in the HC group, which is in accord with the collaborative function of the liver and kidneys to filter blood and process chemicals from food, medication and toxic substances. However, the kidneys could be damaged by BD, and some BD patients present with IgA nephropathy and amyloidosis. The results can be reflected in our correlation analysis in which the liver and renal function modules were separate in all BD groups (BD, BD-C, BD-I and BD-C&I).
Furthermore, the nonhierarchical clustering analyses revealed many physiological variable linkages that persisted in all HC and BD groups. For example, a high positive correlation (r > 0.6) was observed for three physiological variable linkages (HCT-HGB, MCH-MCV and PLT-PCT), confirming the functional association of these variables in oxygen transportation and coagulation. Conversely, A/G and ESR displayed a negative correlation in all HCs and BD patients (r = − 0.96~− 0.73), which confirmed their functional association with inflammation.
In addition, we noticed pathological and pharmacological variable linkages that were shown in BD patients without and with immunomodulatory treatment compared to the HCs. For example, four linkages (A/G-MCH, A/G-MCHC, Alb-uSG, TP-uSG) were changed from none to positive correlations (r = 0.339-0.834) in the BD patients regardless of treatment, indicating the role of nutrition in BD pathogeneses. As important inflammatory and thrombosis markers in BD, WBC (r = 0.532, P = 0.04) and NEUT (r = 0.593, P = 0.02) showed a significantly positively correlation with PLT in BD patients without immunomodulatory treatment, data not shown), suggesting their role of interactions in chronic infection, inflammation and tissue lesions in BD.
The pharmacological linkages were clustered into five modules in BD patients under treatment. The correlation of linkages in the BD group was lower in modules #1-#4 and higher in module #5 than in the other groups. Compared to the HC group, the negative correlation of pharmacological linkages in module #2 (See figure on previous page.)Changes in clinical variables in HC and BD patients. a-d. Beeswarm plot analysis of the changes in clinical variables associated with inflammation, coagulation, nutrition and the ratios of inflammatory cells in HCs and BD patients with and without treatments, respectively; e. Comparison of the changes in inflammation, coagulation, nutrition and the ratios of inflammatory cells in HCs and BD patients with and without treatments, respectively. HC: healthy control; BD: Behcet's disease was decreased in BD patients and then increased in patients with treatments. The same results were observed in module #3 with positive correlations in HCs and treated patients. The linkages in module #1 changed from positive correlations in the HC group to negative correlations in treated groups, which is in contrast to module #4. Notably, the inflammation variables (CRP and ESR) showed no correlation with the nutritional variables (Alb and PA) in BD patients. However, their linkages were changed to positive correlations in BD patients after treatment with corticosteroids or immunosuppressants, which is in accord with previous reportsand confirms the association between the increase in inflammation and poor nutrition. However, the clinical utility of these pharmacological linkages has to be validated in different cohorts of BD patients with the follow-up information.
## Analysis of ratio changes between clinical variables in patients with bd and healthy and disease controls
Based on the linkage analysis, we further calculated the ratios between every two clinical variables to investigate their coordinated changes. In total, there were 152 pairs of variables with significant fold changes of more than one between BD-N and HC .
To validate the significant changes between two clinical variables, the immunoglobulin expression and clinical variables in a validation set consisting of BD-N (n = 27), BD-C (n = 5), BD-I (n = 16), HC (n = 30), AAV (n = 18), and TA (n = 18)were measured. Significant ratio changes for 8 pairs of variables were ultimately validated in the discovery set , among which TP/MCV, PCT/MONO, TP/MCH, and TP/ MCHC were found to be significantly increased in BD compared with HC, AAV and TA in the discovery and validation sets (P < 0.05,,, regardless of immunomodulatory therapy (P > 0.05,; . In addition, these biomarkers were not affected by corticosteroids and immunosuppressants (P > 0.05 for BD-N vs. BD-C and P > 0.05 for BD-N vs. BD-I, . Furthermore, we investigated changes in the ratio of different BD subsets and found that due to the decreased TP levels, TP/MCV, TP/MCH and TP/MCHC were decreased in BD patients with gastrointestinal involvement compared with those without gastrointestinal involvement and healthy controls. Conversely, significant increases in TP/MCV, TP/MCH and TP/MCHC were found in BD patients without gastrointestinal involvement compared to healthy controls. MPV/HCT and MPV/HGB were also increased in BD patients , especially the BD subset with blood system involvement, including five patients with anemia or myelodysplastic syndrome , indicating anemia and potential disorder of blood cell morphology.
# Discussion
BD is an inflammatory disease of unknown etiology that affects the epidermal, mucocutaneous, vascular, ophthalmologic, gastrointestinal, pulmonary, and central nervous systems. Corticosteroids and immunosuppressants are frequently employed clinically to treat BD patients by regulating inflammation and immune disorders. As an option for long-term treatment for autoimmune diseases, immunosuppressants have an inhibitory effect on the immune response to weaken attacks on body's own tissue by inhibiting the proliferation and function of T cells or B cells. In contrast, corticosteroids work quickly but have significant side effects. Corticosteroids can affect almost all kinds of immune cells and multiple points of the immune response. For example, they prevent lymphocyte recycling and the production of antibody-producing and cytotoxic effector cells, but they also have significant anti-inflammatory effects. They inhibit the adhesion of neutrophils to vascular endothelium in inflammatory sites and inhibit monocyte function, among other effects. However, the pathogenesis of BD and its therapeutic influence by immunomodulatory medication are largely unknown. To address this question, we comprehensively measured and analyzed the changes in clinical variables related to immunity, inflammation, coagulation and nutrition in HCs and BD patients without and with immunomodulatory medication.
We observed an overall increase in immunoglobulin proteome expression in BD patients without treatment , which demonstrates the existence of an immune disorder during BD development. However, the expression of the immunoglobulin proteome, especially IgG1, IgG2 and IgG4, can be suppressed by corticosteroids and immunosuppressants. The results can be further confirmed by the correlation analysis, in which the correlations of pathological linkages (IgA-IgG3 and IgA1-IgG3) were increased in the BD group and decreased under immunomodulatory therapy. The results demonstrate that corticosteroids and immunosuppressants exert their effects by inhibiting immune and inflammatory responses.
The same results were observed in inflammation, in which WBC and NEUT were significantly increased in the BD groups, which was consistent with the functions of WBC and NEUT in mediating vessel damage through enhanced migration in the circulatory system. While corticosteroids are used to inhibit inflammation and the immune response in certain clinical situations, they may also cause an increase in the WBC count and predominantly neutrophils (NEUT) mainly by the demargination of the neutrophils from the endovascular lining. In addition, the use of corticosteroids may promote the maturation of neutrophils in the bone marrow and mobilization into the blood circulation by expression of key receptors such as Annexin A1, as also observed for the pathological link of BASO-LY% and LY%-WBC. Although immunosuppressants such as azathioprine are reported to causes dose-related bone marrow suppression and leukopenia, we did not observe significant difference in blood cells in this study. All these results suggest that clinical evaluations of inflammation should consider medication use as well as the clinical symptoms and signs of the patients. Inflammation may cause damage to the vessel wall and initiate the coagulation pathway and thrombosis. Platelets could be hyperactivated under inflammation, after which granules are released to further promote coagulation and inflammation. In this study, upregulation of MPV in BD patients and its downregulation by corticosteroids were observed. MPV reflects alterations in the morphology of platelets. Elevated MPV means larger platelets with more dense granules that are therefore more thrombogenic than smaller ones, and it is a marker of platelet function and is involved in thrombosis and vascular damage in BD. In contrast, the change in PLT was not obvious, as changes in MPV and PCT can be observed before detectable changes in platelets. There is also growing evidence that platelets are not only involved in fatal vascular events but also function in disease progression by interacting with neutrophils. Schrottmaier et al. proposed that direct interaction of platelets with neutrophils leads to neutrophil activation, recruitment and formation of neutrophil extracellular traps, further promoting the progression of vascular pathologies. Pamuk et al. found significantly higher levels of platelet-neutrophil complexes in BD patients with major vascular involvement than in those without vascular involvement and healthy controls. Consistently, interaction between WBCs or neutrophils and platelet was observed in this research in BD patients based on their positive correlation. Platelets also showed the highest affinity for other innate and adaptive immune cells, including monocytes (as discussed in the next section) and lymphocytes, by soluble mediators. In this study, the pharmacological linkages of LY%-PLT and CRP-PLT further suggest that integrative analysis of granulocytes, platelets and related variables is likely to provide a comprehensive understanding of disease activity, thrombotic potential and potential tissue damage.
Based on linkage analysis, we further constructed a novel method according to the ratio changes between two clinical variables and demonstrated that four ratios -TP/MCV, PCT/MONO, TP/MCH, and TP/ MCHChave higher value in BD than in HC, TA and AAV, suggesting these four ratios as potential diagnostic indicators for BD. PCT, which is produced from PLT and MPV, reflects the total platelet mass. In our research, PCT and MPV were significantly increased in BD. Platelets play an important role in the pathogenesis of thromboembolic diseases. Platelets are more reactive in BD patients than in normal controls, which may contribute to the tendency for thrombosis. Moreover, increased MPV in an inflammatory state contributes to thrombosis, which may be an independent risk factor for vascular involvement in BD. Evidence has shown that monocytes in BD patients are activated and produce proinflammatory cytokines, causing increased adhesion of neutrophils to endothelial cells and chronic inflammation. Interactions between platelets and monocytes is also reported to relate to major vascular involvement in BD, and platelets may induce monocyte differentiation into a more inflammatory phenotype. The higher value of PCT/MONO, consisting of platelets and monocytes, confirms the potentially close interaction between platelets and monocytes. This has been highlighted as an important pathophysiological link between inflammation, thrombosis and endothelial activation, such as the concordance of platelets and monocytes in immune-thrombosis. Moreover, platelets are reported to interact with monocytes to propagate their differentiation into macrophages, and when activated, platelets stimulate monocytes to leave the blood vessel and enter tissues, causing a higher level of PCT/ MONO. We propose that a higher level of PCT/ MONO, representing aggregates and interaction between platelet and monocyte, is a potentially attractive and easily accessible marker in BD.
MCV, MCH, and MCHC are useful biomarkers in the evaluation of anemia. MCV indicates the mean size of red blood cells, while MCH and MCHC indicate the mean amount and the mean concentration of hemoglobin in each red blood cell, respectively. It has been reported that chronic anemia is common in BD patients, especially with intestinal involvement, with contributors like bone marrow failureor serum prohepcidin and hepcidin, whose levels are also closely associated with disease activity. It is likely that the increasing trend of total protein and/or decreases in MCV, MCH or MCHC lead to high levels for the three ratios. However, our study demonstrated that corticosteroids and immunosuppressants do not function by decreasing these higher ratios, illustrating the stability of these indicators. However, other factors involved and the specific mechanisms of these interactions remain to be elucidated in future studies.
There are several limitations in our research. First, the coregulatory mechanisms of clinical variables through physiological, pathological and pharmacological linkages are not well understood and should be carefully interpreted according to the clinical symptoms of BD patients. Second, the numbers of samples and patients' information employed in this study were limited. In the future, we will include more information to match the backgrounds of control patients and verify the utility of these functional linkages in diagnosis and prognosis in larger cohorts.
# Conclusion
In this work, we measured and performed a comprehensive correlation analysis of clinical variables for BD patients with appropriate therapeutic treatment. It is important for clinicians to be aware of the effect of immunomodulatory therapy on laboratory tests for an appropriate interpretation of patients' conditions and to reduce unnecessary medical examinations or therapies. Moreover, we conceptually defined the physiological, pathological and pharmacological linkages of these variables and elucidated their functions for clinical application. The linkages found in our study highlight the close interactions between several markers in BD. Mutual regulation of platelets and different immune cells or other inflammatory markers promotes the development and exacerbation of vascular abnormalities in the pathogenesis of BD, suggesting the potential of combined anti-coagulation and anti-inflammation therapy in BD. The ratio changes among PLT/MONO, TP/MCV, TP/ MCH and TP/MCHC are stable diagnostic indicators for BD regardless of the medication status. These results demonstrate the utility of our approach in elucidating the potential mechanism of BD pathogenesis and therapeutic effects as well as in identifying potential biomarkers to assist BD diagnosis and therapies in the future.
## Supplementary information
The online version contains supplementary material available at https://doi. org/10.1186/s12865-021-00403-1.
Additional file 1:Abbreviations and full names of the laboratory tests used in this study.Variables with significant differences between healthy controls and BD patients not treated with corticosteroids and immunosuppressants therapy.Spearman correlation (r and P value) analysis of the immunoglobulin proteome in HCs and BD patients with and without immunomodulatory therapy. Ratios with significant differences and fold change more than one between healthy controls and BD patients not treated with corticosteroids and immunosuppressants therapy in the discovery set. Ratios with significant differences between BD patients not treated with corticosteroids and immunosuppressants therapy, healthy control and disease controls in both the discovery set and validation set.. Standard curves for the quantification of eight immunoglobulin isotypes. . The assay performance of the plasma microarray in the detection of the immunoglobulin proteome.. Correlation analysis between immunoglobulins and age in HC(A) and BD(B).. Beeswarm plots of immunoglobulin proteome expression between sex groups in HC(A) and BD(B).. Comparison of laboratory tests in HC and BD patients with and without immunomodulatory therapy. |
Gender and Age Impact on the Association Between Thyroid-Stimulating Hormone and Serum Lipids
The relationship between thyroid-stimulating hormone (TSH) and hyperlipidemia is still a topic of debate. We aimed to explore the impact of gender and age on the association between serum TSH and lipid profile in a large cohort of Chinese.This cross-sectional study enrolled 13,915 participants (8565 male, 5350 female), who self-reported as healthy without any known previous diseases. Clinical data including anthropometric measurements, thyroid function, and other serum parameters were collected. The associations between TSH and hyperlipidemia of males and females were analyzed separately after dividing TSH and age into subgroups. Odds ratio for hyperlipidemia was calculated by binary logistic regression models.Young males had significantly higher prevalence of hypercholesterolemia, hypertriglyceridemia, and high serum low-density lipoprotein-cholesterol than females, yet after menopause, females had higher prevalence than males. TSH was positively associated with hyperlipidemia independent of thyroid hormones. Males showed more reduced risks of hyperlipidemia in low TSH concentrations, while females demonstrated more enhanced risks of hyperlipidemia in high TSH concentrations. For instance, if TSH was lower than 0.3 mIU/mL, the risks of developing hypercholesterolemia and hypertriglyceridemia in males were only 0.198 (P < 0.01) and 0.425 (P < 0.05) of the reference TSH risks (between 2.0 and 3.0 mIU/mL), while in females the risks were 0.553 (P < 0.05) and 0.642 (P > 0.05), respectively. If TSH was higher than 4.0 mIU/mL, women displayed significantly higher risks of developing hypertriglyceridemia than the reference TSH risks (P < 0.05), yet, men did not demonstrate such significances.Our results showed thyroid hormone independent positive associations between serum TSH and lipids, which were substantially influenced by gender and age. Males demonstrated more protective effects of low TSH against hyperlipidemia, while females showed more detrimental effects of high TSH on hyperlipidemia.(Medicine 94(49):e2186)Abbreviations: BMI = body mass index, Cr = creatinine, FT3 = free triiodothyronine, FT4 = free thyroxine, LDL = low-density lipoprotein-cholesterol, TC = total cholesterol, TG = triglycerides, TSH = thyroid-stimulating hormone.Editor: Patrick Wall.FIGURE 3. Prevalence of dyslipidemia in different age subgroups. Definition of dyslipidemia: hypercholesterolemia (HyperTC), TC ! 5.18 mmol/L; hypertriglyceridemia (HyperTG), TG ! 1.70 mmol/L; high serum level of low-density lipoprotein-cholesterol (HyperLDL), LDL ! 3.37 mmol/L; low serum level of high-density lipoprotein-cholesterol (LowHDL), HDL < 1.04 mmol/L. Age subgroups 1 to 7 referred to the followings: age < 25 years, 25 years age < 35 years, 35 years age < 45 years, 45 years age < 55 years, 55 years age < 65 years, 65 years age < 75 years, age ! 75 years. HDL ¼ high-density lipoprotein-cholesterol, LDL ¼ low-density lipoprotein-cholesterol, TC ¼ total cholesterol, TG ¼ triglycerides. # Difference of prevalence between gender was significant at 0.05; Ã difference of prevalence between gender was significant at 0.01.Medicine Volume 94, Number 49, December 2015 TSH and Lipids
# Introduction
T he world faces a burden of thyroid disease that has reached epidemic proportions. It is estimated that around 200 million individuals worldwide have various kinds of thyroid dysfunctions. [bib_ref] Subclinical thyroid disease, Cooper [/bib_ref] The association between thyroid function and serum lipid status has become a popular area of research in recent years. It is well recognized that thyroid function can influence the synthesis, mobilization, and degradation of lipids. [bib_ref] Thyroid functions and serum lipids in older women: a population-based study, Bauer [/bib_ref] [bib_ref] Thyroid dysfunction and serum lipids: a community-based study, Walsh [/bib_ref] Recent studies have indicated that thyroid-stimulating hormone (TSH) is associated with adverse changes of lipid metabolism and increased cardiovascular risks as well. [bib_ref] Thyroid dysfunction and serum lipids: a community-based study, Walsh [/bib_ref] [bib_ref] Thyroid stimulating hormone, independent of thyroid hormone, can elevate the serum total..., Xu [/bib_ref] [bib_ref] Subclinical hypothyroidism and its association with cardiovascular risk factors, Sharma [/bib_ref] [bib_ref] TSH and free thyroxine concentrations are associated with differing metabolic markers in..., Garduno-Garcia Jde [/bib_ref] [bib_ref] Thyroid-stimulating hormone levels within the reference range are associated with serum lipid..., Wang [/bib_ref] [bib_ref] The association between TSH within the reference range and serum lipid concentrations..., Asvold [/bib_ref] [bib_ref] Thyroid function is intrinsically linked to insulin sensitivity and endotheliumdependent vasodilation in..., Fernandez-Real [/bib_ref] [bib_ref] Thyroid function is associated with components of the metabolic syndrome in euthyroid..., Roos [/bib_ref] [bib_ref] The Colorado thyroid disease prevalence study, Canaris [/bib_ref] For instance, Xu et al 4 proved that TSH can increase total cholesterol (TC) level independent of thyroid hormones. Several other studies did not observe such an association, [bib_ref] Subclinical hypothyroidism and the risk of heart failure, other cardiovascular events, and..., Rodondi [/bib_ref] [bib_ref] Thyroid status, cardiovascular risk, and mortality in older adults, Cappola [/bib_ref] [bib_ref] Low-density lipoprotein cholesterol in subclinical hypothyroidism, Vierhapper [/bib_ref] [bib_ref] Association between thyroid dysfunction and total cholesterol level in an older biracial..., Kanaya [/bib_ref] leading to a controversy. For example, Rodondi et al [bib_ref] Subclinical hypothyroidism and the risk of heart failure, other cardiovascular events, and..., Rodondi [/bib_ref] indicated that subclinical hypothyroidism was not associated with increased risk for coronary heart disease, stroke, peripheral arterial disease, or cardiovascular-related or total mortality. Therefore, confirmative studies with large recruited subjects are needed.
Gender and age can influence the relationship between thyroid function and lipid profiles. [bib_ref] Age and gender substantially influence the relationship between thyroid status and the..., Tognini [/bib_ref] Yet, on the one hand, this aspect has not been thoroughly investigated; on the other, the available literature is still controversial. [bib_ref] Age and gender substantially influence the relationship between thyroid status and the..., Tognini [/bib_ref] [bib_ref] Serum lipid levels in relation to serum thyroid-stimulating hormone and the effect..., Iqbal [/bib_ref] [bib_ref] Initial thyroid status and cardiovascular risk factors: the EPIC-Norfolk prospective population study, Boekholdt [/bib_ref] For example, by using stepwise regression analysis, Tognini et al [bib_ref] Age and gender substantially influence the relationship between thyroid status and the..., Tognini [/bib_ref] revealed that age was the most crucial factor influencing serum cholesterols, and TSH was the second important factor following age. Furthermore, this research also found that both age and TSH could influence either TC or low-density lipoprotein-cholesterol (LDL) significantly in females. Yet, in males, TSH only strongly affected TC. Nevertheless, in males, both TSH and body mass index (BMI) greatly affected triglycerides (TG) concentrations independently of age, while in women, TSH did not show any obvious influence on TG. The Tromsø study, [bib_ref] Serum lipid levels in relation to serum thyroid-stimulating hormone and the effect..., Iqbal [/bib_ref] which recruited 5143 participants, displayed a significantly positive correlation between TC and TSH, as well as LDL and TSH, in both sex. However, in women, if adjustment was made by age and BMI, these relationships became not significant. Therefore, these discordant results warrant a large-scale investigation with the emphasis on both age and gender.
The aim of the current study was to evaluate the impact of gender and age on the association between TSH and lipid profile and the burden of dyslipidemia in a large cross-sectional Han Chinese population sample.
# Methods
## Design
This cross-sectional study was conducted in Tianjin Medical University General Hospital in China, under collaboration from the departments of Health Management, Endocrinology & Metabolism, and Nuclear Medicine. During the period from September 2011 to April 2014, a total of 13,915 eligible subjects (8565 male, 5350 female) participated in this mainly community-based health check program. This work was a part of our continuous project for the general public health of Tianjin Municipality, and the protocol was developed and executed previously by our group. [bib_ref] Relationship between lifestyle choices and hyperuricemia in Chinese men and women, Liu [/bib_ref] [bib_ref] Gender and age impacts on the correlations between hyperuricemia and metabolic syndrome..., Zhang [/bib_ref] [bib_ref] Gender impact on the correlations between subclinical thyroid dysfunction and hyperuricemia in..., Zhang [/bib_ref] All participants were asked to complete a self-reported questionnaire and provide an overnight fasting blood sample. All participants were self-reported as healthy without any known previous diseases. In order to avoid the influence of confounding factors, the following criteria were used for exclusion: subjects with thyroid, hepatic, renal, gastro-intestinal diseases, or oncology; subjects with any diseases or taking any medicine that might affect their thyroid status or lipid metabolism (eg, antithyroid drugs, thyroid hormone, amiodarone, iodine, estrogen, androgen, statins, steroid hormones, etc.); and pregnancy. Written consents were obtained, and the institutional review board and ethic committee of Tianjin Medical University General Hospital approved this study.
## Measurements
Anthropometric measurements and fasting blood tests of the participants were performed during their visits to our institution. Body height and body weight were measured in centimeters and kilograms. BMI was calculated by dividing body weight (kg) by the square of body height (). [bib_ref] Thyroid functions and serum lipids in older women: a population-based study, Bauer [/bib_ref] Fasting blood samples were obtained between 7 AM and 10 AM. TSH, free triiodothyronine (FT3), and free thyroxine (FT4) were analyzed on a fully automated ADVIA Centaur analyzer (Siemens Healthcare Diagnostics, Erlangen, Germany) by chemiluminescent reaction principle. TC, TG, LDL, HDL, alanine aminotransferase, total bilirubin, blood urea nitrogen, uric acid, creatinine (Cr), and fasting glucose were determined enzymatically by an auto-analyzer (Hitachi Model 7600 analyzer, Hitachi, Tokyo, Japan).
## Definition
The diagnostic criteria for dyslipidemia were in accordance withthe National Cholesterol Education Program Adult Treatment Panel III criteria as follows: TC ! 5.18 mmol/L, TG ! 1.70 mmol/ L, LDL ! 3.37 mmol/L, and HDL < 1.04 mmol/L.Participants were divided into subgroup 1 to 8 according to TSH levels: TSH < 0.3 mIU/mL, 0.3 mIU/mL TSH < 1.0 mIU/mL, 1.0 mIU/mL TSH < 2.0 mIU/mL, 2.0 mIU/mL TSH < 3.0 mIU/mL, 3.0 mIU/mL TSH < 4.0 mIU/mL, 4.0 mIU/mL TSH < 5.0 mIU/mL, 5.0 mIU/mL TSH < 10.0 mIU/mL, and TSH ! 10.0 mIU/mL. Participants were also divided into subgroup 1 to 7 based on ages: age < 25 years, 25 years age < 35 years, 35 years age < 45 years, 45 years age < 55 years, 55 years age < 65 years, 65 years age < 75 years, and age ! 75 years.
# Statistical analysis
Statistics were done as our prior protocols. [bib_ref] Relationship between lifestyle choices and hyperuricemia in Chinese men and women, Liu [/bib_ref] [bib_ref] Gender and age impacts on the correlations between hyperuricemia and metabolic syndrome..., Zhang [/bib_ref] [bib_ref] Gender impact on the correlations between subclinical thyroid dysfunction and hyperuricemia in..., Zhang [/bib_ref] All data were presented as mean AE standard deviation. Differences of Data presented as mean AE standard deviation, and analyzed by independent sample's t-test. ALT ¼ alanine aminotransferase, BMI ¼ body mass index, BUN ¼ blood urea nitrogen, Cr ¼ creatinine, FG ¼ fasting glucose, FT3 ¼ free triiodothyronine, FT4 ¼ free thyroxine, HDL ¼ high-density lipoprotein-cholesterol, LDL ¼ low-density lipoprotein-cholesterol, TBIL ¼ total bilirubin, TC ¼ total cholesterol, TG ¼ triglycerides, TSH ¼ thyroid stimulation hormone, UA ¼ uric acid, .
indices between groups were analyzed by independent sample's t-test or one-way analysis of variance. For multiple comparisons among different subgroups, the least significant difference test was applied. In order to compare differences of intergroup prevalence, Chi-square test was performed. Pearson bivariate correlation was conducted between TSH and other variables. After data stratification according to different thyroid functional states, binary logistic regression models were executed to calculate odds ratio for hyperlipidemia with 95% confidence interval (CI). Statistical Package for Social Sciences (SPSS version 17.0, Chicago, IL) was used for statistics. And a P value of <0.05 was defined as significance.
# Results
## The characteristics of the participants in different gender
The characteristics of the participants were summarized [fig_ref] TABLE 1: Population Characteristics Based on Different Genders [/fig_ref]. There were significant differences in all parameters with respect to opposite gender. Males were younger than females, yet BMI in males was higher than in females. TSH was lower in males than in females, while FT3 and FT4 were higher in males than in females. Serum lipids, hepatic, and renal functions were also different between genders.
Age cast differences among the participants . For males, TC, TG, and LDL showed the tendency of increase from FIGURE 1. Serum lipid distribution in different age subgroups. Box plots were drawn based on different age subgroups in males (A-D) and in females (E-H). Age subgroups 1 to 7 referred to the followings: age < 25 years, 25 years age < 35 years, 35 years age < 45 years, 45 years age < 55 years, 55 years age < 65 years, 65 years age < 75 years, age ! 75 years. HDL ¼ high-density lipoproteincholesterol, LDL ¼ low-density lipoprotein-cholesterol, TC ¼ total cholesterol, TG ¼ triglycerides.
the youngest to the age range of 65 to 75, and then decreased in the eldest participants. Inversely, HDL demonstrated a decrease trend. For females, TC, TG, and LDL showed constant trend of increase from the youngest till the eldest, and HDL displayed constant decrease trend.
The differences of serum lipids based on TSH levels were demonstrated in [fig_ref] FIGURE 2: Serum lipid distribution in different TSH subgroups [/fig_ref]. TC in both sex, TG in female, as well as LDL in male, showed consistent increase from the lowest TSH level to the highest TSH level. The differences of HDL levels among the TSH subgroups were relatively subtler in both genders.
## Prevalence of hyperlipidemia in different gender
The prevalence of hypercholesterolemia and high serum LDL showed the same increasing tendency in males from the youngest to the age range of 65 to 75, and then decreased in the eldest participants. Yet, in females, this prevalence tendency increased from the youngest to the eldest . Young males (younger than 45) had significantly higher prevalence than females. However, after menopause (older than 55), females had significantly higher prevalence than males. This crisscross pattern was also discovered in hypertriglyceridemia, yet the converging point was around 65 to 75 years of age. For low-serum HDL, males always showed higher prevalence than females indiscriminate of age.
Prevalence of hyperlipidemia rose when TSH concentration rose, yet no crisscross pattern was found [fig_ref] FIGURE 4: Prevalence of dyslipidemia in different TSH subgroups [/fig_ref]. The prevalence of hypercholesterolemia was higher in females if TSH was less than 0.3 mIU/mL, or between 4.0 and 10.0 mIU/ mL, otherwise no significant differences existed. The prevalence of high serum LDL was higher in females if TSH was between 5.0 and 10.0 mIU/mL, this prevalence was higher in males if TSH was between 1.0 and 4.0 mIU/mL. For the prevalence of hypertriglyceridemia and low-serum HDL, males always showed higher prevalence than females.
## Incidence of thyroid dysfunction in different gender
Females had significantly higher thyroid dysfunction incidence than males [fig_ref] TABLE 2: Incidence of Hypothyroidism and Hyperthyroidism on Different GendersIncidence of hypothyroidism and/or hyperthyroidism... [/fig_ref] , hypothyroidism and hyperthyroidism were defined as TSH > 5.0 mIU/mL and TSH < 0.3 mIU/ mL, respectively. Most age subgroups demonstrated the same pattern of differences in opposite sex, except for the youngest and the eldest. There existed a significant increasing tendency of hypothyroidism incidence with aging (except for the youngest) for both gender (Chi value for male, 86.911, P ¼ 1.326E-16; Chi value for female 58.561; P ¼ 8.818E-11).
## Correlations between tsh and serum lipid levels in different gender
Significant negative relationships were demonstrated for FT3 and FT4 for both genders. In males, positive relationships were demonstrated for age, TC, LDL, and Cr. In females, positive relationships were demonstrated for age, BMI, TC, TG, LDL, alanine aminotransferase, blood urea nitrogen, uric acid, and Cr [fig_ref] TABLE 3: Pearson Bivariate Correlations Between TSH and Other Variables Based on Different GendersALT... [/fig_ref].
## Risks of developing hyperlipidemia in different tsh concentrations in opposite gender
Binary logistic regression models were utilized on different thyroid functional state. Thyroid functional states were designated as the categorical variables, and the mid-normal TSH group of ''2.0 mIU/mL TSH < 3.0 mIU/mL'' was set as the reference. Model 1 had FT3 and FT4 as covariates. Model 2 had all parameters as covariates [fig_ref] TABLE 5: The Likelihood of Hyperlipidemia Among Different Thyroid Functional States in Logistic Regression... [/fig_ref]. In males, we demonstrated significantly reduced risks of hyperlipidemia while TSH decreased from the reference level, indicating protective effects of low TSH against hyperlipidemia. For instance, in Model 2, the risks of hypercholesterolemia, hypertriglyceridemia, and high serum level of LDL in TSH subgroup 1 were only 0.198 (P < 0.01), 0.425 (P < 0.05), and 0.219 (P < 0.01) of the corresponding reference TSH subgroup risks. In females, the reduced risks of hyperlipidemia along with the decreasing TSH level did not reach such significance as in males. For example, in Model 2, the risks of hypercholesterolemia, hypertriglyceridemia, and high serum level of LDL were just 0.553 (P < 0.05), 0.642 (P > 0.05), and 0.635 (P > 0.05) of the reference risks. On the other hand, our results displayed that if TSH level rose, the risks of hyperlipidemia generally increased, especially in the subgroup of TSH ! 10.0 mIU/mL, indicating detrimental effects of high TSH on hyperlipidemia. However, this pattern appeared to be much more significant in females than in males, particularly for hypertriglyceridemia. In females, if TSH was higher than 4.0 mIU/mL, risks of developing hypertriglyceridemia were significantly higher in both models than the reference TSH subgroup risks (all P < 0.05). Nevertheless, in males so such significances existed.
# Discussion
Clinical and subclinical thyroid dysfunctions are both very common clinical entities, the latter usually denotes the presence of a disease without obvious symptoms. [bib_ref] Subclinical thyroid disease, Cooper [/bib_ref] Mild thyroid dysfunction often means the evolution of the disease at an early stage. [bib_ref] Subclinical thyroid disease, Cooper [/bib_ref] The incidence of mild hypothyroidism and mild hyperthyroidism were reported to be around 9.5% 11 and 1.8%, [bib_ref] Serum TSH, T(4), and thyroid antibodies in the United States population (1988..., Hollowell [/bib_ref] respectively. Results of the current study not only were in concord with the above-reported data, we also distinguished incidence disparity between genders. Mild hypothyroidism represents early thyroid failure, which is usually progressive, especially when TSH is higher than 10 mIU/mL, in females and with positive thyroid peroxidase antibodies. [bib_ref] Antithyroperoxidase and antithyroglobulin antibodies in a five-year follow-up survey of populations with..., Li [/bib_ref] [bib_ref] Thyrotropin and thyroid antibodies as predictors of hypothyroidism: a 13-year, longitudinal study..., Walsh [/bib_ref] Hypothyroidism is associated with greater cardiovascular risk factors, including hyperlipidemia, [bib_ref] Subclinical thyroid disease, Cooper [/bib_ref] [bib_ref] The Colorado thyroid disease prevalence study, Canaris [/bib_ref] [bib_ref] Clinical review 115: effect of thyroxine therapy on serum lipoproteins in patients..., Danese [/bib_ref] [bib_ref] The clinical significance of subclinical thyroid dysfunction, Biondi [/bib_ref] making it an important topic in people's health.
Recently, a large number of clinical studies have indicated that TSH is associated with a deleterious change of lipid metabolism. And it has been suggested that the proatherogenic influence of TSH on serum lipid profile is in a thyroid hormone independent manner. Abnormal elevation of TC, LDL, and TG, and decrease of HDL have been observed in patients with subclinical hypothyroidism, [bib_ref] Subclinical hypothyroidism and its association with cardiovascular risk factors, Sharma [/bib_ref] [bib_ref] TSH and free thyroxine concentrations are associated with differing metabolic markers in..., Garduno-Garcia Jde [/bib_ref] or even in subject with normal TSH (TSH near the upper limit within the normal reference range). [bib_ref] TSH and free thyroxine concentrations are associated with differing metabolic markers in..., Garduno-Garcia Jde [/bib_ref] [bib_ref] Thyroid-stimulating hormone levels within the reference range are associated with serum lipid..., Wang [/bib_ref] [bib_ref] The association between TSH within the reference range and serum lipid concentrations..., Asvold [/bib_ref] [bib_ref] Thyroid function is intrinsically linked to insulin sensitivity and endotheliumdependent vasodilation in..., Fernandez-Real [/bib_ref] [bib_ref] Thyroid function is associated with components of the metabolic syndrome in euthyroid..., Roos [/bib_ref] The current large population-based study was able to indicate protective effects of low-TSH concentration against hyperlipidemia and enhanced risks of hyperlipidemia in high-TSH concentration; these effects were independent of thyroid hormones. Our results were in line with the above-mentioned reports, confirming a linear and significant increase of hyperlipidemia with elevation of serum TSH levels. [bib_ref] Thyroid stimulating hormone, independent of thyroid hormone, can elevate the serum total..., Xu [/bib_ref] [bib_ref] Thyroid-stimulating hormone levels within the reference range are associated with serum lipid..., Wang [/bib_ref] The exact mechanism for the direct effects of TSH on lipids has not been fully elucidated with sensible explanations. Only some plausible hypotheses were put forward as followed. Tian et al [bib_ref] A novel role for thyroid-stimulating hormone: up-regulation of hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase..., Tian [/bib_ref] found that liver cells could express TSH receptor, and TSH could upregulate the expression of hepatic 3-hydroxy-3-methyl-glutaryl co-enzyme A reductase (a rate-limiting enzyme in cholesterol synthesis) by acting on the TSH receptor. TSH receptor could also play an important role in adipocyte differentiation and adipogenesis, resulting in obesity in mice and increasing BMI in humans. [bib_ref] Role of extrathyroidal TSHR expression in adipocyte differentiation and its association with..., Lu [/bib_ref] TSH could stimulate lipolysis in cultured adipocytes and elevate serum-free fatty acid levels in vivo. [bib_ref] Thyroid-stimulating hormone stimulates lipolysis in adipocytes in culture and raises serum free..., Gagnon [/bib_ref] Besides, the involvement of leptin activation, [bib_ref] Acute exogenous TSH administration stimulates leptin secretion in vivo, Santini [/bib_ref] visceral obesity [bib_ref] Free triiodothyronine and thyroid stimulating hormone are directly associated with waist circumference,..., De Pergola [/bib_ref] and insulin resistance could also be relevant. [bib_ref] The relationship between thyrotropin and low density lipoprotein cholesterol is modified by..., Bakker [/bib_ref] Our study might shed some new light on the pathophysiological role of TSH on lipid in clinical perspective. Gender difference on the association between TSH and lipids was the most important finding in our study. Males were more likely to suffer TSH-related dyslipidemia [fig_ref] FIGURE 4: Prevalence of dyslipidemia in different TSH subgroups [/fig_ref] , although females were more likely to have thyroid dysfunction [fig_ref] TABLE 2: Incidence of Hypothyroidism and Hyperthyroidism on Different GendersIncidence of hypothyroidism and/or hyperthyroidism... [/fig_ref]. Men demonstrated more protective effects of low TSH against hyperlipidemia, while females showed more detrimental effects of high TSH on hyperlipidemia [fig_ref] TABLE 5: The Likelihood of Hyperlipidemia Among Different Thyroid Functional States in Logistic Regression... [/fig_ref]. It is evidently recognized that male gender is associated with a markedly more proatherogenic lipid profile (higher LDL and lower HDL) than female gender. In addition, among women, menopause is associated with a significant shift toward an atherogenic lipid profile. [bib_ref] Effects of menopause, gender and age on lipids and high-density lipoprotein cholesterol..., Anagnostis [/bib_ref] [bib_ref] Sex differences in lipid and lipoprotein metabolism: it's not just about sex..., Wang [/bib_ref] [bib_ref] Differential impact of aging and gender on lipid and lipoprotein profiles in..., Goh [/bib_ref] Besides, there is also a sex dimorphism in the regional distribution of body fat. Evolutionary pressures predispose women to store excess fat in gluteal regions, while men are characterized by a greater accumulation of visceral adipose tissue. [bib_ref] Working Group. Sex differences in obesity and the regulation of energy homeostasis, Lovejoy [/bib_ref] Visceral obesity is associated with increased atherogenic dyslipidemia. [bib_ref] Role of the sex hormone estrogen in the prevention of lipid disorder, Pellegrini [/bib_ref] [bib_ref] Androgen therapy in men with testosterone deficiency: can testosterone reduce the risk..., Saad [/bib_ref] The traditional view of the above female advantage is thought to be due to the differences in the sex hormone milieu, in particular, the availability of estrogens and androgens. [bib_ref] Gender differences in cardiovascular disease: hormonal and biochemical influences, Perez-Lopez [/bib_ref] The endogenous estrogens have been reported to have a lowering effect on TC and LDL. And lipoprotein lipase is higher in women than in men. [bib_ref] Endogenous sex hormones: impact on lipids, lipoproteins, and insulin, Haffner [/bib_ref] Now, it is thought that sexual dimorphism in lipid metabolism should be the result of a complex network of hormone action in combination with other, sexspecific, direct or indirect modulators of lipid metabolism. [bib_ref] Sex differences in lipid and lipoprotein metabolism: it's not just about sex..., Wang [/bib_ref] Besides gender, age should also be taken as a crucial factor for TSH-related dyslipidemia. Generally, aging is associated with deterioration of serum lipid profile. [bib_ref] Effects of age, gender, and menopausal status on plasma low density lipoprotein..., Schaefer [/bib_ref] It is further recognized that women have less risk of atherosclerotic cardiovascular disease compared with men up until midlife (age 50-60), after which the gap begins to narrow, and hyperlipidemia in women even surpass those in men. [bib_ref] Effects of age, gender, and menopausal status on plasma low density lipoprotein..., Schaefer [/bib_ref] [bib_ref] Narrowing sex differences in lipoprotein cholesterol subclasses following mid-life: the very large..., Swiger [/bib_ref] Therefore, after menopause and beyond, lipid profile of women undergoes unfavorable changes, becoming worse than men. [bib_ref] Effects of menopause on intraindividual changes in serum lipids, blood pressure, and..., Torng [/bib_ref] Our results were concordant with the previous studies. We found that young males, with the age of younger than 45 years, had significantly higher prevalence of hypercholesterolemia and high serum LDL than females. However, after menopause (older than 55), females had significantly higher prevalence than males. This crisscross pattern was also discovered in hypertriglyceridemia, yet the converging point was around 65 to 75 years of age. For the prevalence of low-serum HDL, males always showed higher prevalence than females. Limitation of the study deserves comments. First, using a cross-sectional design, causality relationship could not be established. Prospective studies are necessary to truly determine their cause-and-effect relationship. Second, we applied stringent exclusion criteria to rule our relevant disease histories. However, since the annual health checkup policy is not good enough to cover the majority of Chinese, a number of the participants with various diseases might not be aware of them, which could be a confounding factor for our investigation. Third, the power of the present study could have been stronger if a multiethnic population and a much larger number of subjects were recruited.
In conclusion, we found that serum TSH levels were positively associated with the levels of TC, LDL, and TG, and the incidence of hyperlipidemia, these relationships were independent of thyroid hormones. Moreover, these associations were found to be stronger with increasing age. Males demonstrated more protective effects of low TSH against hyperlipidemia, while females showed more detrimental effects of high TSH on hyperlipidemia.
[fig] FIGURE 2: Serum lipid distribution in different TSH subgroups. Box plots were drawn based on different TSH subgroups in males (A-D) and in females (E-H). TSH subgroups 1 to 8 referred to the followings: TSH < 0.3 mIU/mL, 0.3 mIU/mL TSH < 1.0 mIU/mL, 1.0 mIU/ mL TSH < 2.0 mIU/mL, 2.0 mIU/mL TSH < 3.0 mIU/mL, 3.0 mIU/mL TSH < 4.0 mIU/mL, 4.0 mIU/mL TSH < 5.0 mIU/mL, 5.0 mIU/ mL TSH < 10.0 mIU/mL, TSH ! 10.0 mIU/mL. HDL ¼ high-density lipoprotein-cholesterol, LDL ¼ low-density lipoprotein-cholesterol, TC ¼ total cholesterol, TG ¼ triglycerides, TSH ¼ thyroid stimulating hormone. [/fig]
[fig] FIGURE 4: Prevalence of dyslipidemia in different TSH subgroups. Definition of dyslipidemia: hypercholesterolemia (HyperTC), TC ! 5.18 mmol/L; hypertriglyceridemia (HyperTG), TG ! 1.70 mmol/L; high serum level of low-density lipoprotein-cholesterol (HyperLDL), LDL ! 3.37 mmol/L; low serum level of high-density lipoprotein-cholesterol (LowHDL), HDL < 1.04 mmol/L. TSH subgroups 1 to 8 referred to the followings: TSH < 0.3 mIU/mL, 0.3 mIU/mL TSH < 1.0 mIU/mL, 1.0 mIU/mL TSH < 2.0 mIU/mL, 2.0 mIU/mL TSH < 3.0 mIU/mL, 3.0 mIU/mL TSH < 4.0 mIU/mL, 4.0 mIU/mL TSH < 5.0 mIU/mL, 5.0 mIU/mL TSH < 10.0 mIU/mL, TSH ! 10.0 mIU/mL. HDL ¼ highdensity lipoprotein-cholesterol LDL ¼ low-density lipoprotein-cholesterol, TC ¼ total cholesterol, TG ¼ triglycerides, TSH ¼ thyroid stimulating hormone. # Difference of prevalence between gender was significant at 0.05; Ã difference of prevalence between gender was significant at 0.01. [/fig]
[fig] TABLE 4: The Likelihood of Hyperlipidemia Among Different Thyroid Functional States in Logistic Regression Models for Males OR (95% CI) in Different TSH Subgroups, confidence interval, HyperLDL ¼ high serum level of low-density lipoprotein-cholesterol, HyperTG ¼ hypertriglyceridemia, HyperTC ¼ hypercholesterolemia, LowHDL ¼ low-serum level of high-density lipoprotein-cholesterol, OR ¼ odds ratio, TSH ¼ thyroid stimulation hormone. TSH was designated as categorical variable, and the subgroup of ''2.0 TSH < 3.0'' was determined as the reference subgroup. Model 1 included FT3 and FT4 as covariates. Model 2 had all parameters as covariates. [/fig]
[table] TABLE 1: Population Characteristics Based on Different Genders [/table]
[table] TABLE 2: Incidence of Hypothyroidism and Hyperthyroidism on Different GendersIncidence of hypothyroidism and/or hyperthyroidism between males and females compared by Chi-square method. Ã Hypothyroidism defined as TSH > 5.0 mIU/mL, hyperthyroidism defined as TSH < 0.3 mIU/mL. [/table]
[table] TABLE 3: Pearson Bivariate Correlations Between TSH and Other Variables Based on Different GendersALT ¼ alanine aminotransferase, BMI ¼ body mass index, BUN ¼ blood urea nitrogen, Cr ¼ creatinine, FG ¼ fasting glucose, FT3 ¼ free triiodothyronine, FT4 ¼ free thyroxine, HDL ¼ high-density lipoprotein-cholesterol, LDL ¼ low-density lipoprotein-cholesterol, TC ¼ total total cholesterol, TG ¼ triglycerides, TBIL ¼ total bilirubin, TSH ¼ thyroid stimulation hormone, UA ¼ uric acid. Ã P < 0.05. ÃÃ P < 0.01. [/table]
[table] TABLE 5: The Likelihood of Hyperlipidemia Among Different Thyroid Functional States in Logistic Regression Models for Females OR (95% CI) in Different TSH Subgroups (mIU/mL) CI ¼ confidence interval, HyperLDL ¼ high serum level of low-density lipoprotein-cholesterol, HyperTC ¼ hypercholesterolemia, HyperTG ¼ hypertriglyceridemia, LowHDL ¼ low serum level of high-density lipoprotein-cholesterol, OR ¼ odds ratio, TSH ¼ thyroid stimulation hormone. TSH was designated as categorical variable, and the subgroup of ''2.0 TSH < 3.0'' was determined as the reference subgroup. Model 1 included FT3 and FT4 as covariates. Model 2 had all parameters as covariates. [/table]
|
Salmonella enterica Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007
We determined characteristics of Salmonella enterica pulsed-fi eld gel electrophoresis clusters that predict their being solved (i.e., that result in identifi cation of a confi rmed outbreak). Clusters were investigated by the Minnesota Department of Health by using a dynamic iterative model. During 2001-2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of >4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the fi rst 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the fi rst 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-fi eld gel electrophoresis clusters, investigation of clusters of >4 cases and clusters in which the fi rst 3 case isolates were received at a public health laboratory within 7 days may improve outbreak investigations.
S almonellosis is a major foodborne illness that results in ≈1.4 million infections, 15,000 hospitalizations, and 400 deaths each year in the United States [bib_ref] Food-related illness and death in the United States, Mead [/bib_ref]. Salmonella infections are primarily of foodborne origin but can also occur through contact with infected animals, humans, or their feces. The epidemiology of salmonellosis is complex largely because there are >2,500 distinct serotypes (serovars) with different reservoirs and diverse geographic incidences [bib_ref] The changing epidemiology of salmonella: trends in serotypes isolated from humans in..., Olsen [/bib_ref]. Changes in food consumption, production, and distribution have led to an increasing frequency of multistate outbreaks associated with fresh produce and processed foods.
The development of molecular subtyping by pulsedfi eld gel electrophoresis (PFGE) has revolutionized Salmonella spp. surveillance. The National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet) provides state and local public health department laboratories with standardized methods to subtype Salmonella serovars and normalize PFGE patterns against a global reference standard provided by the Centers for Disease Control and Prevention (CDC) [bib_ref] Surveillance for human Salmonella infections in the United States, Swaminathan [/bib_ref]. Molecular subtyping enhances case defi nition specifi city, enabling outbreaks to be detected and controlled at an earlier stage, and enabling detection of geographically dispersed outbreaks [bib_ref] Surveillance for sporadic foodborne disease in the 21st century: the FoodNet perspective, Allos [/bib_ref] [bib_ref] Interpretation of pulsed-fi eld gel electrophoresis patterns in foodborne disease investigations and..., Barrett [/bib_ref].
Although the benefi ts of molecular subtyping, specifically by PFGE, in foodborne disease outbreak detection and investigation have been well established, there is no consensus about when a PFGE cluster warrants further investigation and almost no quantitative analysis about characteristics of PFGE clusters that indicate a common source will be identifi ed [bib_ref] Framework for evaluating public health surveillance systems for early detection of outbreaks:..., Buehler [/bib_ref] [bib_ref] Timeliness of enteric disease surveillance in 6 US states, Hedberg [/bib_ref] [bib_ref] Surveillance for foodborne-disease outbreaks-United States, Lynch [/bib_ref] [bib_ref] Commentary: cluster evaluation, PulseNet, and public health pracitce, Hedberg [/bib_ref]. Cluster size and the number of days from receipt of the fi rst cluster case isolate to the third case isolate received by the public health laboratory were predictors of a source of infection being identifi ed for Listeria monocytogenes clusters in France [bib_ref] evaluation of cluster investigation criteria, Hedberg [/bib_ref]. The objective of this study was to determine characteristics of Salmonella PFGE clusters that could serve as useful predictors for their being solved (i.e., result in identifi cation of a confi rmed outbreak). This information could help public health agencies with limited resources prioritize investigation of Salmonella PFGE clusters.
# Materials and methods
Salmonella infections are reportable to the Minnesota Department of Health (MDH) by state law. Clinical laboratories are required to forward all Salmonella isolates to the MDH Public Health Laboratory (PHL). PFGE sub-typing after digestion with XbaI is conducted on all isolates as soon as they are received according to PulseNet protocols [bib_ref] Standardization of pulsed-fi eld gel electrophoresis protocols for the subtyping of Escherichia..., Ribot [/bib_ref]. PFGE subtypes are uploaded into the national PulseNet database [bib_ref] CDC PulseNet Task Force. PulseNet: the molecular subtyping network for foodborne bacterial..., Swaminathan [/bib_ref]. All Minnesota residents with a culture-confi rmed Salmonella infection are routinely interviewed as soon as possible by MDH staff with a standard questionnaire about symptom history, food consumption, and other potential exposures occurring in the 7 days before onset of illness. The questionnaire contains detailed food exposure questions, including open-ended food histories and objective yes/no questions about numerous specifi c food items, as well as brand names and purchase locations. Clusters are investigated by using an iterative model in which suspicious exposures identifi ed during initial casepatient interviews are added to the standard interview for subsequent cases [bib_ref] Outbreaks of salmonellosis in Minnesota (1998 through 2006) associated with frozen, microwaveable,..., Smith [/bib_ref] [bib_ref] Restaurant Salmonella Enteritidis outbreak associated with an asymptomatic infected food worker, Hedican [/bib_ref]. Similarly, initial cluster casepatients may be reinterviewed to ensure uniform ascertainment of the suspicious exposures. This iterative approach is used to identify exposures for further evaluation with formal hypothesis testing, product sampling, or product tracing [bib_ref] Outbreaks of salmonellosis in Minnesota (1998 through 2006) associated with frozen, microwaveable,..., Smith [/bib_ref].
A cluster was defi ned as >2 cases of salmonellosis in different households with isolates of the same serovar and PFGE subtype and with specimen collection dates within 2 weeks [bib_ref] Use of molecular subtyping in surveillance for Salmonella enterica serotype Typhimurium, Bender [/bib_ref]. Thus, a single cluster would be ongoing as long as a new isolate was collected within 2 weeks after the most recent isolate in the cluster. A cluster was considered solved if the epidemiologic evaluation of that cluster resulted in the identifi cation of a common source of infection for those cases and consequently the documentation of a confi rmed outbreak. Therefore, the terms solved cluster and confi rmed outbreak are equivalent and used interchangeably.
## Inclusion and exclusion criteria
Laboratory-confi rmed cases of nontyphoidal Salmonella enterica infection among Minnesota residents with specimen collection dates from January 1, 2001, through December 31, 2007, for which isolates were received and subtyped by MDH PHL were included in the study. Isolates not received through routine surveillance (i.e., testing was requested or conducted by MDH as a part of an ongoing investigation) were excluded from the analysis.
Solved clusters were included if they were detected and identifi ed solely on the basis of investigation of cases identifi ed through submission of isolates to MDH for routine laboratory surveillance. Solved clusters for which a call to the MDH foodborne disease hotline (www.health. state.mn.us/divs/idepc/dtopics/foodborne/reporting.html) (e.g., from the public or a healthcare provider) directly contributed to the identifi cation of an outbreak were excluded from analysis. Secondary clusters, defi ned as clusters in which the cases were part of a confi rmed outbreak that had been previously identifi ed, were also excluded from analysis. Clusters that were part of a probable outbreak (an epidemiologic evaluation suggested, but did not confi rm, a common source of infection) were also excluded.
## Study variables
Variables incorporated into the analysis were cluster year, cluster size, cluster case density, cluster serovar, cluster subtype, and cluster serovar diversity. Cluster size was defi ned as the number of cases in each cluster and was categorized into cluster sizes of 2, 3, 4, and >5. For clusters in which a common source was identifi ed, only cases received before the cluster was solved were included. Cluster case density was defi ned as the number of days from receipt date of the fi rst cluster isolate at MDH PHL to the receipt date of the third cluster isolate and was categorized into cluster case densities of 0, 1-7, 8-14, and >14 days [bib_ref] evaluation of cluster investigation criteria, Hedberg [/bib_ref].
Cluster serovar was coded as a categorical variable on the basis of serovar frequency. Serovars representing >20% of all isolates (Typhimurium and Enteritidis) were categorized as very common, those representing 3%-20% (Newport, Heidelberg, and Montevideo) as common, and those representing <3% (all other serovars) as uncommon. The relationship between common and uncommon PFGE subtypes and solving a cluster was examined for serovars Typhimurium and Enteritidis. For serovar Typhimurium, clusters with CDC PFGE subtype designations JPXX01.0003, JPXX01.0410, and JPXX01.0111 (each representing >8% of all Typhimurium isolates) were categorized as common, and all other subtypes were categorized as uncommon. For serovar Enteritidis, clusters with CDC PFGE subtype designations JEGX01.0004 and JEGX01.0030 (each representing >20% of all Enteritidis isolates) were categorized as common, and all other subtypes were categorized as uncommon.
Cluster serovar diversity was examined by categorizing the 17 most frequent serovars into highly clonal or low clonality serovars on the basis of the Simpson diversity index [bib_ref] Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-fi eld gel electrophoresis, and..., Boxrud [/bib_ref]. Serovars with a Simpson index score <0.90 were considered highly clonal, and serovars with a Simpson index score >0.90 were considered to have low clonality. Cluster investigation thresholds were examined by comparing the percentage of outbreak clusters meeting a threshold, cluster investigation positive predictive value, and estimated interview burden in hours per year for various investigational thresholds. The time required to interview each patient with a Salmonella infection by using the MDH standard questionnaire was recorded for a 6-month period in 2008, and the median interview time was calculated.
# Statistical analysis
A descriptive analysis was conducted to characterize the frequency of Salmonella serovars and subtypes. Mantel-Haenszel χ 2 test for trend was used to characterize tem-poral trends in the number of Salmonella clusters that were solved. Two-sided Wilcoxon rank-sum tests were used to compare the median cluster size and cluster density of point source and non-point source outbreaks. Univariate analysis was performed to calculate odds ratios (ORs) and 95% confi dence intervals (CIs) characterizing the crude associations between Salmonella cluster serovar, cluster PFGE subtype, cluster serovar diversity, cluster size, and cluster case density and a cluster being solved. Mantel-Haenszel χ 2 tests for trend and interaction terms were used to investigate the linear nature of the relationship between cluster size, cluster case density, and the outcome. SAS software version 9.1 (SAS Institute, Cary, NC, USA) was used for descriptive and univariate analysis. An α value <0.05 was considered signifi cant.
# Results
During 2001-2007, a total of 4,154 nontyphoidal Salmonella isolates from Minnesota residents were received at MDH through routine surveillance; they represented 98% of reported Salmonella cases (n = 4,235, incidence 11.78 cases/100,000 person-years). PFGE subtyping was performed for 4,018 (97%) isolates, which were included in the study. Among these isolates, 194 Salmonella serovars were observed. The 6 most common S. enterica serovars were Typhimurium, 1,004 (25%); Enteritidis, 822 (20.5%); Newport, 314 (7.8%); Heidelberg, 223 (5.6%); Montevideo, 121 (3.0%); and Saintpaul, 81 (2.0%) [fig_ref] Figure 1: Frequency of the 17 most common Salmonella enterica serovars among clinical case... [/fig_ref].
The frequency of PFGE subtypes was examined in detail for serovars Typhimurium and Enteritidis. The 3 most common subtypes of serovar Typhimurium were JPXX01.0003, 107 (11%); JPXX01.0410, 87 (9%); and JPXX01.0111, 85 (8%). The 3 most common subtypes of serovar Enteritidis were JEGX01.0004, 309 (38%); JEGX01.0030, 181(22%); and JEGX01.0005, 106 (13%).
Serovar diversity was examined by comparing Simpson diversity indices for the 17 most frequent serovars [fig_ref] Table 1: Salmonella enterica serovar diversity identified by pulsed-field gel electrophoresis among case isolates... [/fig_ref]. Javiana, Newport, Agona, Infantis, and Typhimurium were low clonality serovars. Heidelberg, Hadar, Enteritidis, Thompson, and I 4,5,12:I:-were highly clonal serovars.
## Cluster and outbreak characteristics
During 2001-2007, a total of 376 Salmonella PFGE clusters were detected; they represented 1,399 (35%) isolates. Thirty-two (8.5%) clusters were excluded from analysis (21 secondary clusters, 7 clusters in which a hotline call directly contributed to identifi cation of an outbreak, and 4 probable outbreak clusters). Forty-three (12.5%) of the 344 clusters included in the analysis were solved.
During 2001-2007, a total of 65 confi rmed Salmonella outbreaks involving Minnesota cases were identifi ed; these represented 502 (12.5%) isolates. Twenty-two (34%) outbreaks were excluded from analysis (6 were multistate outbreaks in which only 1 case was identifi ed in Minnesota; in 7 outbreaks, a hotline call contributed to identifi cation of the outbreak; 1 was an outbreak was not detected by PFGE; 4 were outbreaks that did not have cases that met the cluster defi nition; and 4 outbreaks were considered probable). The remaining 43 outbreaks, representing 287 (7%) isolates, were included in the analysis and were composed of 35 foodborne, 6 person-to-person, and 2 animal contact outbreaks. Of these 43 outbreaks, 30 (70%) involved 1 facility (restaurant, daycare center, school) or event and therefore were classifi ed as point source. Thirteen (30%) involved commercially distributed food items at multiple points of sale (grocery stores, restaurants) and therefore were classifi ed as non-point source. The median cluster size of point source outbreaks was 3 cases, and the median cluster size of non-point source outbreaks was 5 cases (p<0.01, by Wilcoxon rank-sum test). The median cluster density was 6 days for point source and non-point source outbreaks (p = 0.74 by Wilcoxon rank-sum test).
## Temporal trends
During the study period, the median number of Salmonella isolates subtyped per year was 567 (range 507-662 isolates). The median number of Salmonella clusters per year was 50 (range 44-57 clusters). The median number of confi rmed Salmonella outbreaks per year was 6 (range 4-8 outbreaks). There were no statistically signifi cant trends in the proportion of Salmonella clusters that resulted in identifi cation of a confi rmed outbreak (p = 0.20) [fig_ref] Figure 2: Temporal trends in number of Salmonella enterica isolates, number of clusters, and... [/fig_ref]. Clusters of the common Salmonella serovars Newport, Heidelberg, and Montevideo had 2.7× higher odds of being solved than did clusters of the very common serovars Enteritidis and Typhimurium [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. The proportion of uncommon serovar clusters that were solved did not differ signifi cantly from the proportion of very common or common serovar clusters that were solved [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. Low clonality serovar clusters were not signifi cantly more likely to be solved than highly clonal serovar clusters (OR 1.6, 95% CI 0.8-3.1).
## Cluster subtype
No signifi cant associations between the subtype frequency of a cluster and a cluster being solved were observed. Uncommon serovar Enteritidis subtype clusters were not signifi cantly more likely to be solved than were common clusters (OR 1.4, 95% CI 0.4-5.1). Uncommon serovar Typhimurium subtype clusters were not significantly more likely to be solved than were common clusters (OR 0.9, 95% CI 0.3-3.2).
## Cluster size
The probability of a cluster being solved increased signifi cantly as the number of cluster cases increased (Mantel-Haenszel χ 2 for trend 13.7, p<0.001) [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. The odds of solving a cluster of >5 cases were 3.8× higher than the odds of solving a cluster of 2 cases. Clusters of 4 cases were 3.9× more likely to be solved than were clusters of 2 cases. Twenty-four percent of clusters with >4 cases were solved [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. Clusters of 3 cases were 2.1× more likely to be solved than clusters of 2 cases, but the difference was not statistically signifi cant. There was statistical evidence of a nonlinear relationship between cluster size and solving the cluster (Wald χ 2 for interaction 5.0, p = 0.03). The dose response between cluster size and solving a cluster plateaued after a cluster size of 4.
## Cluster case density
The proportion of clusters solved increased significantly as the density of cluster cases increased (Mantel-Haenszel χ 2 for trend, 12.7, p<0.001) [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. The odds of solving a cluster if the fi rst 3 case isolates were received on the same day were 25.8× higher than the odds of solving a cluster in which the fi rst 3 case isolates were received during a period >14 days [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. The odds of solving a cluster if the fi rst 3 case isolates were received within 1-7 days were 5.0× higher than the odds of solving a cluster in which the fi rst 3 case isolates were received during a period >14 days. Clusters in which the fi rst 3 case isolates were received within 8-14 days were 2.8× more likely to be solved than clusters in which the fi rst 3 case isolates were received during a period >14 days, but the difference was not statistically signifi cant [fig_ref] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and... [/fig_ref]. There was statistical evidence of a nonlinear relationship between cluster case density and solving the cluster (Wald χ 2 for interaction, 6.96, p<0.01).
## Cluster investigation threshold
During June-December 2008, 10 MDH staff interviewed 214 persons with Salmonella infections and recorded the time required to complete the MDH standard [fig_ref] Figure 1: Frequency of the 17 most common Salmonella enterica serovars among clinical case... [/fig_ref] , where n is number of isolates of each subtype and N is total number of isolates of a serovar. A value of 1 indicates infinite diversity, and a value of 0 indicates no diversity.
questionnaire. Interview times did not vary between interviewers. The median interview time was 27 minutes (range 13-56 minutes). Therefore, conducting standard interviews of all cases in the 344 clusters of >2 cases (n = 1,182 [31%] cases) required an estimated 76 interview hours/year. This threshold detected all 43 outbreaks identifi ed through routine laboratory surveillance during the study period and resulted in a cluster investigation positive predictive value (percentage of clusters investigated that were solved) of 13% [fig_ref] Table 3: Comparison of Salmonella enterica cluster investigation thresholds, Minnesota, USA, 2001-2007* *PPV, positive... [/fig_ref]. Other cluster investigation thresholds had outbreak detection sensitivities of 53%-81% and positive predictive values of 23%-28% [fig_ref] Table 3: Comparison of Salmonella enterica cluster investigation thresholds, Minnesota, USA, 2001-2007* *PPV, positive... [/fig_ref].
# Discussion
During the study period, 344 Salmonella PFGE clusters were identifi ed and 43 (13%) were solved. Cluster size and cluster case density were the most useful predictors of a cluster being solved. The proportion of clusters that were solved increased as the number of cases in the cluster increased (up to 4 cases). The association was not linear and the percentage solved did not increase further for clusters with >5 cases. The observed association is logical because as the number of cluster cases increases, the amount of epidemiologic data available for evaluation also increases. Our results suggest that public health offi cials should not wait to investigate Salmonella clusters if >4 cluster cases have been received.
The ability to solve a cluster of cases of Salmonella infection was also strongly associated with the density of the cluster cases. The proportion of clusters that were solved increased as the density of the cluster cases increased, but this relationship was not linear. This association is also logical. Dense clusters increase the likelihood that the cluster cases are epidemiologically linked rather than unrelated sporadic cases. In addition, dense clusters also likely signal larger outbreaks. Our results demonstrated a clear increase in the success of solving clusters in which the fi rst 3 case isolates were received within 7 days.
In theory, PFGE subtyping is less useful for recognizing clusters of unusual serovars worth investigating. In the current study, clusters of the common serovars Newport, Montevideo, and Heidelberg were statistically Emerging Infectious Diseases - www.cdc.gov/eid - Vol. [bib_ref] evaluation of cluster investigation criteria, Hedberg [/bib_ref] more likely to be solved than clusters of the very common serovars Enteritidis and Typhimurium. However, clusters of uncommon serovars were not more likely to be solved than were clusters of common or very common serovars. It has been suggested that uncommon serovar clusters may be associated with uncommon food vehicles, which makes them more diffi cult to solve by using standard methods [bib_ref] The growing burden of foodborne outbreaks due to contaminated fresh produce: risks..., Lynch [/bib_ref]. The relationship between serovar frequency and the likelihood of solving a cluster is unclear and warrants further study. The limited number of solved clusters prevented multivariate analysis from being used to characterize the independent effect of predictors and possible effect modifi cation between predictors. However, comparing the magnitude of the estimated effect of cluster size and cluster case density suggests that cluster case density may be a more useful predictor of a cluster being solved.
The 22 confi rmed outbreaks that were excluded from the analysis demonstrate the value for national collaboration such as PulseNet and use of outbreak detection methods in addition to PFGE clustering within a given state. Six outbreaks were solved in which Minnesota only had 1 case, which demonstrated the utility of molecular subtyping in detecting geographically dispersed outbreaks. For 7 confi rmed outbreaks, a call placed to the MDH foodborne illness hotline contributed to identifi cation of the outbreak and demonstrated the utility of complaint systems in detecting outbreaks.
Interviewing all persons with Salmonella infection required a median of 27 minutes per person with Salmonella infection when the MDH standard questionnaire was used. By extrapolation, MDH staff spent ≈244 hours/year conducting routine interviews of persons with Salmonella infections. This fi gure does not include time spent attempting to reach persons, gathering demographic information from clinicians, or reinterviewing persons for cluster investigations. We recommend interviewing all persons with Salmonella infection and investigating all PFGE clusters to identify as many outbreaks as possible. However, many health departments do not have the resources to interview all persons with Salmonella infection or investigate all small clusters. Rather, they must balance the time required for these efforts and the ability to detect outbreaks [bib_ref] Capacity of state and territorial health agencies to prevent foodborne illness, Hoffman [/bib_ref].
Incorporating a cluster investigation threshold on the basis of cluster size and cluster case density can decrease the number of unsuccessful cluster investigations and conserve public health resources. However, this approach would also reduce the number of outbreaks that would be identifi ed. One reason for this fi nding is that outbreaks that are manifested as smaller, less dense clusters would not be investigated. Another potential disadvantage of a cluster threshold approach is that delay of interviews until a cluster is solved can decrease the quality of exposure information obtained and therefore the likelihood that the cluster will be solved [bib_ref] Timeliness of enteric disease surveillance in 6 US states, Hedberg [/bib_ref].
Four confi rmed outbreaks during the study did not meet the cluster defi nition, and many confi rmed outbreaks had cases that were outside the cluster defi nition. This fi nding is an important reminder that lack of temporal clustering does not eliminate the possibility of an outbreak. Increasing the period covered by a cluster defi nition will yield the benefi t of solving more outbreaks. However, more resources will be expended conducting unsuccessful cluster investigations. The results of this study suggest that the use of a 2-week cluster window is suffi ciently sensitive to detect most outbreaks. However, in practice, MDH epidemiologists do not use a strict 2-week cluster window when investigating clusters. Instead, all persons with Salmonella infection are interviewed and cases with matching PFGE patterns are often compared even if the second case is received >2 weeks after the fi rst case.
The potential utility of the cluster investigation thresholds reported is based on the characteristics of the population of Minnesota and MDH surveillance methods: conducting real-time PFGE subtyping of all Salmonella isolates, interviewing all case-patients in real time by using a detailed exposure questionnaire from a central location for the entire state, and investigating clusters by using an iterative model [bib_ref] Outbreaks of salmonellosis in Minnesota (1998 through 2006) associated with frozen, microwaveable,..., Smith [/bib_ref] [bib_ref] Restaurant Salmonella Enteritidis outbreak associated with an asymptomatic infected food worker, Hedican [/bib_ref]. These factors aid in the timeliness of outbreak detection and investigation in Minnesota. These re- sults may not be applicable in jurisdictions in which PFGE is not conducted in real time or batching of PFGE isolates occurs. Additional studies at the national level and in other states are needed to understand surveillance characteristics in other states and determine useful predictors of multistate clusters being solved. Although successful cluster investigations will depend on the experience and ability of public health staff involved, this study demonstrates the increased probability of a cluster being solved as the number of cases in a cluster increases and as the cluster density increases. Specifi cally, investigation of PFGE clusters of >4 Salmonella case isolates and clusters in which the fi rst 3 cases were received at the MDH PHL within 1 week yielded a major benefi t in terms of outbreak identifi cation. These results establish a benchmark for surveillance of Salmonella infections, and may provide a basis for investigating clusters of Salmonella cases for public health agencies with limited resources.
[fig] Figure 1: Frequency of the 17 most common Salmonella enterica serovars among clinical case isolates submitted to the Minnesota Department of Health, 2001-2007. Typ, Typhimurium; Ent, Enteritidis; New, Newport; Hei, Heidelberg; Mon, Montevideo; Sai, Saintpaul; S.I4, S.I 4,5,12:I: -; Inf, Infantis, Ago, Agona; Mue, Muenchen; Ora, Oranienburo; Par, Paratyphi B var. L; Tho, Thompson; Bra, Braenderup; Had, Hadar; Jav, Javiana; Ana, Anatum. [/fig]
[fig] Figure 2: Temporal trends in number of Salmonella enterica isolates, number of clusters, and number of clusters solved (i.e., result in identifi cation of a confi rmed outbreak), Minnesota, USA, 2001-2007. [/fig]
[table] Table 1: Salmonella enterica serovar diversity identified by pulsed-field gel electrophoresis among case isolates submitted to the Minnesota Department of Health, 2001-2007* [/table]
[table] Table 2: Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and cluster being solved, [/table]
[table] Table 3: Comparison of Salmonella enterica cluster investigation thresholds, Minnesota, USA, 2001-2007* *PPV, positive predictive value.†A total of 215 isolates associated with excluded clusters were removed from study isolate total (n = 4,018). ‡Based on a 27-min median interview time per case-patient. §Density defined as the number of days from receipt of first cluster case isolate to third case isolate received at the Minnesota Department of Health Public Health Laboratory. [/table]
|
Antifungal activity of an artificial peptide aptamer SNP-D4 against Fusarium oxysporum
Fusarium oxysporum f. sp. cubense (FOC4) is a pathogen of banana fusarium wilt, which is a serious problem that has plagued the tropical banana industry for many years. The pathogenic mechanism is complex and unclear, so the prevention and control in agricultural production applications is ineffective. SNP-D4, an artificial peptide aptamer, was identified and specifically inhibited FOC4. To evaluate the efficacy of SNP-D4, FoC4 spores were treated with purified SNP-D4 to calculate the germination and fungicide rates. Damage of FOC4 spores was observed by staining with propidium iodide (PI). Eight proteins of FOC4 were identified to have high affinity for SNP-D4 by a pull-down method combined with Q-Exactive mass spectrometry. Of these eight proteins, A0A5C6SPC6, the aldehyde dehydrogenase of FOC4, was selected as an example to scrutinize the interaction sites with SNP-D4. Molecular docking revealed that Thr66 on the peptide loop of SNP-D4 bound with Tyr437 near the catalytic center of A0A5C6SPC6. Subsequently 42 spore proteins which exhibited associations with the eight proteins were retrieved for proteinprotein interaction analysis, demonstrating that SNP-D4 interfered with pathways including 'translation', 'folding, sorting and degradation', 'transcription', 'signal transduction' and 'cell growth and death', eventually causing the inhibition of growth of FOC4. This study not only investigated the possible pathogenic mechanism of FOC4, but also provided a potential antifungal agent SNP-D4 for use in the control of banana wilt disease.
# Introduction
Banana fusarium wilt is a soil-borne vascular disease caused by Fusarium oxysporum f. sp. cubense (FOC4). It infects banana roots and causes devastating damage to the banana industry [bib_ref] Fusarium wilt of banana is caused by several pathogens referred to as..., Ploetz [/bib_ref]. The virulence and pathogenicity of FOC4 is far higher than other banana pathogens [bib_ref] A. Recent occurrence of Fusarium oxysporum f. sp. cubense tropical race 4..., Molina [/bib_ref]. FOC4 has strong tolerance to stress and survives in soil for 40 years [bib_ref] Knockout on Fusarium oxysporum f, Olivia [/bib_ref] , causing extensive destruction of fields. FOC4 adheres to root epidermal cells in the form of mycelium, macroconidia, and small conidia, and destroys the membrane structure of roots by secreting a series of toxins. Subsequently FOC4 directly invades plant vascular bundles and induces vascular bundle ligation [bib_ref] Panama disease: cell wall reinforcement in banana roots in response to elicitors..., De Ascensao [/bib_ref]. There is still no effective protection against banana fusarium wilt [bib_ref] Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp...., Guo [/bib_ref]. The relevant prevention strategies of FOC4 are mainly dependent on the screening of chemical pesticides, however, the excessive dependence and abuse of chemical pesticides brings great harm to the environment and can even endanger human health. So, environmentally friendly alternatives need to be developed for the control of FOC4.
Peptide aptamers (or pepaptamers) are short peptides that bind to target proteins with high specificity and affinity [bib_ref] Peptide aptamers: specific inhibitors of protein function, Hoppe-Seyler [/bib_ref]. Their structures consist of a stable scaffold protein and a variable peptide loop with both ends immobilized on the scaffold protein [bib_ref] Peptide aptamer libraries, Borghouts [/bib_ref]. The variable peptide is usually composed of 8-20 random amino acids, which recognizes and binds to the specific target protein. The development of the artificial pepaptamers are currently used as biological antibacterial agents as they have the following three advantages. First, its target specificity is extremely high so that the pepaptamer distinguishes the different members from the same protein family. Second, its molecular weight is smaller than an antibody, while the binding ability is similar to that of the antibody. Third, it folds into a stable tertiary conformation in vivo, which is more conducive to maintain the higher biological activity than general polypeptides.
At present, the pepaptamer has been exploited in tumor therapy [bib_ref] The Hsp70 inhibiting peptide aptamer A17 potentiates radiosensitization of tumor cells by..., Schilling [/bib_ref] , food safety [bib_ref] Aptamer and its potential applications for food safety, Dong [/bib_ref] , and plant biotechnology [bib_ref] Use of peptide aptamers, cationic peptides and artificial zinc finger proteins to..., Sera [/bib_ref]. The pepaptamer is widely used for the controls of plant viruses, which hinders the viral cycle in the infected plants. Using geminivirus replication protein (Rep) as the target, a pepaptamer library was screened by yeast two-hybrid analysis, and then the pepaptamer coding sequence was transformed into tomato plants. Transgenic plants were resistant to yellow leaf curl virus and tomato mottle virus [bib_ref] Peptide aptamers that bind to geminivirus replication proteins confer a resistance phenotype..., Reyes [/bib_ref].
The use of pepaptamers to control plant fungal diseases remains to be explored. Previously we constructed a pepaptamer library using Staphylococcal nuclease (SNase, SN) as the scaffold, wherein 16 random amino acids were inserted into the plasmid pTRG (abbreviated as pTRG-SNP). After the plasmids were transformed into E. coli, they expressed the fusions of SNase and various peptides acting with diverse structures and functions (abbreviated as SNPs, hereinafter) . One artificial peptide aptamer, designated as SNP-D4, was tested to interact with calmodulin for inhibiting Magnaporthe oryzae [bib_ref] Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe..., Xu [/bib_ref]. In this study, SNP-D4 was found capable of specifically impeding the spore germination of FOC4. Afterwards, a total of eight proteins were identified to directly interact with SNP-D4. Of these eight proteins, A0A5C6SPC6, the aldehyde dehydrogenase of FOC4, was chosen as an instance to examine the interaction sites with SNP-D4. Subsequently 42 spore proteins which exhibited high affinities with the eight proteins were retrieved for protein-protein interaction analysis, demonstrating that SNP-D4 interfered in the pathways including 'translation', 'folding, sorting and degradation', 'transcription', 'signal transduction' and 'cell growth and death', eventually resulting in the inhibition of growth of FOC4.
This study helps to further understand the pathogenic mechanism of FOC4 and contribute to the development of transgenic plants or an antifungal agent against FOC4, bringing a new breakthrough in the control of banana wilt disease.
# Materials and methods
# Materials
## Plasmids and strains
The peptide aptamer library pTRG-SNP was constructed , which expressed both scaffold protein and the random exposed loop of 16 random amino acids. The plasmid pTRG-SN only containing the scaffold was used as the negative control. The vector pET-28a was used to express SNP or SN for antifungal assays.
Escherichia coli XL1-Blue MR was used as the host for checking the activity of pTRG-SN expression. E. coli BL21(DE3) strain was chosen as the host for pET-28a derivative expression. The pathogen Fusarium oxysporum f. sp. cubense tropical race4 (FOC4) strain was stored at 4 C for use.
## Medium
LB liquid medium contained 1% tryptone, 0.5% yeast extract, and 0.5% NaCl. LB solid medium was made by supplementing 1.5% agarose into liquid medium. LB medium was adjusted to pH 7.0 and sterilized at 121 C for 20 min. Potato Dextrose Broth (PDB) medium was purchased from Solarbio (Solarbio Science & Technology Co., Ltd., Beijing, China). PDA medium was appended with 3.7% agar into PDB. PDA and PDB media were adjusted at pH 6.5 and sterilized at 115 C for 20 min.
# Methods
## Selection of peptide aptamer for the inhibition of foc4
The plasmids pTRG-SNP was transformed into E. coli XL1-Blue MR, and the clones were picked randomly on the plate and cultured at 37 C overnight in LB medium supplemented with 5 mM IPTG. The cells were then harvested and suspended in 10 mM PBS (pH 7.4) for ultrasonic disruption. The lysate was mixed with 10 mL of 4 × 10 6 cells/mL spores of FOC4, and the percent of spore germination was calculated after 6 h incubation at 28 C. The germination rate was counted using the following formula: Germination rate = (germinated spores/total spores) × 100%. The colonies conferring the inhibition activity to FOC4 were selected and the extracted plasmids were sent for DNA sequencing [bib_ref] Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe..., Xu [/bib_ref].
## Prokaryotic expression
The functional peptide aptamer SNP-D4 was inserted into pET-28a vector and transformed into E. coli BL21 . The final concentration of 100 mM IPTG (isopropyl β-D-thiogalactoside) was added for induction after the clone was cultured until the optical density (OD) value of 0.4 at 580 nm wavelength. The lysate was loaded onto the nickel column for purification with a gradually increased elution using imidazole . Likewise, the scaffold protein SN was inserted into pET-28a which enabled His-tagged expression as the negative control.
## Antifungal assay
The purified SNP-D4 was diluted to the ultimate concentrations of 2-8 mM. Each 10 mL of purified protein and 10 mL of PDB spore suspension with a final concentration of 4 × 10 6 cells/mL were mixed and incubated at 28 C for 12 h. Both PBS buffer and SN served as the controls. The spore germination was observed under an optical microscope with a 40× objective lens. Spore germination rates were calculated as described above.
## Fungicidal assay
The mixtures including both SNP-D4 and 3 × 10 4 cells/mL spore suspension were cultured in PDB medium at 28 C for 12 h, followed by plating on PDA medium for an additional 48 h. The number of colonies growing on the plate was observed and the fungicidal rate was calculated as follows: Fungicidal rate % = [(number of negative control coloniesnumber of treated colonies)/number of negative control colonies] × 100%. Each treatment was repeated three times.
## Pi staining
Both 5 mM SNP-D4 and 2 × 10 6 cell/mL spores were incubated in the volume of 800 mL PBS at 28 C for 3 h. The spores were collected by centrifugation at 14,000 rpm for 10 min and resuspended in 200 mL PBS. Prior to washing with 800 mL PBS buffer three times, the spores were incubated with 20 mL PI (100 mg/mL) for 5 min. Thereafter the spores were observed whether they were damaged with a fluorescence microscope equipped with 535 nm excitation and 615 nm emission light sources [bib_ref] Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde..., Chen [/bib_ref] [bib_ref] Measuring cell death by propidium iodide uptake and flow cytometry, Crowley [/bib_ref].
## Pull-down assay and mass spectrometry
The purified SNP D4 protein was incubated with unpurified spore lysate to isolate the interacting proteins [bib_ref] Determination of Rab5 activity in the cell by effector pull-down assay, Qi [/bib_ref]. FOC4 was cultured in PDB medium, and the hyphae was filtered by gauze to collect the spores. Subsequently the spores were disrupted with liquid nitrogen and resuspended in 50 mM Tris-HCl (pH 7.0) supplemented with 0.1% Triton X-100. The supernatant was incubated with SNP-D4 for 2 h and loaded on the nickel column. After the nonspecific binding proteins were eluted with 20 mM imidazole, the target proteins interacting with SNP-D4 were pulled down eluted using 80 mM imidazole. Consequently, the elution was performed by Q-Exactive Mass Spectrometry analysis (Thermo Fisher Scientific, Waltham, MA, USA).
## Molecular docking model of snp-d4 and spore proteins
To obtain the interactive sites, the three-dimensional model of the protein was created by I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Molecular docking was performed using Hex 8.0.0 to evaluate and optimize the docking model. The interactive sites between the ligands and receptors molecules, and the formations of hydrogen bonds were analyzed using the Discovery Studio software.
## Prediction of protein-protein interactions
The interolog method was used to infer the interactions between spore proteins. Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Escherichia coli, and Homo sapiens were selected as reference organisms. Their protein sequences were downloaded from the UniProt (UniProt, 2019) database, and the experimentally verified protein-protein interactions (PPIs) were collected from the BioGrid [bib_ref] The BioGRID interaction database: 2019 update, Oughtred [/bib_ref] , IntAct [bib_ref] The MIntAct project-IntAct as a common curation platform for 11 molecular interaction..., Orchard [/bib_ref] , DIP [bib_ref] The database of interacting proteins: 2004 update, Salwinski [/bib_ref] and MINT [bib_ref] MINT, the molecular interaction database: 2012 update, Licata [/bib_ref] databases. In addition, A. thaliana and H. sapiens PPIs were also collected from the TAIR [bib_ref] The Arabidopsis information resource: Making and mining the "gold standard" annotated reference..., Berardini [/bib_ref] and HPRD [bib_ref] Human protein reference database and human proteinpedia as resources for phosphoproteome analysis, Goel [/bib_ref] databases, respectively. Orthologs between FOC4 and the six reference organisms were identified using Inparanoid Version 4.1 [bib_ref] InParanoid 8: orthology analysis between 273 proteomes, mostly eukaryotic, Sonnhammer [/bib_ref] , of which the ones with an Inparanoid score < 1.0 were removed. The experimentally verified PPIs of the six reference organisms were identified in FOC4 on the basis of the orthologs, and eventually the interactions between spore proteins were obtained.
# Statistical analysis
Each treatment was performed in triplicate under the same conditions. The experimental data were documented as the mean and standard deviation (mean ± SD). P values of less than or equal to 0.05 or 0.01 represented significant or extremely significant differences, respectively.
# Results
## Identification of peptide aptamers for the inhibition of foc4
Among 150 peptide aptamer clones, five candidates were found to have inhibitory effects on FOC4 spores, and SNP-D4 had the best inhibitory effect. Both bacterial protein extract of the strain expressing pTRG-SN and PBS treatment promoted significant mycelium growth, indicating that these had no inhibitory effects on the germination of spores. Most of the spores in the SNP-D4 treatment did not germinate, and the hypha length of a few germinated spores were significantly shorter than that of the negative control; The inhibitory effect of SNP-D4 was almost equal to that of 0.3% hymexazol [fig_ref] Figure 1: Microscopic analysis of antifungal activity of SNP-D4 against FOC4 [/fig_ref] , indicating that SNP-D4 itself had a certain inhibitory effect on the germination of spores. The sequencing results showed that the peptide aptamer encoded an exposed loop except for the scaffold protein SN, whose amino acid sequence consisted of "VTFLVNTYPNGVQSRA".
## Antifungal assay of the functional peptide aptamer
The peptide aptamer SNP-D4 and the scaffold protein SN were purified using nickel column . To analyze the inhibition activity on the spores of FOC4, SNP-D4 or SN was diluted to different concentrations and incubated with the spores. The spore germination of FOC4 was significantly inhibited (p < 0.05) at 2 mM SNP-D4, and the extremely significant inhibition occurred when elevating the concentration of peptide aptamer to 4 or 8 mM; an antifungal effect was not found by altering SN concentrations .
## Fungicidal assay on snp-d4
To verify whether SNP-D4 had fungicidal activity, the spores of FOC4 were treated with SNP-D4 and then plated on nutrient-rich PDA medium. The positive control, 0.3% pesticidehymexazol, caused 80% of growth defects on spores, while the scaffold protein SN incurred 20% of the abnormal growth without the concentration dose dependence. SNP-D4 showed fungicidal effects on the spores of FOC4 , i.e., in comparison with the SN groups, the fungicidal rate had a significant difference (p < 0.05) at 4 mM SNP-D4, and it reached extremely significant levels (p < 0.01) when treated with 8 mM and 10 mM SNP-D4.
## Snp-d4 damages foc4 spores
The method of PI staining is used to detect cell death since it enters the interior of the cell and intercalates into double-stranded DNA when the cell membrane is damaged [bib_ref] The new antimicrobial peptide SpHyastatin from the mud crab scylla paramamosain with..., Shan [/bib_ref]. The spores were stained with PI dye to determine the survival status of spores. FOC4 spores treated with PBS buffer and SN did not emit red light using fluorescence microscopy, indicating that they had no damage. However, about 50% of the FOC4 spores treated with SNP-D4 (10 mM) emitted red fluorescence, suggesting that the spores were severely damaged and died [fig_ref] Figure 4: SNP-D4 destroyed FOC4 spores [/fig_ref]. Identification of spore proteins interacting with SNP-D4 by mass spectrometry
The collected elution was analyzed by Mass Spectrometry to identify the unique proteins interacting with SNP-D4. A total of eight proteins were identified by searching in the UniProt database. Seven of the proteins were classified with exact functions, while the other one was unknown [fig_ref] Figure 1: Microscopic analysis of antifungal activity of SNP-D4 against FOC4 [/fig_ref].
## Verification of snp-d4 and the protein a0a5c6spc6 interaction
To further validate the association between SNP-D4 and the protein A0A5C6SPC6, a three-dimensional model of the protein was constructed, and molecular docking was developed. Since the crystals of SNP-D4 and FOC4 A0A5C6SPC6 were not found in the PDB database (Protein data bank, https://www.rcsb.org/), three-dimensional structures were predicted by I-TASSER, yielding the five greatest possibilities of structure. The highest score model of SNP-D4 had C-score of −1.34, Estimated TM-score of 0.55 ± 0.15, and Estimated RMSD of 8.2 ± 4.5 Å [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref] ; the highest score model of FOC4 A0A5C6SPC6 had C-score of −0.10, Estimated TM-score of 0.70 ± 0.12, and Estimated RMSD of 7.6 ± 4.3 Å [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref] ; all the values met the requirements for further structure analysis. By I-TASSER homology comparison of the crystal structure of A0A5C6SPC6, the enzymatic site of A0A5C6SPC6 was Gly424, which formed a protein active pocket with other amino acids in the region, as shown in [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref]. Molecular docking between SNP-D4 and A0A5C6SPC6 was achieved via Hex 8.0.0. E total of the best conformation was −832.66, and the estimated energy was below 0 indicating that the docking result was reliable [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref]. Through the molecular docking model (Figs. 5F and 5G), the residue Thr66 on the peptide loop of SNP-D4 formed hydrogen bonds with Tyr437 in A0A5C6SPC6 at 1.94 Å. The amino acids Arg79 and Thr66 in SNP-D4 formed two hydrogen bonds with distances of 2.35 Å and 2.54 Å, respectively, stabilizing the structure of the peptide loop and helping SNP-D4 to bind with A0A5C6SPC6. Although there was competition for space between Pro421 and Tyr437 in A0A5C6SPC6, the hydrogen bond between Thr66 in SNP-D4 and Tyr437 in A0A5C6SPC6 was much stronger, and the bonds between Arg79 and Thr66 in SNP-D4 could assist in binding. Besides, the residues Ser420 and Pro421 in the catalytic active center region of A0A5C6SPC6, were close to the crucial site Tyr437 in A0A5C6SPC6. SNP-D4 might contend with Tyr437 at the entrance of the active pocket in A0A5C6SPC6, preventing the substrate from entry. Therefore, SNP-D4 could achieve an antifungal effect.
## Protein-protein interactions effect on the functional pathways in foc4
Two of eight proteins networking with SNP-D4, A0A5C6SNW8 and A0A559KXS2, had no interaction partners in FOC4 based on the interolog method. The remaining six proteins were associated with 42 spore proteins, forming 89 PPIs [fig_ref] Figure 1: Microscopic analysis of antifungal activity of SNP-D4 against FOC4 [/fig_ref]. The 42 spore proteins were involved in 'translation', 'folding, sorting and degradation', 'transcription', 'signal transduction' and 'cell growth and death' pathways by KEGG Automatic Annotation Server (https://www.genome.jp/kaas-bin/kaas_main). In the six proteins networking with SNP-D4, A0A559LNA3, A0A5C6SPC6, A0A5C6TEI9 were
# Discussion
Banana wilt disease is one of the important fungal diseases that endangers the banana industry and has long attracted people's attention [bib_ref] Fusarium wilt of banana is caused by several pathogens referred to as..., Ploetz [/bib_ref]. With the rapid mutation of pathogenic fungi, traditional control methods have been challenged. To effectively prevent the blight that is harmful to the banana industry, it is still necessary to explore new and effective environmental protection techniques. Peptide aptamers, as a new type of biomaterial, are increasingly being applied in drug development and as antiviral agents in biological pesticides [bib_ref] Peptide aptamers that bind to a geminivirus replication protein interfere with viral..., Lopez-Ochoa [/bib_ref]. The peptide aptamers are screened for the purpose of interfering with virus function such as the assembly of viral DNA replication complexes [bib_ref] Peptide aptamers that bind to geminivirus replication proteins confer a resistance phenotype..., Reyes [/bib_ref]. Previously, the peptide aptamer SNP-D4 was verified to target calmodulin for the growth inhibition of Magnaporthe oryzae [bib_ref] Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe..., Xu [/bib_ref]. In this study, SNP-D4 was identified to specifically repress the pathogen of Fusarium oxysporum f. sp. cubense. To evaluate the antifungal activity, SNP-D4 was expressed in E. coli, purified, and the inhibitory effect on F. oxysporum was performed, revealing that the spore germination of F. oxysporum was dependent on the concentration of SNP-D4 . Furthermore, the functional region of the peptide aptamer was the variable peptide segment, not the scaffold protein (Figs. 5A and 5B). A fungicide assay was performed, which determined that SNP-D4 effectively killed pathogenic spores . Besides, PI staining experiment revealed that the outer membrane of F. oxysporum spores was damaged after treating with SNP-D4 [fig_ref] Figure 4: SNP-D4 destroyed FOC4 spores [/fig_ref].
To figure out the action of SNP-D4 on FOC4, a pull down assay was executed to capture the target proteins in spores using the purified SNP-D4 with a histidine tag as the prey. The proteins binding with SNP-D4 were separated and analyzed for mass spectrometry. Eight proteins were identified to interact characteristically with SNP-D4 in FOC4 [fig_ref] Table 1: The proteins interacting with SNP-D4 by mass spectrometry identification [/fig_ref]. Of these eight proteins, A0A5C6SPC6, which is annotated as acetaldehyde dehydrogenase, contributes to the metabolism of alcohol, oxidizing acetaldehyde and converting it into acetic acid [bib_ref] Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde..., Chen [/bib_ref] [bib_ref] How does fusarium oxysporum sense and respond to nicotinaldehyde, an inhibitor of..., Anand [/bib_ref]. Aldehydes can inhibit spore germination and have sporicidal activity, which is one of the key factors leading to spore death [bib_ref] Antibotulinal properties of selected aromatic and aliphatic aldehydes, Bowles [/bib_ref]. The inhibition of acetaldehyde dehydrogenase can cause the accumulation of the intermediate product acetaldehyde, which results in biological poisoning and even death [bib_ref] Alcohol and medication interactions, Weathermon [/bib_ref]. So, aldehyde dehydrogenase has been considered as the target of some antibiotics [bib_ref] Alcohol and medication interactions, Weathermon [/bib_ref]. In order to evaluate the credibility of the interaction, [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref] (continued) (near the catalytic center of A0A5C6SPC6) via Thr66 on the peptide loop. The red atoms and structure represented the peptide aptamer scaffold protein of SNP-D4, the green atoms and structure represented the peptide loop of SNP-D4, the purple atoms and structure represented A0A5C6SPC6 protein, the blue atoms and structure represented possible binding sites, the yellow atoms represented the predicted active site.
Full-size DOI: 10.7717/peerj.12756/ [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref] A0A5C6SPC6 was selected as an example to analyze the collaboration sites with SNP-D4 [fig_ref] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6 [/fig_ref] , suggesting that SNP-D4 may bind to the catalytic center of A0A5C6SPC6, thereby inhibiting acetaldehyde dehydrogenase activity, and leading to the impaired metabolism of ethanol and possibly contributing to the antifungal effect. Next the spore proteins interacting with the eight proteins, which were trapped by SNP-D4, were subjected to protein-protein interaction analysis [fig_ref] Figure 1: Microscopic analysis of antifungal activity of SNP-D4 against FOC4 [/fig_ref] , indicating that the interference of SNP-D4 was implicated in 'translation', 'folding, sorting and degradation', 'transcription', 'signal transduction' and 'cell growth and death' pathways, and impairedthe growth of F. oxysporum broadly.
Collectively, Fusarium oxysporum f. sp. cubense (FOC4) is a devastating pathogen that causes rotted roots in banana and damage the banana industry. Pepaptamer SNP-D4 was identified to impede the spore germination, possibly by interfering with protein-protein A protein-protein interaction network formed by SNP-D4 and spore proteins. The network was visualized using Cytoscape. In the network, SNP-D4, the proteins interacting with SNP-D4, and the partners were shown as circle, diamonds, and triangles, respectively. The spore protein partners involved in different pathways were displayed in different colors. The interactions between proteins were presented as edges. The raw data were in [fig_ref] Table 1: The proteins interacting with SNP-D4 by mass spectrometry identification [/fig_ref].
Full-size DOI: 10.7717/peerj.12756/ interactions and blocking the target protein function in FOC4. In our future plan, a specific FOC4-resistant banana plant will be engineered that confers a high level of specificity to inhibit FOC4 by expressing SNP-D4. Another alternative biological control is to directly spray the bacterial products expressing SNP-D4 on banana plants suffering from banana wilt, so to achieve the therapeutic effect of biological pesticides. These are of positive significance for reducing the incidence of FOC4 in the banana industry and ensuring the safety of bananas for humans.
## Additional information and declarations funding
This work was supported by the grants from the National Natural Science Foundation of China (Nos. 32060153 and 31860676), and the Hainan Natural Science Foundation (No. 319QN161). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[fig] Figure 1: Microscopic analysis of antifungal activity of SNP-D4 against FOC4. The ungerminated spores were marked by red circles, and the germinated spores were marked by red arrows. (A) PBS: Phosphate buffer saline, (B) 0.3% hymexazol, (C) SN: Scaffold protein, (D) SNP-D4: Functional peptide aptamer. Full-size DOI: 10.7717/peerj.12756/fig-1 [/fig]
[fig] Figure 2 2, Figure 3: Antifungal assay of the functional peptide aptamer. (A) The purified SNP-D4 and SNP were detected by 12% SDS-PAGE gel. Lane M, molecular mass marker; lanes 1-2, the purified SN; lanes 3-4, the purified SNP. The purification of SN and SNP-D4 were inFig. S1Aand S1B. (B) Effects of SN and SNP-D4 on the spore germination of FOC4. PBS, the treatment with PBS as the negative control; The raw data was inTable S2. An asterisk ( Ã ) represented the significant difference at the 0.05 level, ÃÃ represented the significant difference at the 0.01 level. Mean values of the treated spores were compared to PBS treatment group.Full-size DOI: 10.7717/peerj.12756/fig-Effects of SN and SNP-D4 on the fungicidal rate of FOC4 spores. The fungicidal rate was measured after different treatments. The raw data was inTable S3. An asterisk ( Ã ) represented the sig nificant difference at the 0.05 level, ÃÃ represented the significant difference at the 0.01 level. Mean values of the treated spores were compared to SN (2 mM) group. Full-size DOI: 10.7717/peerj.12756/fig-3 [/fig]
[fig] Figure 4: SNP-D4 destroyed FOC4 spores. FOC4 spores were treated with different chemicals, stained with PI, and observed with a fluorescence microscope. The concentrations of SNP-D4 and SN were 10 mM. The damaged spores were marked by red circles. The length of the tuler is 50 mm.Full-size DOI: 10.7717/peerj.12756/fig-4 [/fig]
[fig] Figure 5: Molecular docking of SNP-D4 and A0A5C6SPC6. (A) and (B) are the protein model of SNP-D4 and A0A5C6SPC6. (C) With G424 as the active center, it formed an enzyme catalytic domain with the surrounding amino acids. (D) Front view and (E) side view of A0A5C6SPC6. (F) The structure of the molecular docking model, and the predicted binding sites. (G) SNP-D4 interacted with Tyr437associated with each of the five pathways. While for A0A5C6THD3, A0A5C6SMF1, A0A5C6TD26, they were only connected to four of the five pathways. [/fig]
[table] Table 1: The proteins interacting with SNP-D4 by mass spectrometry identification. [/table]
|
Cerebral autoregulation and response to intravenous thrombolysis for acute ischemic stroke
We hypothesized that knowledge of cerebral autoregulation (CA) status during recanalization therapies could guide further studies aimed at neuroprotection targeting penumbral tissue, especially in patients that do not respond to therapy. Thus, we assessed CA status of patients with acute ischemic stroke (AIS) during intravenous r-tPA therapy and associated CA with response to therapy. AIS patients eligible for intravenous r-tPA therapy were recruited. Cerebral blood flow velocities (transcranial Doppler) from middle cerebral artery and blood pressure (Finometer) were recorded to calculate the autoregulation index (ARI, as surrogate for CA). National Institute of Health Stroke Score was assessed and used to define responders to therapy (improvement of ≥ 4 points on NIHSS measured 24-48 h after therapy). CA was considered impaired if ARI < 4. In 38 patients studied, compared to responders, non-responders had significantly lower ARI values (affected hemisphere: 5.0 vs. 3.6; unaffected hemisphere: 5.4 vs. 4.4, p = 0.03) and more likely to have impaired CA (32% vs. 62%, p = 0.02) during thrombolysis. In conclusion, CA during thrombolysis was impaired in patients who did not respond to therapy. this variable should be investigated as a predictor of the response to therapy and to subsequent neurological outcome. open
The key objective of current acute ischemic stroke (AIS) treatment is based on rapid blood flow restoration by thrombolysis, using intravenous recombinant tissue plasminogen activator (r-tPA), and/or mechanical arterial recanalization techniques. Several factors predict stroke outcome including age, initial stroke severity, arterial blood pressure (BP), site of occlusion, collaterals, and others. Nevertheless, complete or partial successful recanalization may not necessarily result in favorable outcome, with a number of predictors hypothesized. In particular, BP control may affect penumbral lesion size, with an optimal strategy still lacking evidence. Therapeutic BP manipulation may further impact on microvascular autoregulatory failure, as a consequence of an increase in lactate and free oxygen radicals in the occluded and/or reperfused tissues.
Cerebral autoregulation (CA) refers to a set of physiological mechanisms that maintain the constancy of cerebral blood flow (CBF) despite wide variations in arterial BP. CA can be impaired within the first hours of ischemic stroke onset; as a consequence, BP control may be important for improving both the ischemic area in the brain and clinical outcome. Therefore, assessment of CA during recanalization therapy for AIS is relevant, and may influence future strategies for personalized BP control and associated neuroprotection.
The aims of the present study were to assess CA status of responder and non-responder AIS patients to intravenous r-tPA during the therapy and after 24-48 h, and to test the hypothesis that CA during thrombolysis is associated with early response to therapy. www.nature.com/scientificreports/
# Results
Forty-five patients (34 from São Paulo, 11 from Leicester) met the inclusion criteria. Seven patients were excluded due to absence of a temporal window or poor quality of acquired data from both hemispheres, leaving 38 patients for further analysis. From these, eight hemisphere's data (5 affected and 3 unaffected) during thrombolysis and 3 (2 affected and 1 unaffected) after 24-48 h had to be discarded due to proximal occlusion of MCA (3 patients) or poor quality of data (5 patients). In addition, in one affected hemisphere, data were not included at both time-points because the patient had a vertebrobasilar stroke and the data from both hemispheres were averaged. Demographics and patient characteristics for responders and non-responders groups are given in, with a higher 24-48 h NIHSS in the non-responder group being the only significant difference. Regarding the cerebral hemodynamic data, CBFV from both groups was significantly higher at 24-48 h than during thrombolysis. ARI during thrombolysis was significantly lower in non-responders in both AH and UH, though no significant differences were seen at 24-48 h,. No significant differences were seen in values for phase or gain between responders and non-responders during thrombolysis or at 24-48 h in either the VLF or LF bands.
The number of AIS patients with impaired ARI during thrombolysis, defined by ARI < 4, was significantly greater in the non-responder group, but again no significant differences were seen at 24-48 h. ROC analysis confirmed that ARI during thrombolysis predicts good response to therapy (AUC 0.66, p = 0.02;, and an ARI cut-off value of 4.0 had the best sensitivity and specificity (0.68 and 0.62, respectively). www.nature.com/scientificreports/
# Discussion
To our knowledge, this is the first study reporting the status of CA during intravenous thrombolytic therapy. The main finding is that ARI was lower during thrombolytic therapy in non-responders, and thus may be considered as a potential evaluation tool to predict response to therapy and also could be important in planning neuroprotective strategies for the ischemic penumbra. Due to its limited energy reserve, the brain is highly dependent on stable blood flow, and has evolved a number of protective mechanisms to maintain cerebral perfusion 12 . However, these regulatory mechanisms may be impaired during and following ischemia, and this has particular importance for BP management in the hyperacute period following AIS, when associated BP disturbances may increase the risk of further damage. Few studies have assessed CA acutely (< 24 h) following AIS, and only one study in a small sample of AIS patients with major anterior circulation stroke (NIHSS ≥ 10) undergoing thrombolytic therapy, was performed an average of 20 h post-symptom onset. This study reported that severe CA impairment in the AH (assessed by Mx index and phase shift) was associated with poor outcome. Though our study found less severe impairment, it has extended these observations to a broader AIS population (NIHSS 4-24), for both AH and UH and with recordings undertaken during thrombolysis providing a potential opportunity for future intervention at an earlier stage in the evolution of the ischemic penumbra. In addition, previous studies have demonstrated that CA could be associated with infarction size 13,16,17 but it is not clear if the ischemic lesion determines the CA impairment or vice versa; although our study did not evaluate infarct volume, the difference of CA in the very early phase amongst the two groups, (responders x non-responders) with no difference after 24-48 h, suggests that in our population, CA impairment was not influenced by infarct extension. We also could not demonstrate a direct link between intracranial haemorrhage and CA during thrombolytic therapy because this complication only occurred www.nature.com/scientificreports/ in one patient; however it is plausible to assume that impaired CA during therapy could have impacted on this complication as demonstrated in previous work.
Whilst the main goal of acute stroke treatment is recanalization of the occluded vessel, a significant percentage of patients do not have good neurological outcome despite successful recanalization, so-called 'futile recanalization'. Different hypotheses have been put forward trying to explain this phenomenon, such as collateral status and/or microvascular occlusion 7 ; however it also seems plausible to consider the role of impairment of CBF regulatory mechanisms in worsening neurological outcome in patients with futile recanalization. For example, CA impairment could lead to further penumbral damage secondary to reperfusion, as reported in a previous study within 6 h of AIS. Interestingly, in the pivotal NINDS trial 1 , there was no difference in early neurologic improvement amongst treated and placebo patient groups; this finding corroborates our hypothesis that there are other factors apart from recanalization that influences clinical outcome in the early stages after ischemic stroke. Thus, our findings are important to drive the investigation of strategies aimed to preserve the penumbral tissue and improve neurological recovery, irrespective of successful recanalization or not.
The ROC analysis in our study revealed that a cut-off value of 4.0 had the best sensitivity and specificity for predicting response to therapy and is in agreement with previous publications suggesting an ARI < 4 to define impaired CA. This cut-off value is a new finding in a stroke population, and should be further investigated and replicated in larger studies to be implemented as a valid assessment tool for detection of CA impairment.
Our study has a number of limitations. First, there is a known limitation of TCD when used as a surrogate of CBF 24 . Secondly, we included patients with carotid stenosis, which may also impair CA, though we found no difference in the prevalence or severity of carotid stenosis between responders and non-responders. Thirdly, there was no evaluation of CA before thrombolysis, therefore it was not possible to assess if pre-existing CA impairment can predict poor response despite recanalization; however, it would be challenging, and potentially unethical, to delay treatment to allow pre-thrombolysis CA assessment. Fourthly, CA assessment was not possible during thrombolysis in patients with proximal MCA occlusion (n = 3) and the effects of recanalization, intracranial stenosis and stroke mechanism were not accounted for; this may have over-or under-estimated the predictive effect of impaired peri-thrombolysis ARI. In addition, this study only included thrombolysis-treated patients and not those with mechanical recanalization therapies. Nonetheless, this therapy remains in limited use in both developed and developing countriesand it may be possible that this group of patients with higher rates of recanalization have greater influence of CA status on neurologic outcome. Noteworthy, the use of medical reperfusion is increasing in stroke patients with no major vessel occlusion, after publication of trials in wake-up stroke and extended therapeutic window. Fifthly, our study investigated only the early response to therapy; thus, the effects of CA in long-term neurologic outcome should be the object of future studies. Finally, the number of patients included in this study is relatively small with heterogeneity of patients and the results obtained are hypothesis generating; in addition it could be argued that the inclusion of one patient with vertebrobasilar stroke (responder group) may have biased the results. We intentionally included this subgroup to try to demonstrate that CA change is more a global than a local phenomenon. Removing this patient from the analysis did not change our results.
In conclusion, the present study has revealed that impaired CA in the very early phase of AIS increases the likelihood of poor response to thrombolytic recanalization therapy, as assessed by NIHSS score at 24-48 h. Based on our results, further studies with larger populations, including different stroke mechanisms and severities, should be planned to corroborate these findings. If our findings are replicated in larger studies, strategies to www.nature.com/scientificreports/ preserve CBF after recanalization therapies should be implemented to minimise secondary damage in patients with CA impairment.
# Methods
This was a collaborative research project between Hospital das Clinicas, São Paulo University Medical School, Sao Paulo, Brazil, and the Cerebral Haemodynamics in Ageing and Stroke Medicine (CHiASM) Group at the University of Leicester, Leicester, United Kingdom. The researchers involved in the data collection were trained at the same laboratory (Department of Cardiovascular Sciences, University of Leicester, UK) and used a standard protocol for data collection and analysis. The local ethics committee of the University of Sao Paulo and University of Leicester approved the study and informed consent was obtained in compliance with local ethics committee regulations. Both study centres applied the same inclusion criteria: AIS eligible for r-tPA therapy, aged ≥ 18 years, no premorbid disability, ability to monitor systemic and cerebral hemodynamic data without interfering with thrombolytic therapy and any related procedures, and informed consent (or relative assent). Exclusion criteria were: ineligibility for r-tPA thrombolysis and absence of an acoustic window for transcranial Doppler ultrasound (TCD) monitoring. National Institutes of Health Stroke Scale (NIHSS) scores were measured by neurologists blinded to cerebral hemodynamic data at the following time-points: (1) before therapy (NIHSS initial ),immediately at the end of therapy (NIHSS end ), and (3) after 24-48 h (NIHSS ). The scale was used to assess the early response to therapy, which was defined as improvement of ≥ 4 points on NIHSS 24-48 1 . The same protocol for monitoring systemic and cerebral hemodynamic data was used for all studies. Briefly, beat-to-beat BP was recorded continuously using a Finapres or Finometer device (FMS, Finapres Measurement Systems, Arnhem, Netherlands). Heart rate (HR) was recorded using a 3-lead electrocardiogram (ECG) and end-tidal CO 2 (etCO 2 ) was measured via nasal prongs (Salter Labs) by an infrared capnograph (Capnocheck Plus and Transmai MX-200 in Leicester and São Paulo, respectively). Bilateral insonation of the middle cerebral arteries (MCA) was performed using TCD (Viasys Companion III, Viasys Healthcare, and Doppler box, DWL, respectively, for Leicester and São Paulo) with 2 MHz probes, which were secured in place using a head-frame. Hemispheres were classified as affected (AH, side with ischemia) and unaffected (UH, side without ischemia), based on clinical symptoms and confirmed retrospectively with control imaging. If ischemia related to the vertebrobasilar system, both sides were considered as unaffected and they were averaged. Data were collected: (1) during thrombolysis, within the last 30 min of r-tPA infusion; and (2) 24-48 h after the treatment.
Assessment of dynamic cerebral autoregulation. Data were simultaneously recorded onto a data acquisition system (PHYSIDAS, Department of Medical Physics, University Hospitals of Leicester) for subsequent off-line analysis. Mean BP and CBF velocity (CBFV) values were calculated for each cardiac cycle. Beatto-beat data were spline interpolated and resampled at 5 samples/s to produce signals with a uniform time-base.
Dynamic CA was calculated by transfer function analysis using spontaneous fluctuations of mean BP as input and corresponding changes in CBFV as output as described previously; then the frequency-dependent estimates of phase were averaged for the very low-(VLF, 0.02-0.07 Hz) and low-frequency (LF, 0.07-0.20 Hz) ranges according to previous guidelines. The autoregulation index (ARI) was extracted by using the best leastsquares fit between the CBFV step response, and one of the 10 model ARI curves proposed by Tiecks et al.. To evaluate the frequency of impaired CA amongst the two groups ARI values were dichotomized into impaired (ARI < 4) and unimpaired CA (ARI ≥ 4) 22 .
Statistical analysis. Statistical software SPSS version 20 was used for all statistical tests. Mean values of each variable were calculated from the entire baseline recording. Tests of normality were performed using the Shapiro-Wilk normality test. To test for differences between AH & UH from responders and non-responders during thrombolysis, and 24-48 h after, a general linear model was used with the following factors: time (thrombolysis × 24-48 h), side (AH x UH), and response (responders × non-responders). Post-hoc comparisons were performed when appropriate, and Bonferroni correction was applied to multiple comparisons. Receiver operator characteristic curve (ROC) analysis was performed to test the prognostic value of ARI in this cohort. Differences amongst categorical data were assessed with Fisher's exact test and a p value < 0.05 indicating statistical significance.
## Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. |
The construction and validation of the Severe Asthma Questionnaire
@ERSpublications Validating a new severe asthma-specific quality of life questionnaire, the SAQ ABSTRACT The US Food and Drug Administration's procedure for scale validation requires a documented stepwise process of qualitative and quantitative data. The aim of this paper is to provide final quantitative validating data for the Severe Asthma Questionnaire (SAQ).The SAQ, Asthma Control Test, Mini Asthma Quality of Life Questionnaire and EQ-5D-5L were completed by 160 patients attending a severe asthma clinic; 51 patients completed the SAQ on two occasions for test-retest reliability analysis. The SAQ produces two scores, a SAQ score based on the average of 16 items and a SAQ-global score from a single 100-point global quality of life scale.Construct validity was demonstrated by factor analysis of the 16 items, convergent validity by correlations of >0.6 between the SAQ, SAQ-global and other questionnaires, and discriminant validity by the ability of the SAQ and SAQ-global to distinguish between different treatment levels. Test-retest reliability (intra-class correlation) was 0.93 for the SAQ and 0.93 for the SAQ-global, and the alpha coefficient for the SAQ was 0.93.The SAQ was developed using recommended qualitative and quantitative procedures for scale development, and can be used to gain insight into patients' perceptions of how severe asthma and its treatment affects their lives. This article has supplementary material available from
# Introduction
Patients with severe and difficult-to-treat asthma comprise a small proportion (5-10%) of all asthmatic patients, yet are responsible for a disproportionate degree of asthma morbidity and costs [bib_ref] International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma, Chung [/bib_ref] [bib_ref] The cost of treating severe refractory asthma in the UK: an economic..., O'neill [/bib_ref]. Quality of life assessment forms an essential part of asthma assessment because measures such as respiratory symptoms and lung function may not convey the true limitations caused by the disease from the patient's perspective [bib_ref] Asthma outcomes: quality of life, Wilson [/bib_ref]. Compared to patients with mild and moderate asthma, patients with severe asthma have additional quality of life deficits caused by comorbidities, higher treatment burden ( particularly oral corticosteroids) and multiple and more severe exacerbations that disrupt the lives of patients and their relatives [bib_ref] Comorbidity in severe asthma requiring systemic corticosteroid therapy: cross-sectional data from the..., Sweeney [/bib_ref].
The US Food and Drug Administration (FDA) published recommendations for validating Patient Reported Outcome Measures (PROMs) in 2009. The US FDA recommends a stepwise process of validation: first, content validity is established by documented qualitative research; second, construct and other validity is established through quantitative methods.
The three most commonly used asthma-specific health-related quality of life scales [bib_ref] Measuring quality of life in asthma, Juniper [/bib_ref] [bib_ref] Development and validation of the Mini Asthma Quality of Life Questionnaire, Juniper [/bib_ref] [bib_ref] A self-complete measure of health status for chronic airflow limitation, Jones [/bib_ref] were published prior to the US FDA's 2009 guidelines and include symptom items that are perceived negatively in this context by patients [bib_ref] Validity of three asthma-specific quality of life questionnaires: the patients' perspective, Apfelbacher [/bib_ref]. These and other published asthma scales fail to assess quality of life deficits specific to severe asthma [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref]. Thus, although existing scales are valid in terms of earlier definitions of validity [bib_ref] Fit for purpose and modern validity theory in clinical outcomes assessment, Edwards [/bib_ref] or for mild and moderate asthma, they are not valid in terms of the US FDA's 2009 guidelines when used in people with severe asthma.
The qualitative stage of the development and content validation of the Severe Asthma Questionnaire (SAQ) has been reported in two studies [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref] [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref]. The first study [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref] showed that in addition to deficits experienced in mild and moderate asthma, those with severe asthma also had problems caused primarily by two factors. First, the side effects of medication produced a variety of problems including mood changes, changes in self-perception, problems with eating, sleep disturbance and changes in appearance. Second, exacerbations (in particular those leading to hospitalisation) created problems for the patient and the wider family.
Using our findings from the first study we drafted a severe asthma-specific questionnaire, and subsequently conducted four focus groups in which patients with severe asthma provided iterative changes to the design and content of the draft questionnaire [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref]. The aim of this second phase was to ensure that the questionnaire was able to correctly capture the kind of information patients felt important in relation to their quality of life. Patients defined the response scale, combined two items, split one item to assess different impacts on family lives, advised on recall period and optimised the wording of individual items. In addition, patients expressed a desire to provide an overall assessment of their quality of life (supplementary material).
The SAQ comprises 16 questions about different aspects of life and a global question that assesses their quality of life overall. The SMOG (simple measure of gobbledegook) grade [bib_ref] Using the SMOG formula to revise a health-related document, Hedman [/bib_ref] for the SAQ is 5.9, indicating suitability for a reading age of at least 11-12 years. The unweighted aggregation of the 16 questions produces a score similar in methodology to other quality of life questionnaires. The additional global question is used for two reasons. The first is because patients express a strong preference for providing an overall score in contrast to those requiring differentiation between different aspects of life. The second reason is psychological. Patients make judgements, including judgements about their lives, using either one or a combination of two cognitive processes, called System 1 and System 2. System 1 judgements are fast and automatic, and the process is unavailable to consciousness. System 2 judgements are slow and deliberative, and people can introspect the process. A patient's response to a question in clinic of "how are you?" will be influenced primarily by a System 1 judgement. Using two types of measure, the SAQ score based on the 16 items and the SAQ-global score, provides a way of capturing these different kinds of judgement [bib_ref] Assessing the performance of the EQ-VAS in the NHS PROMs programme, Feng [/bib_ref]. Single-item scales are less preferred in clinical trials because the use of multiple items reduces error variance.
The aim of this study was to provide the final stage of validation required by the US FDA, i.e. a quantitative study that establishes construct and other types of validity.
## Questionnaires
The SAQ comprises 16 items, with response options on a seven-point scale averaged to produce the SAQ score (scores 1-7) and a 100-point Borg-type scale [bib_ref] Psychophysical bases of perceived exertion, Borg [/bib_ref] producing the SAQ-global score (scores 0-100). Quantifiers are indicated against all seven of the SAQ response options. A Borg scale is a category rating scale with quantifiers at either end and additional empirically placed quantifiers along the categories of the scale. The SAQ-global was adapted from an existing global quality of life scale [bib_ref] Development of a new type of global quality of life scale, and..., Hyland [/bib_ref] (more information is available from www.saq.org.uk).
The Mini Asthma Quality of Life Questionnaire (MiniAQLQ) [bib_ref] Development and validation of the Mini Asthma Quality of Life Questionnaire, Juniper [/bib_ref] comprises 15 items with response options on a seven-point scale with responses averaged (scores 1-7).
The Asthma Control Test (ACT) [bib_ref] Development of the asthma control test: a survey for assessing asthma control, Nathan [/bib_ref] comprises five symptom and medication items (five response options per item) totalled to produce an asthma control score.
EQ-5D-5L comprises five quality of life items (five response options per item), which were averaged to produce an EQ-5D-5L score and the EQ-5D-VAS, which is a 100-point visual analogue scale (scores 1-100) [bib_ref] Development and preliminary testing of the new five-level version of EQ-5D (EQ-5D-5L), Herdman [/bib_ref].
Criteria for missing data are shown in the supplementary material.
## Clinical data
Clinical data collected were spirometry (forced expiratory volume in 1 s (FEV1)), prednisolone dose (mg·day −1 ), number of severe exacerbations in the last 12 months requiring systemic steroids, Global Initiative for Asthma (GINA) severity, body mass index (BMI) and estimated cumulative oral corticosteroid (OCS) dose per year. The latter was calculated by multiplying the participant's maintenance steroid dose by 365 days, and adding an estimate of OCS use per exacerbation in the previous 12 months. Based on British Thoracic Society and GINA guidance, one exacerbation was judged to comprise prednisolone 40 mg·day −1 multiplied by 7 days, which equates to 280 mg of prednisolone per exacerbation [bib_ref] Date last accessed, Gina [/bib_ref].
## Procedure
Patients were recruited to the validation study or the reliability study or both studies. Questionnaires were completed either at home ( postal return of questionnaire) or in clinic. Participants' data were included in an analysis of test-retest reliability if their ACT scores did not change by the minimum clinically important difference of ⩾3 points and they reported stable asthma (see supplementary material, supplementary figure E1).
# Analysis
Analysis was on an intention-to-treat basis. Convergent validity between questionnaires was established using Pearson correlations. Groups of patients were identified by 1) level of maintenance dose of OCS (mg·day −1 ) and 2) estimated cumulative OCS dose per year. Differences between groups (discriminant validity) were tested by one-way ANOVA and where significant followed by least significant difference (LSD) post hoc tests. Construct validity was tested by exploratory factor analysis using principal axis factoring following recommended practice [bib_ref] Best practices in exploratory factor analysis: four recommendations for getting the most..., Costello [/bib_ref]. Evidence that the items could be aggregated into a single scale score was tested by examining whether the scree test indicated a unifactorial solution and whether items loaded significantly (>0.3) on the first factor. Test-retest reliability was calculated by intra-class correlations. Internal consistency was calculated by Cronbach's alpha coefficient. All analyses were conducted using IBM SPSS statistics version 24 (Chicago, IL, USA).
# Ethical approval
The study was approved by the University Hospitals Plymouth NHS Trust and Research Ethics Committee/Health Research Authority (ethical approval number 16/NE/0188, IRAS ID: 207601). All patients provided informed written consent.
# Results
For the validation study, 260 participants were invited to participate: 20 declined to participate, 54 failed to return questionnaires by post and 26 failed to attend their clinic appointment and provide written informed consent, leaving 160 participants. For the test-retest reliability study, 115 patients were invited to participate, of whom 10 declined. Of the 105 who consented for the test-retest reliability study, 67 returned questionnaires and 16 patients were excluded because their ACT score had changed by ⩾3 points, leaving 51 participants (37 female) (supplementary figure E1). One patient at GINA step 2 was enrolled in the study in error; they did not meet the criteria for severe asthma but were at higher risk. Their data have been included in the analysis because this study used intention-to-treat criteria. Participant demographics are displayed in table 1.
Of the 160 patients who participated in the validation study, 154 (96%) completed the SAQ, 146 (91%) the MiniAQLQ and 159 (99%) the ACT. Of the 100 patients asked to complete the EQ-5D-5L, 96 (96%) complied. [fig_ref] TABLE 2: Pearson's correlations between all six scales, BMI and FEV1 % predicted [/fig_ref] shows the correlations between the different questionnaires. All correlations were >0.60, showing convergence between the different questionnaires. The correlations between all questionnaires, BMI and FEV1 % predicted are also shown. [fig_ref] TABLE 3: Mean±SD, factor loadings and the percentage of participants responding "very difficult" or... [/fig_ref] shows the mean±SD of responses to the 16 items of the SAQ. Absence of floor or ceiling effects is indicated by no SAQ item having a standard deviation less than one point away from an end point. In addition, participants used the full range of response options for all items with some using every option between 1 and 7 for all items [fig_ref] TABLE 1: Demographic information for all patients and those at GINA step 4 and... [/fig_ref]. These results show that no item should be rejected on the basis of poor distribution. The SAQ scores ranged from 5 (extremely bad quality of life) to 100 (perfect quality of life) (supplementary table E2).
When testing whether it is valid to aggregate the 16 items into a single scale score, exploratory factor analysis of the SAQ revealed a one-factor solution using the scree test (the first four eigenvalues were 9.91, 1.11, 0.92 and 0.72) accounting for 60% of the variance. The factor scores are shown in table 3. All factor scores are >0.6, showing that despite difference in content all items were related to the same latent variable. Cronach's alpha coefficient was 0.96. Test-retest reliability as measured by intra-class correlation was 0.93 (95% confidence interval (CI) 0.87-0.96) for the SAQ and 0.93 (95% CI 0.86-0.96) for the SAQ-global, showing that the SAQ is a reliable scale. Test-retest reliability was also calculated individually for each item of the SAQ [fig_ref] TABLE 3: Mean±SD, factor loadings and the percentage of participants responding "very difficult" or... [/fig_ref]. [fig_ref] TABLE 4: Mean, confidence intervals and n-values for questionnaire scores at different levels of... [/fig_ref] shows the mean score values for each of the six scales as a function of four groups of patients stratified by maintenance prednisolone dose: no maintenance prednisolone, 1-9 mg·day −1 , 10 mg·day −1 and >10 mg·day −1 (ranges were selected to achieve groups of approximately equal size). All scales were significantly different across the four groups ( p<0.001), showing that the SAQ can discriminate between groups that are theoretically predicted to be different based on asthma severity. To compare the discrimination of different questionnaires between the 10 mg·day −1 and >10 mg·day −1 groups as a function of maintenance dose, we conducted LSD tests. Significant differences were found for the SAQ score ( p=0.01), SAQ-global score ( p=0.01) and EQ-5D-VAS score ( p=0.02), but not the MiniAQLQ score ( p=0.13), ACT score ( p=0.34) or EQ-5D-5L score ( p=0.23). With regard to other paired comparisons, there was a pattern of poorer outcome with increasing dose of OCS except for the comparison between those on zero dose and those on 1-9 mg·day −1 . For these comparisons (using LSD tests), the patients taking 1-9 mg·day −1 had better quality of life than those on zero dose according to the MiniAQLQ ( p=0.008), ACT ( p=0.001) and SAQ ( p=0.017) but not the SAQ-global ( p=0.19), EQ-5D-5L ( p=0.2) or EQ-5D-VAS ( p=0.4). Because the 1-9 mg·day −1 group had better quality of life than the zero dose group, we examined possible differences in biologic prescription between the four groups. In the zero dose group, 17 out of 101 (17%) were on biologics, in comparison to nine out of 17 (53%) in the 1-9 mg·day −1 group, six out of 20 (30%) in the 10 mg·day −1 group and six out of 22 (27%) in the >10 mg·day −1 group. provides a visual comparison of the SAQ and MiniAQLQ as a function of maintenance prednisolone dose.
Participants were allocated to estimated annual cumulative OCS dose groups: 0-1119 mg·year −1 , 1120-1460 mg·year −1 , 1461-3650 mg·year −1 , 3651-6595 mg·year −1 and >6595 mg·year −1 . These doses were chosen to be clinically relevant, e.g. 1120 mg·year −1 equates to approximately four courses of OCS, 3650 mg·year −1 to ∼10 mg·day −1 prednisolone and >6595 mg·year −1 to ∼20 mg·day −1 prednisolone. [fig_ref] TABLE 5: Mean, confidence intervals and n-values for questionnaire scores at different levels of... [/fig_ref] shows the mean scores for these five groups for each of the six scales. provides a visual comparison of the SAQ and MiniAQLQ as a function of cumulative prednisolone dose. One-way ANOVA showed a significant difference ( p<0.001) between the five estimated cumulative OCS dose groups for all questionnaires. In order to carry out post hoc tests that are equivalent to those of table 4, we combined the two groups with the highest burden, i.e. those on 3651-6595 mg·year −1 and those on >6595 mg·year −1 . ANOVA was repeated on the four groups (all questionnaires were significant at p<0.001). Significant differences were found between those on 1461-3650 mg·year −1 and >3650 mg·year −1 for the SAQ score ( p<0.001), MiniAQLQ score ( p<0.01), ACT total ( p=0.004), EQ-5D-5L score (p=0.003), SAQ-global score ( p=0.006) and EQ-5D-VAS ( p=0.04).
# Discussion
The US FDA's guidance for valid questionnaire constructionrequires documentation of a qualitative stage of research followed by a quantitative stage. The SAQ was developed on the basis of qualitative research reported elsewhere [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref] [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref]. In this paper we provide details of the quantitative stage.
People with severe asthma experience difficulty in a number of different domains of life experience, some of which are not experienced by those with mild and moderate asthma. For example, 28% felt that irritability, a side effect of OCS, made life either very difficult or very, very difficult, and 27% experienced this level of difficulty with food. "Problems at night" were reported by 39% to make life at least very difficult; the sleep disturbance can be caused by either asthma symptoms (which cause waking) or the side effects of OCS (which cause difficulty getting to sleep, waking and additional problems during the night). Two family items are included in the SAQ as a result of the earlier qualitative research [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref] patients rated their family lives as at least very difficult for themselves, and 18% rated it at least very difficult for other family members. These results confirm the earlier qualitative research that severe asthma impacts family life for not only the patient but also the patient's family. Our descriptive results show that people with severe asthma can experience nontrivial disutility in domains of experience which are not included in questionnaires designed for mild and moderate asthma, with particularly high disutility recorded for "the way I look", "getting tired" and the two items assessing different worries about medication.
Although some people with severe asthma experience very poor quality of life, this is not a universal experience. For example, although 32% of people in this study felt they had moderately bad quality of life or worse, 24% felt they had good quality of life or better. Although some of this difference can be explained by severity and treatment differences (those with greater OCS burden report poorer quality of life), the relationship between severity, treatment and quality of life is complex.
In addition to providing descriptive data on people with severe asthma, the data provide construct validity for the SAQ. Statistical analysis showed that all 16 items could be aggregated into a single score and that no item should be rejected on purely statistical grounds. The intra-class test-retest reliability of the SAQ and SAQ-global of 0.93 compares well with other questionnaires, e.g. 0.83 for the MiniAQLQ [bib_ref] Development and validation of the Mini Asthma Quality of Life Questionnaire, Juniper [/bib_ref]. The SAQ score and the SAQ-global score correlate with other asthma and generic scales, thereby providing convergent validity. The SAQ score discriminates between groups of patients defined by maintenance prednisolone dose and defined by estimated cumulative OCS burden per year, thereby providing discriminant validity. There is a trend for the SAQ to demonstrate greater quality of life impairment with increasing OCS burden compared to the MiniAQLQ but the study is underpowered to make statistical comparisons between scales.
All outcome measures showed a trend towards poorer quality of life with greater doses of OCS, except for the comparison between those on no prednisolone versus those prescribed 1-9 mg·day −1 . We do not know why the 1-9 mg·day −1 group had better quality of life than those without a maintenance dose, but it may be due to the higher use of biologics in the 1-9 mg·day −1 group. Further studies are needed to clarify the complex relationship between biologics and OCS on quality of life versus asthma symptoms [bib_ref] Understanding asthma-specific quality of life: moving beyond asthma symptoms and severity, Stucky [/bib_ref].
The SAQ differs from other asthma-specific scales in that it includes a single-item 100-point scale of global quality of life, the SAQ-global, which is included because of patient request. In this respect the SAQ is similar to the EQ-5D, in which there is also a single-item 100-point scale, the EQ-5D-VAS. The SAQ-global is a 0-100 Borg-type scale adapted from another scale [bib_ref] Development of a new type of global quality of life scale, and..., Hyland [/bib_ref] , and research shows the use of additional quantifiers is more reliable than the format used in the EQ-5D-VASin which only the end points have quantifiers. With the exception of the EQ-5D and EQ-5D-VAS, all questionnaires show weak to moderate correlations with lung function, indicating that lung function has a modest causal effect on these measures. The absence of a significant correlation for the EQ-5D and EQ-5D-VAS suggests that these two measures might not be valid for assessing the quality of life in patients with severe asthma.
Multiple-item scales often have better reliability than single-item scales for statistical reasons. Consistent with other research showing Borg scales to be more reliable than visual analogue scales [bib_ref] Development of a new type of global quality of life scale, and..., Hyland [/bib_ref] , our data show that the SAQ-global is highly reliable and as reliable as the SAQ score. The SAQ-global also correlates well with other scales. Comparison between the EQ-5D and EQ-5D-VAS suggests that the EQ-5D-VAS measures a broader concept than the EQ-5D [bib_ref] Assessing the performance of the EQ-VAS in the NHS PROMs programme, Feng [/bib_ref] , and the same difference may apply to the SAQ score and SAQ-global. Additionally, response to these two different parts of the SAQ may reflect different cognitive processes, a fast automatic process for the SAQ-global and a slow deliberate process for the SAQ score. Our study shows that ratings of "good quality of life" on the SAQ-global (i.e. those in the 70-79 range) equate to "makes my life slightly difficult" (i.e. a mean score of 4.8 on the SAQ; supplementary table E2). This demonstrates that the way a question is asked affects the patient's response. Both types of measure can be useful, but it is likely that the SAQ-global provides a value more consistent with patients' immediate response when clinicians ask about their asthma.
# Limitations
The estimated cumulative OCS dose might be an underestimate of OCS burden because patients with severe asthma often require a longer course of OCS for an exacerbation than the 7 days used in our calculation. The use of biologics was not considered in this analysis and could affect quality of life assessments. The study was carried out in the South West of the UK, and although the demographic data of our study population are not dissimilar to the UK demographic data presented within the UK severe asthma registry [bib_ref] Refractory asthma in the UK: cross-sectional findings from a UK multicentre registry, Heaney [/bib_ref] , the population was predominantly Caucasian and further validation in ethnically and culturally diverse populations is required. A reading age of at least age 11-12 years old is required to use the SAQ, but this is consistent with current literacy requirements for patient communication [bib_ref] Readability standards for informed-consent forms as compared with actual readability, Paasche-Orlow [/bib_ref]. We used the 15-item MiniAQLQ rather than the 32-item AQLQ as a comparison scale to reduce questionnaire fatigue and because the MiniAQLQ is similar in length to the SAQ. Nine patients participating in this study also participated in an earlier qualitative work [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref].
The SAQ is structured so that the global measure appears after the 16 context-specific items, and this order of presentation may have an effect. Research shows that prior exposure to a negative event (i.e. asking patients about difficulty in different contexts) will lead to more positive subsequent judgement [bib_ref] The knowledge of what might have been: affective and attributional consequences of..., Johnson [/bib_ref] [bib_ref] When less is more: counterfactual thinking and satisfaction among Olympic medalists, Medvec [/bib_ref]. Further research is needed to explore how contextual factors, including the order of presentation of different questions, influences quality of life judgements.
# Conclusions
The SAQ was designed to detect the impact of both asthma symptoms and treatment on quality of life and has been shown to be content valid in earlier studies [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref] [bib_ref] How patient participation was used to develop a questionnaire that is fit..., Hyland [/bib_ref]. This paper confirms the relevance of items based on the earlier studies, establishes the construct validity of the scale, and shows the scale to be reliable in a group of patients with severe asthma with different types of treatment. These data complete the validation procedures required by the US FDA. Longitudinal studies are required to provide further information about the scale, e.g. to establish sensitivity to change and the minimal clinically important difference in people with severe asthma. The SAQ is available for use in the British English version reported here and is available from the authors or online (www.saq.org.uk). Translations in other languages are required. In addition to providing a scale that is fit for purpose [bib_ref] A qualitative study of the impact of severe asthma and its treatment..., Hyland [/bib_ref] for assessing health-related quality of life in clinical trials, the SAQ can also be used in clinical practice to alert clinicians to the problems experienced by patients, and to gain an initial insight into the patients' own perceptions of the impact on their lives of illness and its treatment.
[table] TABLE 1: Demographic information for all patients and those at GINA step 4 and step 5 of treatment Data are presented as n (range) or n (%), unless otherwise stated. GINA: Global Initiative for Asthma; FEV1: forced expiratory volume in 1 s; BMI: body mass index; ICS: inhaled corticosteroid; BDP: beclomethasone dipropionate. # : one patient at GINA step 2 was included erroneously but included in the intention-to-treat analysis, see Results section. [/table]
[table] TABLE 2: Pearson's correlations between all six scales, BMI and FEV1 % predicted [/table]
[table] TABLE 3: Mean±SD, factor loadings and the percentage of participants responding "very difficult" or "very, very difficult" to each of the 16 items of the Severe Asthma QuestionnaireFactor loading 1. My social life. For example, visiting friends, walking with friends, talking with friends, going to bars/restaurants, and parties. My family lifehow it affects me. For example, caring for children, family responsibilities. Worry that asthma may get worse. For example, medicines no longer help, more frequent attacks. 13. Getting tired. For example, feeling tired for no reason, waking in the morning feeling tired. 14. Problems at night. For example, difficulty going to sleep, being woken very easily, waking often at night. 15. The way I look. For example, my weight, my skin bruises easily, using medicines in public, other people judging me https://doi.org/10.1183/13993003.00618-2018 [/table]
[table] TABLE 4: Mean, confidence intervals and n-values for questionnaire scores at different levels of prednisolone dose [/table]
[table] TABLE 5: Mean, confidence intervals and n-values for questionnaire scores at different levels of estimated annual cumulative oral corticosteroid dose [/table]
|
Skin Intrinsic Fluorescence Is Associated With Coronary Artery Disease in Individuals With Long Duration of Type 1 Diabetes
[bib_ref] Type 1 diabetes and coronary artery disease, Orchard [/bib_ref] [bib_ref] Higher plasma levels of advanced glycation end products are associated with incident..., Nin [/bib_ref] [bib_ref] Glycation research in amino acids: a place to call home, Rabbani [/bib_ref] [bib_ref] Advanced glycation end products and the kidney, Bohlender [/bib_ref] [bib_ref] Advanced glycation end products and the kidney, Bohlender [/bib_ref] [bib_ref] Advanced glycation endproducts and atherosclerosis, Vlassara [/bib_ref] [bib_ref] Simple non-invasive assessment of advanced glycation endproduct accumulation, Meerwaldt [/bib_ref] [bib_ref] Autofluorescence spectrum of skin: component bands and body site variations, Na [/bib_ref]
## Research design and
METHODSdParticipants from the Epidemiology of Diabetes Complications (EDC) study and the MedStar Health Research Institute Diabetes Complications (MDC) Study were enrolled in this cross-sectional study. The EDC cohort is a well-defined population (n = 658) with type 1 diabetes diagnosed before the age of 17 years at the Children's Hospital of Pittsburgh and followed since their baseline examinations between 1986 and 1988, when mean age was 28 years and mean duration of diabetes was 19 years (12). The MDC cohort represents a population of patients with type 1 diabetes followed for their clinical care for a minimum of 4 years and mean of 10 years. A convenience sample of 172 participants with type 1 diabetes was enrolled at both sites. The EDC participants (n = 112) had noninvasive SIF assessment performed during their 20th year of follow-up, ;2 years after their last clinical exam, and were selected on a first-to-respond basis from those living within 50 miles of Pittsburgh with a goal of 100 participants. Though they were marginally older (P = 0.02) and more likely to have CAD (P = 0.02), no differences in diabetes duration, most recent A1C, or renal disease were seen compared with nonparticipants living within the 50mile range. MDC participants (n = 60) were offered participation in this study following their routine clinical visit; no MedStar patients declined to participate. All study procedures were approved by the institutional review boards of the University of Pittsburgh and MedStar Health Research Institute.
Blood samples were assayed for lipids, lipoproteins, glycated hemoglobin, and creatinine. For the EDC study population, stable HbA 1 was originally measured in saline-incubated samples by microcolumn cation exchange chromatography (Isolab, Akron, Ohio). On 26 October 1987, the method was changed to high-performance liquid chromatography (HPLC) (Diamat; Bio-Rad Laboratories, Hercules, CA). The two methods were highly correlated (r = 0.95; Diamat HbA 1 0.18 6 1.00 Isolab HbA 1 ). Beginning in 1998, A1C was measured using the DCA2000 analyzer. Original HbA 1 CAD was defined as a history of myocardial infarction, revascularization for symptoms (bypass surgery or percutaneous coronary intervention), or stenosis .50%. The albumin-to-creatinine ratio was determined by spot urine collections in both EDC and MedStar, while albuminuria was determined by timed urine collections in EDC and by spot urine collections in MedStar. Estimated glomerular filtration rate was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula [bib_ref] CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration). A new equation to estimate glomerular..., Levey [/bib_ref]. Overt nephropathy was defined as an albumin excretion rate .200 mg/min or a history of dialysis/ kidney transplantation.
SIF was noninvasively measured from the skin of the left volar forearm using the SCOUT DS (an investigational device in the U.S.; VeraLight, Albuquerque, NM) skin fluorescence spectrometer [bib_ref] Noninvasive type 2 diabetes screening: superior sensitivity to fasting plasma glucose and..., Maynard [/bib_ref] [bib_ref] Noninvasive optical screening for diabetes, Ediger [/bib_ref]. SIF was excited with a light-emitting diode (LED) centered at 405 nm and was detected over the emission range of 441-482 nm. The skin reflectance at the excitation wavelength was used to compensate for absorbance owing to melanin and hemoglobin [bib_ref] optical detection of diabetes: model studies with porcine skin, Hull [/bib_ref]. The intrinsic fluorescence correction is expressed in the following equation:
[formula] f xm ¼ F xm R kx x R km m [/formula]
where the measured fluorescence (F xm ) is divided by reflectance values at the excitation and emission wavelengths (R x and R m ), respectively. The reflectance values are adjusted by the dimensionless exponents (k x and k m ). For these analyses, k x was set to 0.9 and k m was set to 0.0. The resulting intrinsic fluorescence (f xm ), was integrated over the 441-482 nm spectral region to give the SIF sum. The interday within-subject skin variation in SIF assessed by the SCOUT DS had previously been determined in a large diabetes screening study of 421 subjects at risk for developing type 2 diabetes [bib_ref] Noninvasive Pre-diabetes and Diabetes Screening Using Skin Fluorescence: Clinical Validation in an..., Maynard [/bib_ref]. The interday Hoorn coefficient of variation (fasting versus nonfasting) was 5.1% for SCOUT DSmeasured SIF. Student t and x 2 tests were used to examine univariate correlates of CAD. Logistic regression analysis was used to determine the independent association of SIF with the prevalence of CAD. Odds ratios (ORs) are expressed as per-SD change in continuous variables. Statistical analysis was conducted using SAS version 9.3 (Cary, North Carolina). Model fits were compared using Akaike information criteria [bib_ref] A note on the generalized information criterion for choice of a model, Atkinson [/bib_ref].
RESULTSdEighteen percent (n = 30) of the study participants had CAD. Characteristics of the study participants by CAD status are presented in . However, as formal analyses revealed no significant effect modification by sex (P = 0.52) and there was not a significant difference in SIF distribution with sex stratification (P = 0.55), the sexes were combined for the remainder of the analyses.
The independent relationship of SIF with CAD is presented in . Model 1 shows the relationship of SIF with CAD before any adjustment (OR 3.5), while models 2-6 show the effect of progressively accounting for age, duration, updated mean A1C, and sex. As can be seen, the OR for SIF was particularly reduced by the additions of age and duration to 2.4, while further control for updated mean A1C and sex had a smaller impact and the model fit did not improve (models 4 and 5). Model 7 (full adjustment but without sex) provided the best fit, but this was not materially better than without overt nephropathy. Each SD change in the natural log of SIF was therefore univariately associated with a 3.5-greater likelihood of having CAD, and after full adjustment there was still a 1.7-greater risk. Using model 7 (best fit), we further explored other CAD risk factors in type 1 diabetes, namely, blood pressure, lipids, and smoking. Individually adding these variables to model 7 provided no additional information.
The discriminative ability of SIF to detect CAD is depicted in receiver operator characteristic curves in . SIF alone accounted for 79% of the area under the curve for CAD. Addition of age, diabetes duration, updated mean A1C, and overt nephropathy to the model with SIF increased the area under the curve slightly to 84% with a small gain in the equal error rate (sensitivity = specificity).
CONCLUSIONSdIn individuals with type 1 diabetes, SIF was strongly associated with the presence of CAD. This association was closely linked to age and duration of diabetes and less so to updated mean A1C and nephropathy. Even though it was statistically nonsignificant in the fully adjusted model, the OR remained increased at 1.7. These results suggest that SIF may therefore be a useful overall marker of CAD risk in type 1 diabetes. It may also represent part of the biological basis for the associations of these factors with CAD and potentially contribute above and beyond these factors. These observations may thus help explain the increased CAD risk in those with type 1 diabetes.
AGEs are products formed in laterstage reactions of the glycation of proteins, amino acids, basic lipids, and nucleotides by monosaccharides and related monosaccharide derivatives (3). AGEs form in tissues and accumulate over time when attached to proteins like collagen; however, increased exposure to hyperglycemia and oxidative stress, such as occurs in diabetes, accelerates their rate of formation. AGEs are also known to increase with decreasing renal function and increasing renal damage (probably reflecting accumulation of AGE adducts) [bib_ref] Advanced glycosylation end products in patients with diabetic nephropathy, Makita [/bib_ref] , due to decreased renal clearance of AGEs, another mechanism by which AGEs may be elevated in diabetes. Furthermore, derivatives of AGEs not cleared by the kidneys form highly reactive second-generation AGEs that are thought to contribute to the progression of renal damage [bib_ref] Low-molecular weight advanced glycation end products: markers of tissue AGE accumulation and..., Thomas [/bib_ref]. That our findings were not independent of overt nephropathy when both age and duration were controlled for suggests that AGEs may contribute to the strong risk that renal disease plays in CAD in individuals with long duration of type 1 diabetes.
AGEs accumulate in the heart and large blood vessels, forming cross-links between adjacent proteins in the basement membrane of the extracellular matrix as well as activating the AGE receptor on vessel wall membranes. AGE cross-linking in vessel wall collagen increases the area of the extracellular matrix, resulting in increased stiffness of the vessel wall. AGEs have also been shown to interact with NO, reducing its bioavailability and activity [bib_ref] Advanced glycation end products quench nitric oxide in vitro, Uhlmann [/bib_ref] , and to increase the adhesion of macromolecules, such as macrophages and LDL, to the vessel wall [bib_ref] Cross-linking of glycated collagen in the pathogenesis of arterial and myocardial stiffening..., Aronson [/bib_ref] , and the adhesion of leukocytes to coronary fibroblasts [bib_ref] Diabetesinduced oxidative stress and low-grade inflammation in porcine coronary arteries, Zhang [/bib_ref]. AGEs have been observed in atherosclerotic plaques, foam cells, and fatty streaks [bib_ref] Oxidative stress, AGE, and atherosclerosis, Schleicher [/bib_ref]. Glycated LDL particles may be more atherogenic than regular LDL cholesterol [bib_ref] Oxidative stress, AGE, and atherosclerosis, Schleicher [/bib_ref] [bib_ref] Advanced glycation end products: sparking the development of diabetic vascular injury, Goldin [/bib_ref]. Finally, activation of the AGE receptor by AGE stimulates secretion of inflammatory proteins via activation of nuclear factor-kB [bib_ref] The role of AGEs in cardiovascular disease, Jandeleit-Dahm [/bib_ref]. While AGEs and their pathophysiologic effects are likely important components of the SIF/CAD association reported, it should also be noted that SIF reflects much more than AGEs, e.g., the fluorescent enzymes nicotinamide adenine dinucleotide and flavin adenine dinucleotide. To what degree these latter factors add to the association is unclear but merits further investigation. Our results are in agreement with studies that have shown increased levels of certain AGEs [bib_ref] Increased serum levels of advanced glycation endproducts predict total, cardiovascular and coronary..., Kilhovd [/bib_ref] in association with cardiovascular disease in individuals with diabetes. Nin and colleagues prospectively followed 339 individuals with type 1 diabetes for incident cardiovascular disease and all-cause mortality and found that baseline levels of two plasma AGEs [N9-(carboxyethyl)lysine and pentosidine] were predictive of outcomes 12 years later, independently of markers of renal function, inflammation, endothelial dysfunction, or arterial stiffness [bib_ref] Higher plasma levels of advanced glycation end products are associated with incident..., Nin [/bib_ref]. These data suggest the association is not purely cumulative, as serum AGEs are reflective of plasma protein turnover [bib_ref] Processing of protein glycation, oxidation and nitrosation adducts in the liver and..., Ahmed [/bib_ref] and renal function [bib_ref] Advanced glycosylation end products in patients with diabetic nephropathy, Makita [/bib_ref]. found that higher levels of serum AGEs were predictive of CAD mortality in women, but not men, with type 2 diabetes. This apparent contrast to our findings of a stronger association in men of SIF with CAD morbidity is intriguing. Unfortunately, we do not have mortality follow-up at this time to address SIF and mortality, and it should be noted that risk predictors of CAD mortality (as in Kilhovd et al. [bib_ref] Increased serum levels of advanced glycation endproducts predict total, cardiovascular and coronary..., Kilhovd [/bib_ref] and morbidity (current report) are often different. For example, we have shown that glycemic control is more strongly related to CAD mortality than to morbidity (30), a finding also apparent in the Wisconsin Epidemiologic Study of Diabetic Retinopathy [bib_ref] Association between glycosylated hemoglobin level and cardiovascular and all-cause mortality in type..., Shankar [/bib_ref] [bib_ref] Cardiovascular disease, mortality, and retinal microvascular characteristics in type 1 diabetes: Wisconsin..., Klein [/bib_ref]. Nonetheless, in the EDC study glycemic control was a stronger predictor in men than women [bib_ref] Is glycaemia or insulin dose the stronger risk factor for coronary artery..., Conway [/bib_ref] , consistent with our current findings. These findings therefore raise the possibility that glycemia, serum AGEs, and SIF may differ in their metabolism and/or impact on CAD risk by sexda finding that may also reflect the non-AGE-related determinants of SIF.
Our results are also in agreement with others showing a relationship between skin autofluorescence and heart disease in European populations [bib_ref] Skin autofluorescence provides additional information to the UK Prospective Diabetes Study (UKPDS)..., Lutgers [/bib_ref] [bib_ref] Skin autofluorescence is elevated in acute myocardial infarction and is associated with..., Mulder [/bib_ref] [bib_ref] Skin autofluorescence is a strong predictor of cardiac mortality in diabetes, Meerwaldt [/bib_ref] [bib_ref] Skin autofluorescence is elevated in patients with stable coronary artery disease and..., Mulder [/bib_ref]. Skin autofluorescence was cross-sectionally associated with coronary heart disease and predicted coronary heart disease mortality in 48 individuals with type 1 diabetes and in 69 with type 2 diabetes (35), and with stable CAD independent of diabetes status and renal function in 68 participants [bib_ref] Skin autofluorescence is elevated in patients with stable coronary artery disease and..., Mulder [/bib_ref]. We have previously shown a strong association between SIF and the severity of coronary artery calcification in the EDC population [bib_ref] Skin fluorescence correlates strongly with coronary artery calcification severity in type 1..., Conway [/bib_ref]. This association was strongest in those with a coronary artery calcification score of at least 400, a threshold associated with clinical CAD [bib_ref] Coronary calcium in adults with type 1 diabetes: a stronger correlate of..., Olson [/bib_ref]. Our current results support this finding.
This study has a number of strengths, including the well-documented risk factor status of participants and the confirmation of strong CAD/SIF associations in two independent cohorts. However, a major limitation of this study is the cross-sectional nature of the study design. SIF measurements were taken on average 2 years after clinical exams in the EDC population. Unfortunately, the SCOUT technology became available during a phase of EDC when only surveys were being collected. Another limitation of these analyses is that our population was made up of middle-aged, predominantly Caucasian, adults of long diabetes duration, and thus these results are not generalizable to children, young adults, other ethnic groups, or individuals with short diabetes duration. Felipe et al. [bib_ref] Skin intrinsic fluorescence is associated with hemoglobin A(1c) and hemoglobin glycation index..., Felipe [/bib_ref] found a significant difference in SIF when stratifying a cohort of 110 pediatric subjects by sex. We hypothesize that the effects of much older age (48.9 6 8.6 vs. 13.2 6 3.8 years) and longer duration of diabetes (36.1 6 10.5 vs. 5.9 6 3.6 years) in the EDC/MDC cohort overwhelmed the SIF sex difference observed in that pediatric cohort.
Some limitations arise from the measurement of SIF itself. In a study of 421 subjects at risk for type 2 diabetes, the interday, within-subject Hoorn coefficient of variation was 5.1% [bib_ref] Noninvasive optical screening for diabetes, Ediger [/bib_ref]. In addition, in the SCOUT substudy of 1,036 patients with long-duration of type 1 diabetes from the DCCT/Epidemiology of Diabetes Interventions and Complications Study, the SIF within-day, withinsubject Hoorn coefficient of variation was 4.3% [bib_ref] Association of skin intrinsic fluorescence with cumulative glycemic exposure in the DCCT/EDIC..., Cleary [/bib_ref]. A factor that contributes to SIF measurement variation is skin heterogeneity from freckles, hair follicles, sweat glands, and wrinkles. Skin pigmentation differences between patients and within a patient over time (more or less tan) are mitigated by measuring the reflectivity of the skin and using the measured reflectance to correct the distortion of the melanin. A further limitation is the potential for selection bias, as not everyone in the 50-mile catchment area of the EDC clinic was studied. As shown, the EDC population with SIF measurements was older, and this may have introduced a bias in terms of general health and CAD prevalence, though it is difficult to conceive a selection bias that would falsely strengthen a SIF-CAD association. Indeed, as age is strongly related to both CAD and SIF, the older age of this population would tend to make it more difficult to show an independent association of SIF with CAD, as the current data demonstrate. We thus feel that selection bias is unlikely to have led to a spurious association of SIF with CAD. Finally, owing to sample size limitations, we were unable to more fully explore potential differences by sex in the relationship of SIF with CAD. operator characteristic curves of the discriminative ability of unadjusted and adjusted SIF to detect CAD in individuals with type 1 diabetes. The adjusted receiver operator characteristic curve was derived from the best-fit model and depicts the discriminative ability of SIF, age, diabetes duration, updated mean A1C, and overt nephropathy to detect CAD in individuals with type 1 diabetes.
In conclusion, we have demonstrated a strong association between SIF and CAD in middle-aged individuals with type 1 diabetes. SIF partially reflects the influence of skin AGEs, skin markers of oxidative stress and cell metabolism, subject age, diabetes duration, long-term glycemic control, and renal disease, which are associated with increased CAD risk.
[table] Table 1dCharacteristics: of study participants by CAD status Data are means (SD), median (interquartile range), or n (%). AU, arbitrary units; eGFR, estimated glomerular filtration rate. *Natural logarithmically transformed before analysis. **Fisher exact test used. [/table]
[table] Table 2dMultivariable -: adjusted correlates of CAD ORs expressed as per-SD change in the continuous variables. AIC, Akaike information criteria. *Natural logarithmically transformed before analysis. †OR for SIF when this variable is added to Model 7. [/table]
|
Hippo Pathway-independent Restriction of TAZ and YAP by Angiomotin*
The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.The Hippo pathway is emerging as an important signaling pathway to regulate organ size in vivo and cell contact inhibition in vitro through control of cell proliferation and apoptosis. The Hippo pathway was originally defined in fly Drosophila melanogaster and is structurally, functionally, and mechanistically conserved in mammals (1-5). In the mammals, the upstream regulators such as NF2-Merlin activate the core kinase machinery composed of Mst1/2, WW45, LATS1/2, and Mob1, leading to inactivation of transcriptional co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). YAP and TAZ are homologous to each other, and they represent the mammalian counterpart of fly Yorkie. YAP and TAZ contain an N-terminal region responsible for interaction with TEAD1-4 transcriptional factors followed by one (for TAZ) or two (for YAP) WW domains that enable them to interact with proteins containing PPXY motifs (4, 6 -11). The WW domain has been shown to interact with several PPXY motifcontaining proteins such as Wbp2(12), p73 (13), Runx2 (14 -16), and Smads(17). The C-terminal region of TAZ and YAP contains a transcriptional activation domain able to stimulate the transcriptional outcome of the cognate transcriptional factors. The C terminus of YAP and TAZ also contains a PDZ-binding motif enabling them to interact with PDZ domain-containing proteins. Among the many transcriptional factors that have been reported to interact with YAP and TAZ, TEAD1, -2, -3, and -4 (and the fly Scalloped) are mostly involved in regulating the transcriptional outcome to govern cell proliferation and apoptosis. Many potential downstream target genes for TAZ-YAP-TEADs have been revealed by microarrays (11, 18), and CTGF and Axl are experimentally shown to be direct target genes(12,19).The Hippo pathway represents a major regulatory control to restrict the activity of YAP and TAZ by affecting their subcellular distribution and stability (10, 11, 18, 20 -23). Upon activation, the protein kinase complex LATS1/2-Mob1 phosphorylates multiple Ser residues within the HXRXXS motif of YAP and TAZ. When Ser 89 of TAZ and Ser 127 of YAP are phosphorylated, YAP and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Furthermore, phosphorylation of Ser 314 of TAZ and Ser 381 of YAP primes them for subsequent phosphorylation by casein kinase 1 followed by ubiquitination and proteasomal degradation(21,22). Whether there exist other regulatory pathways to restrict the activity of TAZ and YAP is not known.Angiomotin (Amot) 2 was initially identified as a binding protein of angiostatin to regulate endothelial cell migration (24) and is expressed as two isoforms (p130 and p80) with p130 containing an N-terminal extension (25). Two proteins homologous to Amot are termed AmotL1 and AmotL2 (25-27). Amot family members have been shown to interact with the actin cytoskeleton (25) and be part of the cell junctional complex (25-28). An extensive proteomic study has shown that Amot interacts with Rich1, a Cdc42 RhoGAP, as well as the Patj-Pals junctional complex to coordinate epithelial cell polarity (28). In this study, we describe our results showing that Amot and AmotL1 are novel regulators that interact with TAZ and YAP, leading to their cytoplasmic retention and inhibition of their transcriptional outcome and oncogenic property. Because Hippo refractory mutant S89A of TAZ is also *
The Hippo pathway restricts the activity of transcriptional co-activators TAZ and YAP by phosphorylating them for cytoplasmic sequestration or degradation. In this report, we describe an independent mechanism for the cell to restrict the activity of TAZ and YAP through interaction with angiomotin (Amot) and angiomotin-like 1 (AmotL1). Amot and AmotL1 were robustly co-immunoprecipitated with FLAG-tagged TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N terminus of Amot. Amot and AmotL1 also interact with YAP via the first WW domain of YAP. Overexpression of Amot and AmotL1 caused cytoplasmic retention of TAZ and suppressed its transcriptional outcome such as the expression of CTGF and Cyr61. Hippo refractory TAZ mutant (S89A) is also negatively regulated by Amot and AmotL1. HEK293 cells express the highest level of Amot and AmotL1 among nine cell lines examined, and silencing the expression of endogenous Amot increased the expression of CTGF and Cyr61 either at basal levels or upon overexpression of exogenous S89A. These results reveal a novel mechanism to restrict the activity of TAZ and YAP through physical interaction with Amot and AmotL1.
The Hippo pathway is emerging as an important signaling pathway to regulate organ size in vivo and cell contact inhibition in vitro through control of cell proliferation and apoptosis. The Hippo pathway was originally defined in fly Drosophila melanogaster and is structurally, functionally, and mechanistically conserved in mammals. In the mammals, the upstream regulators such as NF2-Merlin activate the core kinase machinery composed of Mst1/2, WW45, LATS1/2, and Mob1, leading to inactivation of transcriptional co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). YAP and TAZ are homologous to each other, and they represent the mammalian counterpart of fly Yorkie. YAP and TAZ contain an N-terminal region responsible for interaction with TEAD1-4 transcriptional factors followed by one (for TAZ) or two (for YAP) WW domains that enable them to interact with proteins containing PPXY motifs . The WW domain has been shown to interact with several PPXY motifcontaining proteins such as Wbp2, p73, Runx2 , and Smads. The C-terminal region of TAZ and YAP contains a transcriptional activation domain able to stimulate the transcriptional outcome of the cognate transcriptional factors. The C terminus of YAP and TAZ also contains a PDZ-binding motif enabling them to interact with PDZ domain-containing proteins. Among the many transcriptional factors that have been reported to interact with YAP and TAZ, TEAD1, -2, -3, and -4 (and the fly Scalloped) are mostly involved in regulating the transcriptional outcome to govern cell proliferation and apoptosis. Many potential downstream target genes for TAZ-YAP-TEADs have been revealed by microarrays, and CTGF and Axl are experimentally shown to be direct target genes.
The Hippo pathway represents a major regulatory control to restrict the activity of YAP and TAZ by affecting their subcellular distribution and stability . Upon activation, the protein kinase complex LATS1/2-Mob1 phosphorylates multiple Ser residues within the HXRXXS motif of YAP and TAZ. When Ser 89 of TAZ and Ser 127 of YAP are phosphorylated, YAP and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Furthermore, phosphorylation of Ser 314 of TAZ and Ser 381 of YAP primes them for subsequent phosphorylation by casein kinase 1 followed by ubiquitination and proteasomal degradation. Whether there exist other regulatory pathways to restrict the activity of TAZ and YAP is not known.
Angiomotin (Amot) 2 was initially identified as a binding protein of angiostatin to regulate endothelial cell migrationand is expressed as two isoforms (p130 and p80) with p130 containing an N-terminal extension. Two proteins homologous to Amot are termed AmotL1 and AmotL2. Amot family members have been shown to interact with the actin cytoskeletonand be part of the cell junctional complex. An extensive proteomic study has shown that Amot interacts with Rich1, a Cdc42 RhoGAP, as well as the Patj-Pals junctional complex to coordinate epithelial cell polarity. In this study, we describe our results showing that Amot and AmotL1 are novel regulators that interact with TAZ and YAP, leading to their cytoplasmic retention and inhibition of their transcriptional outcome and oncogenic property. Because Hippo refractory mutant S89A of TAZ is also * This work was supported by a grant from the Agency for Science, Technol-
# Results
## Amot and amotl1 as interacting proteins of taz and yap-
In our large scale co-immunoprecipitation (co-IP) experiments, Amot and AmotL1 were preferentially co-recovered with TAZ and S89A but under-represented in the co-IP of WWm, a mutant TAZ with its WW domain mutated. This indicates that the WW domain of TAZ is important for interaction with Amot and AmotL1. To validate the interaction, we have performed analytic co-IP experiments using HEK293 cells expressing FLAG-TAZ [fig_ref] FIGURE 1: -continued [/fig_ref]. Cell lysates were immunoprecipitated with anti-FLAG antibodies to test the co-recovery of endogenous Amot and AmotL1. As shown, endogenous Amot (left panel) and AmotL1 (right panel) were efficiently co-immunoprecipitated with FLAG-TAZ (lane 4).
To evaluate the importance of the Hippo pathway and WW domain of TAZ in mediating the interaction, we compared the interaction of S89A and WWm with wild-type TAZ. Lysates derived from cells transfected with FLAG-TAZ, -S89A, and -WWm were immunoprecipitated, and the co-recovery of endogenous Amot was detected [fig_ref] FIGURE 1: -continued [/fig_ref]. As shown, Amot was co-recovered with TAZ and S89A at high efficiencies (lanes 6 and 7), whereas WWm failed to interact with Amot (lane 8), suggesting that Amot interacts with TAZ in a manner that is dependent on the WW domain of TAZ regardless of whether Ser 89 is subjected to Hippo regulation. YAP is homologous to TAZ and is also a downstream target inhibited by the Hippo pathway, and its Ser 127 residue is the primary site for Hippo-mediated phosphorylation and sequestration by 14-3-3 proteins. As compared with TAZ having one WW domain, YAP has two WW domains. We therefore also tested the interaction of Amot with YAP [fig_ref] FIGURE 1: -continued [/fig_ref]. HEK293 cells were transfected to express FLAG-YAP, -WW1m (mutation of the first WW domain), -WW2m (mutation of the second WW domain), and -WW1ϩ2m (mutation of both WW domains). As shown in [fig_ref] FIGURE 1: -continued [/fig_ref] , HA-Amot was co-immunoprecipitated with FLAG-YAP and -WW2m (lanes 7 and 9, respectively), but not with WW1m or WW1ϩ2m (lanes 8 and 10, respectively), suggesting that Amot interacts with YAP in a manner that is dependent on the first but not the second WW domain of YAP. We also tested the interaction of endogenous AmotL1 with the various mutants of TAZ and YAP ( , suggesting that the WW domain of TAZ and the first but not the second WW domain of YAP are similarly important for interaction with AmotL1. Amot and AmotL1 contain two PPXY motifs in the N-terminal region (Amot: 239 PPEY 242 and 284 PPEY 287 ; AmotL1: 310 PPEY 313 and 367 PPEY 370 ) with the first PPXY motif also being conserved in AmotL2 ( 210 PPQY 213 ). Because PPXY motifs are preferred motifs for interacting with WW domains, it is possible that interaction of Amot and AmotL1 with TAZ and YAP is mediated by the PPXY motifs of Amot and AmotL1 and the WW domain of TAZ and YAP. Interestingly, Amot is expressed in two isoforms (p130 and p80) with the smaller p80 isoform lacking the PPXY motif-containing N-terminal 409-residue region. When FLAG-TAZ was immunoprecipitated from transfected HEK293 cells, the endogenous p130 but not the p80 of Amot was co-recovered [fig_ref] FIGURE 1: -continued [/fig_ref] , left panel), supporting the role of PPXY motifs of Amot in mediating interaction with the WW domain of TAZ. We also generated mutants of Amot having the first (PPXY1m), the second (PPXY2m), or both (PPXY1ϩ2m) motifs mutated. HA-tagged Amot and mutants were expressed in FIGURE 1. Interaction of TAZ and YAP with Amot and AmotL1 is dependent of the WW domain and the PPXY. A, lysates derived from HEK293 cells transfected with vector or FLAG-TAZ expression construct were immunoprecipitated with FLAG antibodies. The precipitates and lysates were analyzed by immunoblot to detect FLAG-TAZ and co-precipitated Amot (left panel) and AmotL1 (right panel). The p130 and p80 forms of Amot were indicated. B, lysates derived from HEK293 cells transfected with the indicated expression constructs were immunoprecipitated with FLAG antibodies. The precipitates and lysates were analyzed by immunoblot to detect FLAG-tagged TAZ proteins (lower panel) and co-precipitated endogenous Amot (top panel). C, lysates derived from HEK293 cells transfected with the indicated expression constructs were immunoprecipitated with FLAG antibodies. The precipitates and lysates were analyzed by immunoblot to detect FLAG-tagged YAP proteins (lower panel) and co-precipitated HA-Amot (top panel). D, lysates derived from HEK293 cells transfected with the indicated expression constructs were immunoprecipitated with FLAG antibodies. The precipitates were analyzed by immunoblot to detect FLAG-tagged proteins (second panel) and co-precipitated endogenous AmotL1 (top panel). The input lysates were analyzed for detection of AmotL1 (third panel) and FLAG-tagged protein (bottom panel). E, lysates derived from cells expressing HA-Amot and its indicated mutants were immunoprecipitated with anti-HA antibodies. The precipitates (upper panels) and lysates (lower panels) were processed for immunoblot to detect HA-Amot and co-recovered endogenous TAZ/YAP. F-H, direct interaction of PPXY motif-containing Amot fragment with the YAP-WW1ϩ2 domain. F, the top panel is the raw heat response obtained after injection of YAP-WW1ϩ2 domain into ITC cell containing the N-terminal fragment of Amot. The bottom panel reflects the integrated peak areas normalized to moles of YAP-WW1ϩ2, and the solid line is the least-squares fit to the binding isotherm. The affinity is 9 Ϯ 1 M. G, no significant heat response and binding when YAP-WW1ϩ2m was injected. In this mutant, the tryptophan in the binding site of both WW domains is mutated to alanine. H, the affinity between the N-terminal fragment of Amot and various WW domains are tabulated. aa, amino acids.
HEK293 cells and tested for their ability to co-recover endogenous YAP and TAZ [fig_ref] FIGURE 1: -continued [/fig_ref]. Endogenous YAP but not TAZ was detected by the antibodies that react with both YAP and TAZ (lower panels for input lysate). Importantly, YAP was co-recovered with HA-Amot (lane 2) and Amot-PPXY2m (lane 4) but not Amot-PPXY1m (lane 3) or Amot-PPXY1-2m (lane 5) (upper panel), suggesting that the first but not the second PPXY motif of Amot is important for interacting with endogenous YAP. Because the first but not the second motif is also conserved in AmotL2, it is likely that, like Amot and AmotL1, AmotL2 will also interact with TAZ and YAP. These results, taken together, suggest that the first PPXY motif of Amot (and likely the cor-responding motif of AmotL1 and AmotL2) is responsible for interacting with the WW domain of TAZ and the first WW domain of YAP. In support of this notion, in vitro interaction assay by the ITC method using the PPXY motifcontaining region (residues 8 -317) of Amot and recombinant fragment of the first and second WW domain of YAP (residues 171-267) revealed a direct and strong interaction of the Amot fragment with the WW domains [fig_ref] FIGURE 1: -continued [/fig_ref]. The interaction was completely abolished by mutation of the WW domains [fig_ref] FIGURE 1: -continued [/fig_ref]. Using isolated first and second WW domains, it was observed that first WW domain of YAP has a significantly stronger interaction with the Amot fragment [fig_ref] FIGURE 1: -continued [/fig_ref]. - VOLUME 286 - NUMBER 9
## Inhibition of taz and yap by angiomotin
## Journal of biological chemistry 7021
Amot Negatively Regulates the Function of TAZ by Causing Its Cytoplasmic Sequestration-To investigate the functional consequence of TAZ interaction with Amot and AmotL1, we have first examined the distribution of TAZ-S89A in response to co-expression of Amot as distribution of TAZ and YAP between the cytoplasm and the nucleus is a major regulatory event executed by the Hippo pathway to restrict the transcriptional co-activator activity. The distribution of TAZ and YAP regulated by the Hippo pathway is also evident in response to cell density. In sparse cells, TAZ and YAP are preferentially distributed in the nucleus, whereas in dense cell culture, when Hippo pathway is activated, TAZ and YAP are restricted more to the cytoplasm. FLAG-TAZ-S89A expressed in sparse cells was primarily detected in the nucleus. When expressed in transfected cells, Amot was predominantly in the cytoplasm with some dotty structures and essentially excluded from the nucleus . In cells without co-expression of HA-Amot, FLAG-TAZ-S89A was essentially in the nucleus (cell 1). When HA-Amot was expressed moderately, some FLAG-TAZ-S89A was seen to be shifted to the cytoplasm (cell 2). FLAG-TAZ was mostly shifted to the cytoplasm when HA-Amot was expressed at high levels (cells 3 and 4). Similar results were obtained when TAZ, YAP, and S127A were co-expressed with Amot or AmotL1 (data not shown). These results suggest that Amot is able to restrict TAZ and YAP as well as their Hippo refractory mutants by sequestering them in the cytoplasm in a manner that is independent of the Hippo pathway. To further validate this, we have examined the expression of TAZ target genes such as CTGF and Cyr61. CTGF is a well defined target gene of TAZ and YAP, and its expression is a functional outcome of interaction of TAZ-YAP with TEAD1-4 as the promoter region of CTGF gene contains several TEAD-binding elements. Cyr61 was shown to be up-regulated by TAZ and YAP in several microarray studies. As shown in , B and C, MCF7 cells were transfected with vector control (columns 1-4) or construct to express S89A (columns 5-8) alone (columns 1 and 5) or together with Amot (columns 2 and 6), AmotL1 (columns 3 and 7), or both Amot and AmotL1 (columns 4 and 8). The expression of endogenous CTGF and Cyr61 genes was robustly increased by exogenous TAZ-S89A (column 5). When Amot and AmotL1 were co-expressed with S89A, enhanced expression of CTGF and Cyr61 was compromised (column 8), suggesting that Amot and AmotL1 can negatively regulate the functional outcome of TAZ-S89A. Amot or AmotL1 alone had noticeable inhibition of S89Ainduced expression of CTGF , respectively). However, Amot and AmotL1 alone had little impact on S89A-induced expression of Cyr61 . These results suggest that in MCF7 cells, Amot and AmotL1 have additive and/or synergistic effect on inhibiting S89A-induced expression of endogenous CTGF and Cyr61 genes. The expression of the exogenous S89A, Amot, and/or AmotL1 in these cells was validated by immunoblot analysis . We and others have shown that TAZ-S89A and YAP-S127A possess potent oncogenic property in NIH3T3 and MCF10A cells in conferring anchorage-independent cell growth in soft agar. This oncogenic property of S89A and S127A in NIH3T3 cells was also suppressed when Amot was co-expressed . These results, taken together, suggest that Amot and AmotL1 can negatively regulate the functional outcome of TAZ-S89A, most likely through Hippo pathway-independent sequestration in the cytoplasm.
## Hek293 cells have high levels of amot and amotl1 expression, and knockdown of amot but not amotl1 enhances expression of endogenous ctgf and cyr61 to levels that are
Induced by S89A-We have recovered Amot and AmotL1 by large scale co-IP using HEK293 cells transiently expressing TAZ and S89A. To evaluate the influence of endogenous Amot and/or AmotL1 on the functional outcome of TAZ, we have examined the expression of Amot and AmotL1 in nine different human cell lines [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref]. As shown, HEK293 (lane 9) has the highest level of expression of Amot and AmotL1 among these cell lines. All other eight cell lines express low or undetectable levels of Amot and AmotL1 [fig_ref] FIGURE 1: -continued [/fig_ref]. We therefore used HEK293 cells to investigate the role of endogenous Amot and AmotL1. We first validated that the expression of endogenous Amot and AmotL1 was efficiently silenced by transfection of specific siRNA as the protein levels of Amot and AmotL1 were significantly reduced in cells transfected with their specific siRNA [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref]. We next examined the impact of knockdown of Amot, AmotL1, or both on the expression of TAZ target gene CTGF [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref] and Cyr61 [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref]. When Amot (column 2) but not AmotL1 (column 3) was silenced, the expression of CTGF and Cyr61 was significantly enhanced. The levels of enhancement are almost comparable with those when the cells were expressing exogenous S89A (column 5). Interestingly, the enhanced expression of CTGF and Cyr61 due to exogenous S89A can be further increased when endogenous Amot (column 6) but not AmotL1 (column 7) was suppressed by its siRNA. Therefore, endogenous Amot but not AmotL1 in HEK293 cells negatively regulates the expression of TAZ-YAP target genes CTGF and Cyr61, and this inhibition is evident both for endogenous YAP (as YAP but not TAZ was detected in this cell line as shown in [fig_ref] FIGURE 1: -continued [/fig_ref] as well as when exogenous S89A was expressed. These results obtained from this loss-of-function approach further support the notion that Amot can negatively regulate the function of TAZ and YAP.
# Discussion
TAZ and YAP function as transcriptional co-factors for many transcriptional factors such as TEAD1-4 , p73 (13), TTF1 (32), Runx2 (14 -16), TBX5 (33), peroxisome proliferator-activated receptor ␥ (PPAR␥), Pax3 (34), Smad2/3, MyoD, and Glis3. TEAD1-4 are the major transcriptional factors involved in the TAZ-YAP ability to regulate cell proliferation and apoptosis. As such, their availability and subcellular localization are tightly controlled to govern their functionality. The major known regulatory mechanism for TAZ and YAP is via the recently defined Hippo pathway. Upon activation, the core components of the Hippo pathway phosphorylate TAZ and YAP at multiple sites harboring the HXRXXS motif. When phosphorylated, phospho-Ser 89 of TAZ and phospho-Ser 127
## Figure 2. amot and amotl1 inhibit taz transcriptional outcome and oncogenic property in promoting anchorage-independent growth.
A, MCF7 cells were co-transfected with FLAG-TAZ-S89A and HA-Amot. Cells were then processed to detect the expressed proteins using mouse anti-FLAG and rabbit anti-HA antibodies followed by secondary antibodies (green and red for FLAG and HA tag, respectively). FLAG-TAZ-S89A, expressed alone, is essentially in the nucleus (cell 1). When Amot was expressed at low moderate levels, some S89A was shifted to the cytoplasm (cell 2), and S89A was largely detected in the cytoplasm when Amot was expressed at high levels [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref]. B, the mRNA levels of endogenous CTGF gene were measured by real-time PCR in cells transfected with vector (columns 1-4) or TAZ-89A-expressing construct (columns 5-8) together with the expression vectors indicated at the bottom (ctrl, vector control; A, Amot coding cDNA; L1, AmotL1 coding cDNA; AϩL1, Amot and AmotL1 coding cDNAs together). The levels of CTGF mRNA were normalized to that detected in column 1, which was arbitrarily set at 1. C, the mRNA levels of Cyr61 were measured in those cells described in panel B. The Cyr61 mRNA levels were normalized to that detected in column 1, which was arbitrarily set at 1. D, the expression of S89A, Amot, or AmotL1 was examined by immunoblot. E, NIH3T3 cells were transduced to express TAZ-S89A (left panel) or YAP-S127A (right panel) along with vector (upper panels) or with Amot-expressing construct (bottom panels). Cells were grown in soft agar, and colonies were stained and photographed. F, the quantitative results derived from three independent experiments similar to D were shown. Error bars in panels B, C, and F indicate S.E.
of YAP serve as the binding sites for 14-3-3 proteins. As such, phospho-Ser 89 and phospho-Ser 127 offer the major regulatory effect of the Hippo pathway on the distribution of TAZ and YAP, respectively. Accordingly, S89A and S127A mutants are largely refractory to this inhibition and are mainly distributed in the nucleus, having much enhanced transcriptional and oncogenic property. Recent studies also reveal that the Hippo pathway can regulate the stability of YAP and TAZ. Phosphorylation of Ser 381 of YAP and Ser 314 of TAZ by the Hippo pathway primes YAP and TAZ, The levels of CTGF mRNA were normalized to that detected in column 1, which was arbitrarily set at 1. D, the mRNA levels of Cyr61 were measured in those cells described in panel C. The Cyr61 mRNA levels were normalized to that detected in column 1, which was arbitrarily set at 1. Error bars in panels C and D indicate S.E. E, a working model for diverse regulatory mechanisms for TAZ and YAP. The Hippo pathway causes cytoplasmic sequestration of TAZ and YAP through phosphorylation of Ser 89 and Ser 127 , respectively. Furthermore, Hippo pathway-mediated phosphorylation of Ser 314 and Ser 381 leads to further phosphorylation, ubiquitination, and proteasomal degradation of TAZ and YAP, respectively. Interaction with TEADs is important for nuclear accumulation and transcriptional outcome of TAZ and YAP. The results presented in this study suggest that Amot and AmotL1 (likely also AmotL2) function as negative regulators of TAZ and YAP through direct interaction with the WW domain of TAZ and the first WW domain of YAP via the first PPXY motif of Amot and AmotL1 that is also conserved in AmotL2, leading to their cytoplasmic retention.
respectively, for subsequent phosphorylation by casein kinase 1, leading to their ubiquitination and proteasomal degradation. Therefore, the Hippo pathway can negatively regulate TAZ and YAP by two different mechanisms through cytoplasmic sequestration and proteasomal degradation. Recently, PP2A phosphatase complex was shown to reverse the inhibition imposed on Yorkie by the Hippo pathway. Whether there exist other mechanisms regulating TAZ and YAP function is not known.
The results presented in our study offer another novel mechanism of regulation of TAZ and YAP function through direct interaction with Amot and AmotL1, leading to their cytoplasmic sequestration that is independent of Hippo pathway-regulated interaction with 14-3-3 proteins. The general working model derived from our experiments is that Amot and AmotL1 (likely also AmotL2) are primary cytosolic proteins that interact with TAZ and YAP through the first PPXY motif of Amot and AmotL1 and the WW domain of TAZ and YAP [fig_ref] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF... [/fig_ref]. This interaction causes cytoplasmic sequestration of TAZ and YAP in a manner similar to but independent of TAZ-YAP interaction with 14-3-3 proteins. In the case of YAP, it is intriguing to note that first but not the second WW domain is important for interaction with Amot and AmotL1, offering the possibility that the second WW domain of YAP can interact with yet another PPXY motif-containing protein, creating more complexity of YAP regulation. The structural basis unique to the first WW domain of YAP that enables its Amot interaction is an interesting issue to be addressed in the future. Because Amot has been shown to be distributed to the junctional complexes of polarized epithelial cells, it is also possible that Amot and AmotL1 may mediate localization of TAZ and YAP to the cell junction in polarized epithelial cells. Because the junctional complex is linked to cell-cell adhesion and part of the sensing mode for cell contact, this potential targeting of Amot-TAZ-YAP to the junction may offer an avenue to investigate the potential cross-talk of Amot-mediated regulation and Hippo pathway-mediated control to coordinate cell contact-induced inactivation of TAZ-YAP by the Hippo pathway in epithelial cells. Future experiments should provide insightful understanding about this hypothesis. Another interesting issue that remains to be investigated is whether Amot and AmotL1 (likely also AmotL2) play independent, overlapping, and/or redundant roles in regulating TAZ and YAP. In our overexpression studies in MCF7 cells, overexpression of either Amot or AmotL1 displayed minor inhibition on TAZ target gene expression, whereas expression of both Amot and AmotL1 caused more significant inhibition, suggesting that Amot and AmotL1 may be synergistically and/or additively inhibiting the transcriptional co-activating function of TAZ in MCF7 cells. However, in HEK293 cells, knockdown of Amot alone enhanced expression of TAZ-YAP target genes CTGF and Cyr61 to levels that are comparable with the overexpression of exogenous Hippo refractory S89A mutant of TAZ, suggesting that Amot is the principal member of Amot family in regulating the transcriptional outcome of endogenous TAZ-YAP in terms of CTGF and Cyr61 genes. This possibility is supported by the observation that knockdown of AmotL1 had little impact on CTGF and Cyr61 ex-pression, whereas simultaneous knockdown of both Amot and AmotL1 had a similar effect as Amot knockdown alone. These results suggest that Amot and AmotL1 may play unique and independent roles in regulating the functional outcome of TAZ-YAP gene expression. As expected, in the presence of S89A, expression of endogenous CTGF and Cyr61 was enhanced. Importantly, under this setting, knockdown of Amot but not AmotL1 further enhanced the expression of CTGF and Cyr61, suggesting that the exogenous S89A is negatively regulated by endogenous Amot but not AmotL1 with regard to the expression of CTGF and Cyr61. Therefore, it seems that Amot has a major role in suppressing the activity of endogenous YAP and exogenous TAZ in HEK293 cells. It is also possible that the relative importance of different Amot family members in regulating the function of TAZ-YAP may depend on the cellular context and thus may be different in different cell types because Amot family members may potentially interact with other cellular proteins that indirectly influence their functionality in regulating TAZ-YAP. Another interesting observation is that the expression level of Amot and AmotL1 is relatively low or undetectable in most cell lines examined. Because they are able to negatively regulate the proliferative properties of TAZ-YAP such as CTGF and Cyr61 expression and anchorage-independent growth, low and undetectable expression of Amot and AmotL1 is more favorable for the proliferative requirement of cell lines. This indicates that suppressing the expression of Amot and AmotL1 is one possible mechanism to uncouple TAZ and YAP from their negative regulation. Whether there exists an active mechanism to suppress the expression of Amot and AmotL1 in these cells and how HEK293 cells overcome the potential inhibition of relatively high levels of Amot and AmotL1 expression will be interesting to examine. Along with these lines, the identification of Amot and AmotL1 as novel negative regulators of TAZ-YAP that act independently of the Hippo pathway will provide an exciting opportunity to gain better understanding about not only the role and mechanism of TAZ-YAP but also the integration of TAZ-YAP with diverse regulatory mechanisms.
The mode of restriction of TAZ and YAP by Amot family members is mechanistically similar to the reported inhibition of Yorkie by expanded (Ex) in the fly (37), although Amot and AmotL1 do not display overall sequence homology with Ex. Ex was firstly defined as an upstream regulator to activate Hippo core kinase machinery to restrict Yorkie, but it was recently shown that Ex can also directly interact with Yorkie and thus prevent Yorkie nuclear accumulation, a mode of action that is independent of the Hippo core kinase machinery. Mechanistically, the direct interaction is mediated by the WW domains of Yorkie and PPXY motifs of Ex, thus resembling the physical interaction of Amot and AmotL1 with TAZ and YAP.
[fig] FIGURE 3: Endogenous Amot but not AmotL1 negatively regulates the expression of endogenous CTGF and Cyr61 genes. A, the lysates derived from the indicated nine different human cell lines were analyzed by immunoblot to detect endogenous Amot (upper panel) and AmotL1 (lower panel). -Actin was used as control. B, Amot and AmotL1 protein levels in cells transfected with their respective siRNA were determined by immunoblot. C, the mRNA levels of CTGF were measured by real-time PCR in HEK293 cells transfected with vector (columns 1-4) or TAZ-89A-expressing construct (columns 5-8) together with siRNA indicated at the bottom (ctrl siRNA, control siRNA; A siRNA, Amot siRNA; L1 siRNA, AmotL1 siRNA; AϩL1 siRNA, Amot and AmotL1 siRNA together). [/fig]
|
Socioeconomic disadvantage as a driver of non-urgent emergency department presentations: A retrospective data analysis
BackgroundGlobally, emergency departments (EDs) are struggling to meet the service demands of their local communities. Across Australia, EDs routinely collect data for every presentation which is used to determine the ability of EDs to meet key performance indicators. This data can also be used to provide an overall picture of service demand and has been used by healthcare planners to identify local needs and inform service provision, thus, using ED presentations as a microcosm of the communities they serve.
# Introduction
Emergency departments have been described as a microcosm of the communities they serve, meaning that they encapsulate features of the wider community. Challenges faced by emergency departments (EDs) can reflect deficits in community-based resources. As increasing demands for ED services continue to be reported globally, it is timely and necessary to identify drivers of ED demand. In Australia, over 8.3 million people accessed ED services between July 2018 and June 2019 (335 per 1000 population), 48% of whom were triaged to the two least urgent triage categories. The Australian Triage Scale (ATS) is a five-tiered triage system with ATS 4 and 5 being the least urgent categories, patients triaged to these categories are assessed as being safe to wait for one or two hours respectively. For the purpose of this study, we refer to ATS 4 and 5 presentations as non-urgent. We are confident that this group of patients included some who could have had their needs met in a primary care setting.
International research investigating these least urgent presentations has identified drivers of ED demand such as: patients' perceived need for urgent attention; age and gender; access to alternative services, and socioeconomic position. Identifying drivers specific to individual EDs can inform service planning. Furthermore, a mismatch between the known causes of ED demand and solutions implemented was identified in a systematic review and highlights the need to develop interventions that address specific causes. These external drivers contribute to the challenge for hospitals and health services in implementing successful and sustainable solutions.
Furthermore, our understanding of the demand for ED services is complicated by contextual differences. These differences challenge the successful implementation of solutions. Variation in demographic profiles, community healthcare needs and service availability influence how and when people access services, including the decision to present to an ED with a 'nonurgent' condition. Socioeconomic position, for example, has been identified as having both a positive and negative correlation with populations accessing EDs. This correlation is observed to vary across contexts, with one study identifying greater representation by populations from mid-high socioeconomic areaswhile others report greater representation from lower socioeconomic areas. Of the studies that reported age and gender, one found a higher incidence among middle aged femaleswhile another found a higher incidence among young males. These studies demonstrate the unique microcosm within EDs and provide an indication of healthcare needs within their respective wider communities.
Tasmania, Australia's smallest State, with a population of 517,000, has the highest rate of non-urgent ED presentations, with 88,000 triaged as ATS 4 or 5 in 2018-19. This island State is separated into three geographic regions with governing health services in the North, Northwest and South all operating under the overarching jurisdiction of the Tasmanian Health Service. The population of Northern Tasmania is older (median age 43 years compared to 38 years nationally) and more socioeconomically disadvantaged (median weekly income $537.00-AU compared to $662.00-AU nationally) than other Australian regions, compounded by inequitable access to primary care services in regional and rural Tasmania. There are considerable regional differences in the profile of ED patients across these three regions highlighting the importance of identifying trends and types of ED presentations to inform service planning. These regional variations in population healthcare trends and the mismatch between identified causes and solutions to address ED demand highlight the importance of bringing together knowledge and understanding of the drivers for ED demand before implementation of sustainable solutions.
In research conducted in Northern Tasmania, 31% of patients who present to the ED with non-urgent conditions would have preferred to be managed by their general practitioner (GP) if they had been available. The limited service optionsin this community and the distance to alternative EDs (the nearest is a smaller rural facility located 90km from the study hospital) contribute to ED demand. Moreover, there are no private EDs or urgent care facilities in Northern Tasmania. Northern Tasmanian residents also have limited access to primary care services within the community once business hours have ended. Business-hours have been defined as between 0800 to 1800 Monday to Friday and 0800 to 1200 Saturdays; public holidays and all other times are considered after-hours. These limited service options indicate potential challenges around timely access to alternative services.
Emergency departments are the 'canary in the coalmine' for health services and the communities they serve. Demands for ED services are reflective of broader population healthcare needsand are influenced by the availability of services within the community. The aim of this paper is to establish a profile of who, when and why ED services were accessed by people with non-urgent conditions. The objectives are to:
1. Develop a profile and identify trends in who is presenting and when;
2. Identify patterns in where people come from, including the socioeconomic position, and;
3. Identify trends in discharge diagnoses. This paper forms part of a larger body of work using an explanatory sequential mixed method to gain a deeper understanding of factors contributing to the decision to present to an ED with non-urgent conditions and develop relevant and sustainable strategies for health service planning.
# Materials and methods
Retrospective analysis of routinely collected hospital data was undertaken for all presentations triaged as ATS 4 or 5 at a single regional ED, between 1 July 2009 and 30 June 2016. This consisted of data entered into the Emergency Department Information Systems (EDIS) by ED staff at time of the patient's presentation, or at the time of discharge. Variables used in this analysis included: date, day of week and time of arrival to the ED; gender; mode of arrival; suburb of residence; discharge diagnosis; discharge destination, and referral on discharge. The first six variables were entered into EDIS by the triage nurse or clerical staff at the patient's time of arrival. The latter three were added by the treating physician or nursing staff at the time of departure. Diagnoses are based on International Diagnostic Codes, revision 10, as outlined by the World Health Organisation. It was beyond the scope of this project to review presentations across all triage categories.
Research ethics approval was granted by the Tasmanian Human Research Ethics Committee (H0016504). Deidentified data were provided by the Tasmanian Department of Health and Human Services (DHHS). This data is not publicly available in Australia and permission was not provided for it to be made publicly available.
## Study setting & participants
This study was undertaken in a large regional hospital in Northern Tasmania with a total bed capacity of 300 and a 26 bed ED. Serving as a referral centre for a population of 143,500dispersed across 20,000 square kilometres. Data used for this analysis was from July 2009 to June 2016, for ATS 4 and 5 presentations. The DHHS also provided the total count of all ED presentations by month across all triage categories so the proportion of ATS 4 and 5 could be calculated. Further explanation of the included study population is provided inWe have included all ATS 4 and 5 presentations who resided in the regional city (Launceston) and its surrounding suburbs. Excluding those from outside this region allowed us to develop a profile of who, when and why the local community choose to access ED services, thus focusing on local drives of ED demand. This area was defined by using statistical area (SA) codes allocated by the Australian Bureau of Statistics (ABS). The greater Launceston area has an SA3 code of 60201. All suburbs with this code were included in the study area and total population was 81,029 in 2016. Population growth in this region was just 2.5% between 2011 and 2016 compared to the national growth of 8.3%.
Data relating to socioeconomic position was derived from ABS data. Five-yearly census data is used to calculate average values of various socioeconomic indexes across geographical areas, known as Socioeconomic Indexes for Areas (SEIFA). One of these is the Index of Relative Socioeconomic Disadvantage (IRSD), which is the preferred measure to use when investigating disadvantage or lack of disadvantage. This index is based on national socioeconomic classification, and takes into account income and additional variables including unemployment, disability, sole-parent status, level of education, employment classification, etc.. Each suburb is given a score based on these variables, the lower the score the greater the disadvantage. The ABS also aggregate suburbs into deciles, dividing Australia's population into ten evenly sized population groups. Ten percent of the Australian population fall into each decile with IRSD 1 being the 10% of those with greatest disadvantage and IRSD 10 being those with the greatest advantage. The histogram of IRSD scores has a long left-tail (at the end of greatest disadvantage), so the difference in disadvantage between decile 1 and decile 2 is larger than between other pairs of adjacent deciles. The IRSD score and deciles were linked to ED data using the suburb of residence in order to determine socioeconomic position.
# Data analysis
Initial review of the data included all presentations to the regional ED triaged as ATS 4 or 5. The patient's suburb/town of residence was used to exclude attendees from outside this regional city. The decision to focus only on presentations from the local area was to gain greater insight and understanding of the local community and to limit outlying factors that may have influenced the decision by non-local attendees to present to the ED.
Descriptive statistics were calculated using SPSS [29] to summarise the profile of patients accessing the ED with non-urgent conditions throughout the seven-year study period. Linear regression was used to explore trends over time by mode of arrival, referral on departure, episode end status, time of arrival (in-hours versus after-hours) and International Classification of Diseases, version 10 (ICD-10). ABS national census data from 2011 and 2016were used to calculate age-standardised presentation rates by suburb (age-standardised to the overall age distribution profile of the Launceston region in 2016), with linear interpolation used to estimate populations in years between 2011 and 2016. Linear regression, weighted by 2016 suburb populations, was used to fit a trend-line showing the association between agestandardized presentation rate and IRSD, with an outlier suburb excluded. RStudiowas used for regression analyses and plots.
# Results
Between 1 July 2009 to 30 June 2016, there were 305,599 ED presentations across all triage categories (ATS 1-5).a summary of how we determined the number (n = 109,633) included as the study population. Our objectives were to: describe the profile of ED attendees and trends over time through retrospective analyses of routinely collected hospital data; identify the usual place of residence and socioeconomic position of people attending the ED with non-urgent conditions, and to summarise the most frequent discharge diagnoses of the study population and trends over time.
## Profile and trends of people presenting with non-urgent conditions
The first objective was to develop a profile and identify trends in who is presenting and when. The number of non-urgent presentations to the ED revealed similar numbers between the first and last 12-month periods, July 2009 to June 2010 (n = 15,322) and July 2015 to June 2016 (n = 15,139). Over the seven-year study period the annual rate of non-urgent presentations among local residents varied between 186 to 205 per 1000 population.average daily rates by month of all non-urgent presentations. While there were short-term fluctuations in presentation numbers, regression analysis did not reveal any long-term linear trend in the number of presentations (p = 0.61). Over the seven-year study period non-urgent presentations by local residents ranged between 38 and 48 per day.
Analysis of age identified that younger people were over-represented among non-urgent presentations. The median age of the study population was 29 years compared to a median age in this regional city of 39 years. provides a summary of presentation and population numbers aggregated by age. The age profile of the local population was recorded to remain stable between census periods, for example, those under 25 years of age continued to contribute to 31-33% of the local population between census periods.
Trends in mode of arrival revealed a consistency in the number and proportion of patients arriving by their own means (87%; . Analysis of presentation outcomes revealed a large proportion of patients either did not require any follow-up or were referred to their GP (74.7%; and were discharged home from the ED (85.3%). For these two variables (arrival mode and presentation outcome), increases were observed in the number of patients with non-urgent conditions who: arrived by ambulance (average increase of 34 annually, p = 0.002); arrived with police (average increase of 56 annually, p<0.001), or who required admission to hospital (average increase of 56 annually, p<0.001).
Time of day and day of week are presented inwith most non-urgent presentations occurring between 0800hrs and 1800hrs with peaks observed on Monday and Sunday mornings. Analysis of presentations occurring in-hours or after-hours revealed that 47.0% arrived in-hours with significant trends to in-hours and after-hours presentation numbers (2c and 2d). Average annual in-hours presentations fell at a rate of 78 per year (95% confidence intervals 18 to 140, p = 0.012). This was offset by a significant increase in after-hours presentations (rate of increase 108 annually, 95% confidence intervals 31 to 184, p = 0.006).
## Non-urgent ed attendees and socioeconomic levels
The second objective was to establish a profile based on the IRSD deciles according to the patient's suburb of residence. This age-standardised analysis revealed an over-representation by residents living in suburbs categorised as having the greatest socioeconomic disadvantage (IRSD decile 1; . Ten percent of the Australian population live in suburbs rated IRSD decile 1 compared to 26.4% of the Launceston population. In this study, residents of IRSD decile 1 suburbs contributed to 36.8% of non-urgent ED presentations. Further analysis using the underlying IRSD score for each suburb revealed a strong negative correlation between IRSD score and the age standardised rate of ED attendance. Presentation rates for people with non-urgent conditions were 4.5 times higher from the most disadvantaged suburb compared to the most advantaged. Residents from the most advantaged suburb (IRSD score 1090) presented at a rate of 96 per 1000 population while residents from the most disadvantaged suburb (IRSD score 591) presented at a rate of 434 per 1000 population.
## Discharge diagnoses and trends over time
The number of presentations for the three most frequent overarching diagnostic groups are summarised in along with the three most frequently recorded sub-diagnostic groups. Median age and results of linear regression analysis to determine trends in diagnostic groups are also reported in .
The most notable results from this analysis were the high proportion of discharge diagnoses falling into the ICD-10 code for injury. One third of non-urgent presentations were diagnosed with an 'injury, poisoning, certain other consequences of external causes', the most frequent sub-diagnostic groups were injuries to distal limbs or head. These patients were younger and there was no significant trend over the study period.
Significant increases in ED attendance were observed in two diagnostic groups, the first being 'symptoms, signs and abnormal clinical and laboratory findings, not elsewhere classified'. The proportion of patients diagnosed into this non-specific group increased from 6.6% in 2009-10 to 9.1% in 2015-16 (p < 0.001), the equivalent of 70 additional presentations per year.
Mental health conditions also increased significantly between 2009-16. These presentations increased from 1.8% of the study population to 3.1% (p < 0.001), a 73.1% increase in diagnoses relating to mental and behavioural disorders over seven years and equivalent to 31 additional presentations annually.
## Table 1. profile of patients by gender, age and index for relative socioeconomic disadvantage irsd) versus profile of local population, ats 4 and 5, july 2009 to june 2016.
## No. % (n = 109 633) % of local population (n = 81,029: abs, 2016) �
## Gender
# Discussion
This research aimed to identify who, when and why people accessed the ED with non-urgent conditions. In the analysis of seven-years' worth of routinely collected ED data, we discovered:
- No increase in total number of non-urgent presentations;
- A significant over-representation by residents from socioeconomically disadvantaged areas and those younger in age;
- Increasing proportion of after-hours presentations;
- Significant increases in presentations for mental health and non-specific symptoms.
## Consistent demand for ed services by patients with non-urgent conditions
The AIHW have consistently reported national increases in the number of annual ED presentations over the past five years, but an increase was not observed in the number of nonurgent presentations recorded to this ED during the study period. Monthly plots of presentation numbers demonstrate short-term fluctuations in ED usage for non-urgent conditions, with presentation numbers between 186 to 205 per 1000 population per year. The simple linear regression we have performed does not adequately model fluctuations. Analysis of the fluctuations was beyond the scope of this publication but is part of an ongoing investigation by the research team. A consistent demand for ED services by patients with non-urgent conditions has also been reported in research conducted in Northwest Tasmania where limited general practices services were identified as a driver. Furthermore, international literature has identified links between the number of ED presentations and timely access to primary care services. Presentation numbers across day of week and time of day were observed to peak between 0900 and 1100hrs and decreasing throughout the day. This indicates that a significant proportion of non-urgent presentations arrive during hours when other services are open. Tuesdays to Saturdays demonstrated similar presentation times and trends, however, peaks were observed on Sunday and Monday mornings. General practice services on a Sunday are minimal in this regional community leaving residents with the ED as the primary option. The peak on a Monday morning is likely to reflect those, who have waited for regular services to open on a Monday morning but been unable to secure an appointment, thus, resulting in an ED presentation. This again highlights the availability of alternative services at the time of need as a driver of non-urgent ED presentations and may be of interest to local service providers aiming to identify peak times and plan services and staffing based on demand.
## Table 2. summary and trends in ed presentations for mode of arrival and outcome of ed presentation, ats 4 and 5, july 2009 -june 2016.
## No. % (n = 109 663) trend: average annual change in presentations per year (95% confidence interval) p-value for trend
## Mode of arrival
## Over-representation by those from lower socioeconomic suburbs and those younger in age
The correlation between IRSD and the number of non-urgent ED presentations per 1,000 head of population demonstrates a striking over-representation by people living in the most disadvantaged areas. The ED is located close to the central business district and is surrounded by suburbs with IRSD deciles between 3 and 7. Furthermore, the suburb with the highest presentation numbers per 1,000 residents is the same distance from the ED as the suburb with the lowest presentation numbers, both being 11km from the ED. This shows that socioeconomic status is a stronger contributor to ED attendance than distance in our region. A higher proportion of non-urgent ED presentations by those living in close proximity has been previously reported; however, this was not the case in this study and highlights the contextual nature of how local populations access health services.
The only exception to the correlation between socioeconomic position and incidence of ED presentationis the city centre. This appears to have occurred when the person providing the patient's details or staff member entering the data has listed the over-arching area of Launceston as the suburb of residence rather than the patient's actual suburb of residence. For example, it is not uncommon for residents from Launceston's lowest IRSD suburbs to list their suburb of residence as Launceston where it shares the same postcode as their actual suburb. These presentations were plotted inthey contribute to the overall number of presentations. However, the data from the city centre were excluded from the weighted regression analysis to fit a trend line due to the recording error.
Findings of over-representation among populations with greater socioeconomic disadvantage are varied across international literature. Some studies report similarly over-represented . Top three diagnostic groups and diagnostic groups with significant trends (based on international statistical classification of diseases and related health problems 10 th Revision: ICD-10). ATS 4 and 5, July 2009 to June 2016.
## Diagnosis, top ten icd-10 in order of frequency most frequent subdiagnoses
## No. presentations (% of subdiagnostic group)
Proportion presentations (n = 109 663) (%)
## Median age (iqr, years)
Trend over time presentations by disadvantaged communitieswhile a Canadian study found midhigh-income communities were over-represented. Additionally, a study from the UKreported that disadvantaged communities had lower ratios of GPs per 1,000 head of population. While it was outside the scope of this study to measure the number of GPs per 1,000 during the study period, it was observed that none of the larger practices with ready access to additional services such as pathology and radiology are located within the most disadvantaged areas of this local community. Furthermore, northern Tasmania was reported to have fewer full-time equivalent GPs in 2014, 70.3 per 1,000 population, versus 85.4 per 1,000 in southern Tasmania. These findings highlight contextual differences in the ability of populations to access health services and demonstrates a disparity in the provision of healthcare services in the most socioeconomically disadvantaged areas of this community. Further supporting this finding, are two studies, one from the US focusing on paediatric presentationsand the other from New South Wales looking at all presentations (adult and paediatric). Both studies found that fewer GPs per 1000 population contributed to higher rates of non-urgent ED presentations. Being younger in age was also a significant factor with a clear over-representation by those in the 0 to 4 and 15 to 24 age groups. These two groups were 1.5 times more likely to present with a non-urgent condition than the rest of the study population. This finding is consistent with international studies from the United States, Canada, Switzerland, the United Kingdom, and Australiaall observing an over-representation in non-urgent presentations by younger populations. Consideration of why this over-representation is occurring may contribute to further understanding of the decision-making processes of young people and access to alternative services for this group.
It is likely that the over-representation of residents from socioeconomically disadvantage areas and by those younger in age is reflective of challenges faced by these populations in accessing the right service at the right time and located in the right place. This information will be of interest to future service planning.
## Increased non-urgent presentations after-hours
An increasing number of people arriving after-hours was also identified. Most GP services in this community are available within normal business hours (0800 to 1800 weekdays and 0800-1200 Saturdays, excluding public holidays). Access to services is limited outside these times. The increase in demand for after-hours services is likely to reflect a lack in available services within the community at the time of need. Two other Tasmanian studies also found increases in after-hours presentationswhile another local study identified 31% of patients attending the ED would have preferred to be managed by their GP if they had been available at the time of need. These findings further support the need for the right services to be available at the right time. As the third Tasmanian project to report a significant increase in the demand for after-hours services it is likely that further research exploring service demand and availability during these hours may assist in informing the provision of timely, patient-centred services and reduce ED demand.
## Increased presentations with non-urgent mental health diagnoses and with non-specific symptoms
The final objective was to identify prominent reasons for presentations through analysis of discharge diagnosis based on ICD-10 codes. Unsurprisingly, presentations as a result of injury were the most common discharge diagnostic group with one third of all nonurgent presentations being as a result of 'injury, poisoning, certain other consequences of external causes'. This is consistent with non-urgent presentations across Australia, the AIHW reporting that in 2017-18, 32.7% of non-urgent ED presentations were allocated into this principle diagnostic group. Other studies have also found similar proportions for this diagnostic group.
A significant increase was observed in diagnoses into the non-specific group of 'signs and symptoms or abnormal clinical findings not elsewhere classified'. This includes people who present to the ED for simple examination, investigation or observation, the proportion found in this study is reflective of nationwide trends for this principle diagnostic group. The significant increase may be explained by international research which clearly identifies the patient's perceived need for urgent medical attention as a major theme when investigating reasons for accessing ED services with non-urgent conditions. The continued high proportion of patients who were discharged home and did not require specialist follow-up in this study raises questions around health literacy, health anxiety and timely access to alternative services.
Diagnoses of 'mental and behavioural disorders' was the only other diagnostic group observed to increase significantly with an additional 30 people per year presenting to this regional ED. To the best of our knowledge, this patient group has not been identified as an increasing proportion of non-urgent ED presentations. In 2017-18 the AIHW recorded 2.6% of ATS 4 and 5 presentations resulting in a mental health or behavioural diagnosis, for the same period this regional ED observed 3.1%. While these are similar proportions to national figures, we were able to identify a concerning increase of 73.1% between 2009-10 and 2015-16 in our regional ED. Limitations in AIHW reporting meant we were not able to compare this increase with earlier national numbers. A patient triaged as an ATS 4 or 5 with a mental health presentation must demonstrate the ability to provide a clear history without signs of restlessness or aggression.
It is not known what has caused this dramatic increase in mental and behaviour diagnoses within the local region. However, if the ED provides an indication of people's healthcare needs and the level of access to services within the community, this increase must be a warning to local service providers. Mental health was identified as the predominant concern for young people in a 2018 national survey of over 28,000 participants aged 15 to 19 years. This report identified for the first time in 17 years that the top concern for youth was mental health. This growing concern among young people and the increasing presentation numbers within this regional community provide policy makers and service providers with a clear local need.
# Limitations
This longitudinal observational study was reliant on routinely collected hospital data; efforts were made to review data for possible discrepancies. The findings are largely reliant upon the quality of data collected at the time of the patients' presentation. Population and socioeconomic position data were based upon ABS data collected in 2011 and 2016 with changes occurring across this time period, to allow for these changes we presumed a direct linear relationship between the two data collection periods. This may not reflect true numbers but provided the closest solution to changes available between these two time periods.
Data provided by the DHHS were for ATS 4 and 5 presentations only, therefore it was not possible to compare presentation trends across all triage categories. This broader analysis was beyond the scope of this project and highlights an area for future enquiry.
# Conclusion
The ED is a 'canary in the coalmine' for the greater health service and community. The overrepresentation of population groups and increases in demand provide clear indicators of the healthcare needs of members of the local community. Patients presenting to this regional ED with non-urgent conditions were younger than the local demographic profile and up to four times more likely to live in the most disadvantaged communities, raising the question of service accessibility and availability in areas of need. In addition, patients are increasingly presenting with non-specific symptoms and with mental health and behavioural issues. These findings will be of use to policy-makers in planning for enhanced primary care service for the young and for people with mental health issues from our most disadvantaged communities.
Supporting information S1 File. |
Chronic Severe Sleep Problems among Non-Nordic Immigrants. Data from a Population Postal Survey in Mid-Sweden
Sweden has a large population of both recent and established immigrants with high prevalence of risk factors for ill health. Here, we aimed to explore the prevalence of chronic severe sleep problems (CSSP) among non-Nordic-born persons, and to evaluate the risk for CSSP when fully adjusted for covariates. Our additional hypothesis was that lengthier time since immigration would reduce the risk for CSSP. We used data from a large-population postal survey covering life and health issues among inhabitants in mid-Sweden. Relationship between different countries of birth and CSSP was assessed in logistic analyses for more severe and longstanding pain, sex, employment, mental disability, gastrointestinal problems, and length of stay (short, middle time, and up to ten years of stay). Persons of non-Nordic birth reported significantly more often CSSP, regardless of short or long-term stay. Our findings indicate that non-Nordic birth, regardless of residence time and covariates, was an independent and significant predictor for CSSP. The findings may contribute to increasing awareness in healthcare personnel to recognize chronic sleep problems among immigrant patients. Thus, our study might contribute to developing strategies to enhance health for minorities.
# Introduction
Satisfactory sleep quality is fundamental for good mental and physical health. Insomnia, or sleeplessness, is a disorder on its own, calling for independent clinical attention, and the diagnostic criteria have recently been made more specific by using frequency and duration for defining the condition.
Longstanding sleep problems increase the risk of mental and physical illness, but have daytime consequences as well, such as slower responses to challenging tasks, all leading to raised costs for health care and accidents. Problematic sleep is often associated with negative lifestyle pattern, weak social network, and poor societal integration. The process of acculturation, unemployment, negative prior life experiences, low self-rated health, and limited access to healthcare are other complicating aspects for many immigrants and refugees. Patients with multiple ill-health problems interfering with good sleep tend, therefore, to be especially common in multicultural primary care settings, and of special interest for their caregivers.
Every sixth person is born outside Sweden (foreign-born or non-Swedish), of which one half or more is born in non-European countries, having insufficient education and small financial margins due to insecure employment or low-income jobs, and pain is a common complaint in this citizen group. Data on longstanding bothering sleep problems among immigrants are few, and show varying results. Based on data from the National Survey in Sweden 1996, found that Bosnian refugee women on a group level had a much higher risk of undefined sleep difficulties than Swedish-born women, and Taloyan et al. found that Kurdish men twice as often reported sleep difficulties, compared with the Swedish-born men. A Swiss study found that the non-Western recent immigrants also, after many years of stay, reported more early awakening and trouble falling asleep, as compared with non-immigrants. This finding was expected to be due to high levels of emotional distress. However, rather new immigrants in a Swedish county reported no inferior physical or psychological health, quality of life, wellbeing, or social functioning, compared with their age-and sex-matched native-born controls. Furthermore, in adjusted analyses, the established immigrants in Canada reported fewer sleep troubles, as compared with non-immigrants.
Because of mentioned differences in findings, we planned another study, based on a large postal survey in Sweden, focusing on severe and longstanding, i.e., more than three months, sleep problems reported by immigrants with more evident cultural distance than Nordic-born immigrants. The Nordic-born populations have, in general, a relation regarding language, tradition, history, and societal organization (Wikipedia), and are less likely to suffer from discrimination, with its negative impact on mental health.
In view of the above, we wished to explore the prevalence of the severe longstanding sleep problems (here: chronic severe sleep problems, CSSP) among foreign-born persons, and if their immigrant status and/or the length of stay in Sweden had independent roles for this serious type of sleep problem.
Our aim here was to explore the data from a large population postal survey conducted in mid-Sweden for the reported prevalence of CSSP by countries of birth, with focus on non-Nordic-born, and subcategories of persons born in Europe and non-Western countries. In addition, we aimed to explore if the number of years since immigration (length of stay) to Sweden would modify this relationship.
We hypothesized that especially non-Nordic born persons would have significantly higher risks of CSSP, and also when fully adjusted for well-known covariates for sleep problems. Our additional hypothesis was that lengthier time since immigration would reduce the risk for CSSP.
# Materials and methods
## Study conduct and study population
We based this paper on a cross-sectional analysis of population-based postal survey data. The postal survey, Life and Health, was distributed in spring (March to May) 2008, to a randomized population-based sample of 68,710 adults age 18 up to 84 years old in the following five counties: Sörmland, Uppsala, Värmland, Västmanland, and Örebro of mid-Sweden. Three subsequent letters reminded all potential participants of the postal survey. The five counties in mid-Sweden include both big cities and smaller communities. The study sample was a randomly chosen sample of the general population with clusters regarding age, sex, and county and city, or parts of the city for larger cities. The survey letter included an information sheet about the study. The participants accepted their approval in the returned survey.
The distributed questionnaire collected data on diseases and disorders, lifestyle, work, and education. The set of questions in this questionnaire was derived from international validated questionnaires such as the Public Health Questionnaire and the General Health Questionnaire (GHQ12). Similar postal surveys were distributed every four years in the five counties. Equivalent questions were also used in the national health surveys distributed by The Public Health Agency of Sweden.
## Outcome variable
Bothering sleep problems was rated by a single item assessing if, and how often, during the last three months, the participants had experienced sleep problems, in four choices. Participants could respond has not been bothered; has been bothered a single time; has been bothered multiple times; or has been bothered almost all the time, during the past three months. For this study, the item was dichotomized as having chronic severe sleep problems (CSSP = sleep problems multiple times, or almost all the time, for three months or more) or not (no CSSP = (no, or occasional, sleep problems).
## Explanatory variables
The main explanatory variable was country of birth, categorized into the region of birth. Nordic countries were Sweden, Norway, Denmark, and Finland. Non-Nordic countries were all countries outside the four Nordic countries. Sub-analyses were performed with the participants categorized as (i) born in Sweden vs. not born in Sweden (non-Swedish); (ii) born in Europe vs. not born in Europe (non-Europeans); and (iii) born in the Western world vs. not born in Europe, the US, or Australia (non-Western).
## Covariates
Pain was measured in the questionnaire by a single item assessing how often, during the last three months, the participants had experienced bothering pain in the shoulders, neck, back, hips, extremities, stomach, or head. Participants could respond never; single time; multiple times; or almost all the time. Bothering pain was summarized into a binary variable-none/single time, and multiple times/almost all the time. Gastrointestinal problems were handled in the same way, and summarized into a binary variable: no/single time and multiple times/almost all the time. Other binary covariates were sex, mental disability, and unemployment. Age was categorized into five categories: 18-34 years, 35-49 years, 50-64 years, 65-79 years, and >80 years. (cf.. Note: # Bothering condition for more than 3 months; ## Number based on which year people immigrated to Sweden.
# Statistical methods
Cross-tabulation was used to describe the study population. Descriptive analyses were performed for CSSP and the region of birth and the confounding variables. Crude analyses were done for the persons born in the Nordic region and the persons born in the non-Nordic countries, for relations between the baseline variables and CSSP. We performed a stepwise forward logistic regression analysis with the most important covariates according to the literature. We also added economic problems and received social welfare, sick leave, self-reported abuse during the last 12 months, anxiety, atopic dermatitis, diabetes, cancer, tiredness, burn-out, sleep apnea, and consumption of sleeping pills.
Independence of the above relationships was tested by logistic regression, and adjusting them for the confounding variables.
We also analyzed if sex modified the relationship between CSSP and countries of birth, as well as CSSP and categories of the length of stay in Sweden. Separate adjusted analyses were performed for three categories of length of stay.
Sensitivity analyses were performed concerning different definitions of immigration: these analyses included CSSP for the Swedish-born as compared with persons born in any other country (non-Swedish), or those born in a country outside Europe (non-European) as compared with persons born in European countries (European), or persons born outside the Western world (non-Western) as compared with immigrants from the Western world (here in Europe, USA, or Australia).
Fewer than 5% of the subjects had missing data for some of the studied factors. They were excluded from the analyses.
Statistical significance was defined as a p-value < 0.05 or a 95% confidence interval (95% CI). The STATA analysis program version 14 was used for statistical analysis.
# Ethical approval
The study followed the Swedish guidelines for studies in social sciences and humanities, and followed the Declaration of Helsinki. According to Swedish regulations, ethical approval by a medical faculty is no longer required for this type of study, with data from anonymous postal surveys where specific persons cannot be identified and are not comprised by the type of research which the Swedish legislations of research ethics have defined as requiring an ethical approval by a Regional Ethical Board (the Ethical Review Act of Sweden 2003:460 and the Personal Data Act). The decision on this study was made accordingly by the ethical committee in Uppsala, 2003, Sweden, EPN 2012/256.
# Results
## Participants
In total, 40,674 adults (45.5% males) returned the questionnaire, corresponding to a response rate of 59.2% (mean age 53.8 years, Std 17.92). The majority was born in Sweden or in the other Nordic countries (37,450; 93.7%). Thus, only 6.3% (n = 2493) of the participants were of non-Nordic birth. A minority of them, n = 1304 (24.2%) had immigrated during the past ten years, and the majority n = 4094 (75.8%) more than ten years previously. Slightly more women than men participated in the study (54.5%). Nearly a fifth of all the participants were unemployed. Swedish-born persons and persons from other Nordic countries had higher response rates (range 59-63% vs. 57-63%, respectively) than persons from non-Nordic countries (41-44%). Women had a higher response rate, 63-67%, vs. men, 51-54%. Persons with higher age, higher education, and employment had higher response rates.shows the distribution of the explanatory and the outcome factors in numbers and percentages. The majority, 78.1%, of the participants had no, or only mild, sleep problems (no CSSP), which was especially common among the youngest participants. One-fifth (21.9%) had CSSP, which was significantly more frequent among the participants of non-Nordic birth (28.5% vs. 21.4%). Furthermore, women, unemployed, and participants with bothering pain, gastrointestinal problems, and/or mental disability had significantly higher frequencies of CSSP. To note, the non-Nordic born participants had still a significantly higher frequency of CSSP after 10 years of residence time.shows that non-Nordic birth predicted CSSP also when adjusted for the female sex, unemployment, bothering pain, gastrointestinal problems, and mental disability. Age did not confound the region of birth/CSSP relationship. Bothering pain was the most important covariate.shows that the adjusted odds ratios for CSSP were increased for all categories of subgroups of persons born outside Sweden. The odds showed insignificant trends with overlapping confidence intervals for different definitions of countries of birth. Persons born in non-European countries had an aOR of 1.33, and persons born outside the Western world had an aOR of 1.37. The category including all persons born outside Sweden had an aOR of 1.21. The covariates for CSSP were similar in all country of birth categories. There was no change in the relationship between country of birth and sleep problems when adding the other potential covariates.shows that the odds ratios for non-Nordic birth and other covariates as having CSSP remained at similarly increased levels over the years since immigration, with no improvement regarding chronic severe sleep problems for the non-Nordic participants with longer stay in Sweden.
## Statistics
# Discussion
This population postal survey in Sweden shows that not being born in a Nordic country was an independent and significant predictor for chronic severe sleep problems (CSSP). It also shows that the increased odds for non-Nordic birth, and the covariates, for CSSP remained at similarly increased levels over the years since immigration.
We believe our findings in this study to be valid and robust. A single question is used to identify insomnia in many surveys. In this population study, there were four alternatives to the question on sleep problems. The rationale here was to study the most severe cases of insomnia. Thus, we defined CSSP as sleep problems multiple times/almost all the time continuously for three months or more, a definition that corresponds with the DSM-5 definition of sleeplessness. The covariates from this population questionnaire are considered as valid and reliable in this context, and used repeatedly in large population studies in Sweden. Furthermore, the Swedish technology assessment institute has expressed a wish of closing some knowledge gaps regarding subjective assessment in self-rated questionnaires by using binary variables instead of multiple choice. We adhered to this advice also, regarding the covariates pain, mental disability, and gastrointestinal problems.
Higher frequencies of undefined problematic sleep are found among the immigrants in some other study samples. Only small differences were found here between the foreign-born participants, but with trends to higher odds for the participants of non-Western birth. However, immigrants are not a homogenous concept, including both refugees, asylum seekers, students, and workforce labor from outside Sweden. Such detailed information was not accessible in this survey material. Here, we used non-Nordic birth as defining immigrants, since the Nordic countries are quite similar societies in many aspects. This definition can, however, be discussed. Furthermore, our other categories only vaguely characterize cultural and linguistic distance from Sweden, and may be here reflected in the trend towards a higher prevalence of CSSP among the more recent immigrants. In clinical practice, this might mean that long-term severe sleep problems should be born in mind also for persons from neighboring countries or long-term immigrants, and that severe longstanding problematic sleep can be an independent problem, and not always a component of ill-health or social malfunction.
Our study showed that both severe pain and mental disability independently predicted CSSP more than short-term and long-term non-Nordic birth, and regardless of sex. However, the non-Nordic birth had a statistically significant, albeit rather small, but yet an independent, impact on longstanding severe sleep problems also in the long term. Hypothetically, this could reflect an adaption to attitudes in communicating health problems in the host country, and/or a withstanding negative acculturation effect with disappointment, segregation, prejudices, and undefined ill-health.
Two novel factors appear in our present paper: non-Nordic birth as an independent risk factor for chronic severe sleep problems, with no actual change over time. Otherwise, we found the same correlates for longstanding severe sleep problems as earlier studies, notably older age, female sex, pain, gastrointestinal problems, mental disability, and unemployment.
Notably, negative socioeconomic factors or higher body mass index seem not to be linked to altered sleep patterns. Both conditions are common among many immigrant groups, and so are unspecified mental disorders when adjusted for socioeconomic factors and knowledge of Swedish. Immigrants on a group level seem overall to have elevated risks independently for depression and psychotic disorders when adjusted for socioeconomic factors and knowledge of Swedish. Further, burnout symptoms are common among foreign-born women in Sweden, again not found to be independently related with sleep problems. An important contributing factor for bad sleep is widespread pain, a condition which tends to be common among foreign-born persons. This type of pain may alter the phase of the deep sleep and increase the pain sensation.
The participants in this postal survey were slightly better educated than the general population in Sweden, and more women and old people participated. Therefore, it is possible that especially younger men with little education are underrepresented in this study cohort, regardless of their country of birth. The potential selection might bias the results towards the null value. Also limiting the transferability of our results is the low participation rate of more recent immigrants, probably due to poor proficiency in Swedish. This study might, therefore, have underestimated, e.g., their mental health problems. To compare, in a small study, the prevalence of insomnia among recent immigrants from the Middle East was as high as among Swedes suffering from chronic stress.
One strength of our study is the large population-based sample of adult individuals. Another strength is the four-level scale for self-reported sleep difficulties. Only the most severe level was in focus for our analysis. The reason was that the most evident risk factors for future ill-health are the chronic severe sleep problems. On the other hand, the simple design of our study has several limitations. First, the cross-sectional nature of self-reported data makes it impossible to conclude the causes and the effects. Second, the level and frequency of chronic sleep problems can be overestimated, or be underreported, by individuals or groups of participants. The extent of this remains unclear. Third, the survey did not include any questions regarding nature or details of sleep problems. In addition, it will be impossible to reproduce our study in later postal surveys, due to differences in alternative answers. Therefore, our conclusions remain to what it is-a study of chronic severe sleep problems and immigrant status framed by its time. Despite all these limitations, it should be possible to transfer our results to Western societies with large, new, and older immigrant populations with varying sociocultural backgrounds.
Large longitudinal epidemiological population studies comparing data over time are justified to give sufficient power to identify the country of birth, length of stay, specific ill-health factors, and sleep quality. Further data of interest in the subject of problematic sleep and immigration could be provided by studies using qualitative methodology in different immigrant groups.
# Conclusions
To conclude, not being born in a Nordic country was an independent and significant predictor for chronic severe sleep problems, regardless of the length of stay in Sweden. The findings may contribute to increasing awareness in healthcare to recognize chronic sleep problems among immigrant patients with, or without, other well-known risk factors. Our study might thus contribute to developing strategies to enhance health for minorities.
Author Contributions: Each author has made substantial contributions to the design of the study, analysis, and interpretation of data. Each author has approved the submitted version and agrees to be personally accountable for the author's own contribution to the study. Conceptualization, methodology, validation and formal analysis, investigation, resources, data curation, writing-original draft preparation, visualization: M.L., L.B., and L.v.K.; supervision, project administration, and funding acquisition: M.L. and L.v.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Uppsala-Örebro Regional Research Council, Sweden. |
Two Cases of Rectal Xanthoma Presenting as Yellowish to Whitish Lesions during Colonoscopy
Two cases of rectal xanthomas are described. One case is that of a 56-year-old Japanese man in whom multiple yellowish spots measuring approximately 3 to 5 mm were observed in the rectum during colonoscopy. The other case is that of a 78-year-old Japanese man in whom colonoscopy showed a whitish plaque of 4 mm in diameter in the rectum. Biopsy examinations performed on both patients revealed the deposition of xanthoma cells within the rectal mucosa. Within the gastrointestinal tract, xanthomas most frequently arise in the stomach, whereas the colorectum is rarely affected. Despite this infrequency, the two cases indicate that xanthomas should be recalled when yellowish to whitish lesions are observed in the colorectum.
# Introduction
Xanthomas in the alimentary tract are benign mucosal lesions resulting from the aggregation of foamy histiocytes within the gastrointestinal mucosa [bib_ref] Xanthomas of the stomach: a report of two cases, Andrejic [/bib_ref]. Within the gastrointestinal tract, the stomach is the most frequently affected by xanthoma, whereas other parts such as the esophagus, duodenum [bib_ref] Xanthoma of the Duodenum, Iwamuro [/bib_ref] , small intestine, and colorectum usually remain unaffected. Macroscopic features of gastric xanthoma are well known as yellow to white well-demarcated plaques or nodules [bib_ref] Gastric Xanthoma: A review of the literature, Basyigit [/bib_ref]. Endoscopic images of colorectal xanthoma have rarely been reported in the literature due to their infrequency.
We recently encountered two patients with rectal xanthomas that were observed as yellowish to whitish lesions during colonoscopy. In this report, we focus mainly on the macroscopic characteristics of the colorectal xanthoma and review previously reported cases of this disease.
## Case report
## Case 1.
A 56-year-old Japanese man was referred to our hospital for investigation of tarry stool. The patient had been consuming lansoprazole, irsogladine, metoprolol, flutoprazepam, and ethyl loflazepate for gastritis, hypertension, and anxiety disorder but had no history of dyslipidemia or diabetes mellitus. A physical examination revealed no abnormalities in his abdomen or xanthomas on his skin, and laboratory findings showed no abnormalities. The levels of cholesterol, triglyceride, and plasma glucose were within normal range. Esophagogastroduodenoscopy showed erosive and atrophic gastritis.
During colonoscopy, multiple yellowish spots measuring approximately 3 to 5 mm were observed in the rectum, in addition to hemorrhoids [fig_ref] Figure 1: Colonoscopy images of case 1 [/fig_ref]. Magnifying observation with narrow-band imaging revealed that the pits of the rectal mucosa were intact [fig_ref] Figure 1: Colonoscopy images of case 1 [/fig_ref]. Indigo-carmine spraying emphasized the whitish to yellowish color of the lesions (Figures 1(c) and 1(d)). Histological analysis of the biopsied samples revealed accumulation of xanthoma cells within the mucosal layer. Consequently, a diagnosis of rectal xanthoma was made.
## Case 2.
A 78-year-old Japanese man had been treated for remitting seronegative symmetrical synovitis with pitting edema. The patient underwent colonoscopy for screening purposes. He had been taking 2 mg/day of prednisone but had no history of dyslipidemia or diabetes mellitus. A physical examination revealed no xanthomas on his eyelid or extremities and a blood test revealed that his levels of cholesterol, triglyceride, and plasma glucose were within normal range. Colonoscopy showed a whitish plaque of 4 mm in diameter in the rectum [fig_ref] Figure 2: Colonoscopy images of case 2 [/fig_ref]. Biopsy examination revealed massive deposition of xanthoma cells within the rectal mucosa [fig_ref] Figure 3: Pathological image of case 2 [/fig_ref] , leading to the diagnosis of rectal xanthoma.
# Discussion
In the two cases presented, rectal xanthomas were observed as yellowish to whitish lesions during colonoscopy. As described above, typical gastric xanthomas are well-demarcated whitish plaques or nodules. Similarly, cases with xanthomas in the sigmoid colon and rectum, showing multiple, well-defined, and whitish-yellow lesions, have been previously reported [bib_ref] Xanthoma disseminatum with large plaques confined to the back, pulmonary involvement and..., Hisanaga [/bib_ref] [bib_ref] Multiple lipid islands of the colonic mucosa. A light and electron microscopic..., Remmele [/bib_ref]. Weinstock et al. reported a case consistent with xanthoma presenting as flat, yellow, and irregularly shaped lesions in the sigmoid colon [bib_ref] Xanthoma of the colon, Weinstock [/bib_ref]. Endoscopic images presented in their report show a hexagonal-shaped appearance, which is not similar to the macroscopy of gastric xanthomas. The case report by Moran and Fogt, which did not present endoscopic images, described a xanthoma in the rectosigmoid colon as polypoid in appearance [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref]. Miliauskas et al. reported that, by reviewing their four cases and nine previously reported cases, papules were observed in eight cases and polyps were noted in three cases [bib_ref] Rectosigmoid (colonic) xanthoma: a report of four cases and review of the..., Miliauskas [/bib_ref] [bib_ref] Colorectal xanthomas with polypoid lesion: report of 25 cases, Nakasono [/bib_ref]. Nakasono et al. summarized 28 colorectal xanthomas biopsied from 25 patients. Xanthomas were located in the sigmoid colon (17/28 lesions) and rectum (11/28 lesions) [bib_ref] Colorectal xanthomas with polypoid lesion: report of 25 cases, Nakasono [/bib_ref]. Macroscopically, 23 lesions presented sessile appearance and the remaining five lesions were pedunculated. Twelve of the xanthoma lesions were reddish, five were whitish, two were yellow-whitish, and one was normal color. Consequently, the morphology of colorectal xanthomas varies from flat, sessile to pedunculated lesions, with yellowish, yellow-whitish, whitish, and even reddish colors. Endoscopists must recall this entity as a differential diagnosis when they observe whitish lesions in the colorectum regardless. In the two cases we presented, narrow-band imaging highlighted lesions that were yellowish to whitish in color. Moreover, observation under magnification revealed intact pits in the rectal mucosa. We propose that the intact pits reflect undamaged epithelial cells observed by histopathological examination of biopsy specimens. However, since this observation is based on only two cases, usefulness of observation under magnification and optical chromoendoscopy techniques such as narrow-band imaging, flexible spectral imaging color enhancement, and i-SCAN for the diagnosis of colorectal xanthomas should be further investigated.
Diseases other than xanthomas that present with yellowish to whitish lesions in the colon include pseudomembranous colitis, lipomas, and lymphomas. Pseudomembranous colitis is a common cause of antibiotic-associated diarrhea. This disease is characterized by elevated yellow-white plaques that coalesce to form pseudomembranes along the colorectal mucosa and can be easily diagnosed based on their endoscopic appearance [bib_ref] Pseudomembranous colitis: not always caused by clostridium difficile, Tang [/bib_ref]. Colonic lipomas are generally observed as solitary, soft, spherical, smooth yellowish lesions [bib_ref] Caecum lipoma: A rare cause of lower gastrointestinal bleeding, Martinez-Mier [/bib_ref]. Pedunculated and semipedunculated lipomas can be easily diagnosed, but those with a slightly elevated appearance may be misinterpreted as xanthomas. Lymphomas, particularly the indolent subtypes, sometimes present as whitish, slightly elevated lesions in the colorectum. For instance, follicular lymphomas in the colorectum are identified as papular, polypoid, or flat elevated lesions [bib_ref] Colorectal manifestation of follicular lymphoma, Iwamuro [/bib_ref]. While the color of the lymphoma lesions is not grossly different than that of the surrounding intact mucosa, colorectal xanthomas, as observed in this report, do show a definite color contrast between the lesions and surrounding mucosa.
Microscopically, foamy macrophages are generally confined to the lamina propria mucosae of the colorectum; the muscularis mucosae or submucosa is rarely affected. Nakasono et al. reported that hyperplastic change was identified in the surface epithelium in 22/28 lesions [bib_ref] Colorectal xanthomas with polypoid lesion: report of 25 cases, Nakasono [/bib_ref]. In addition, thickening of the basement membrane of the surface epithelium, cell debris, and proliferation of the capillaries were frequently observed. In contrast, the two presented cases lack hyperplastic change in the epithelium. In case 2, we speculate that the accumulation of foamy cells itself accounts for the slightly elevated morphology since prominent deposition of foamy cells exists in the lamina propria mucosae [fig_ref] Figure 3: Pathological image of case 2 [/fig_ref].
Although cutaneous and tendinous xanthomas occur in relation to hyperlipidemia, gastrointestinal xanthomas are not associated with dyslipidemia [bib_ref] Gastric Xanthoma: A review of the literature, Basyigit [/bib_ref] [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref] [bib_ref] Gastric xanthomatosis and cholestasis -A causal relationship, Coates [/bib_ref]. reported that, among four cases with colorectal xanthomas, none had hyperlipidemia and only one had diabetes mellitus [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref] [bib_ref] Rectosigmoid (colonic) xanthoma: a report of four cases and review of the..., Miliauskas [/bib_ref]. The two cases presented did not have metabolic disorders, including increased lipid levels and diabetes mellitus. Gastrointestinal xanthomas are asymptomatic and believed to be harmless [bib_ref] Rectosigmoid (colonic) xanthoma: a report of four cases and review of the..., Miliauskas [/bib_ref] [bib_ref] Colorectal xanthomas with polypoid lesion: report of 25 cases, Nakasono [/bib_ref]. Therefore, no specific treatment is considered necessary for colorectal xanthomas.
The etiology of colorectal xanthomas remains unknown. Xanthomas in the stomach are assumed to arise as an inflammatory response to focal mucosal damage and chronic injury such as chronic gastritis [bib_ref] Gastric Xanthoma: A review of the literature, Basyigit [/bib_ref] [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref] [bib_ref] Lipid islands of the stomach: An insular issue, Lechago [/bib_ref] [bib_ref] Histochemical and immunohistochemical characterization of foamy histiocytes (muciphages and xanthelasma) of the..., Bejarano [/bib_ref] [bib_ref] Xanthomatous hyperplastic polyps of the stomach: Clinicopathologic study of 5 patients with..., Bassullu [/bib_ref]. The resident macrophages commonly exist in the subepithelial lamina propria of the gastrointestinal tract. Although macrophages are not pathologically noticeable in normal gut mucosa, they can be identified when they phagocytize and accumulate exogenous or endogenous substances [bib_ref] Macrophage-related diseases of the gut: a pathologist's perspective, Sagaert [/bib_ref]. Content of the foamy macrophages in xanthomas is assumed to come from lipids derived from damaged cell membranes [bib_ref] Gastric Xanthoma: A review of the literature, Basyigit [/bib_ref] [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref] [bib_ref] Lipid islands of the stomach: An insular issue, Lechago [/bib_ref]. Mucosal damage and chronic injury are believed to be associated with the pathogenesis of colorectal xanthomas [bib_ref] 70-year-old female presenting with rectosigmoid (Colonic) xanthoma and multiple benign polypscase report, Moran [/bib_ref] [bib_ref] Histochemical and immunohistochemical characterization of foamy histiocytes (muciphages and xanthelasma) of the..., Bejarano [/bib_ref]. Toxic factors, focal infection, or mechanical damage by peristalsis or contact with feces may cause such injury to the colorectal mucosa [bib_ref] Multiple lipid islands of the colonic mucosa. A light and electron microscopic..., Remmele [/bib_ref] [bib_ref] Colorectal xanthomas with polypoid lesion: report of 25 cases, Nakasono [/bib_ref].
In conclusion, we encountered two patients with rectal xanthomas. Both cases showed yellow to whitish lesions in the rectum. Although colorectal xanthoma can present varied morphology, endoscopists should consider this entity when yellowish to whitish lesions are observed in the colorectum.
## Conflicts of interest
The authors state that they have no conflicts of interest.
[fig] Figure 1: Colonoscopy images of case 1. Multiple yellowish spots measuring approximately 3 to 5 mm are seen in the rectum (a). Magnifying observation with narrow-band imaging reveals intact pits of the rectal mucosa (b). Indigo-carmine spraying emphasizes the whitish to yellowish color of the lesions (c, d). [/fig]
[fig] Figure 2: Colonoscopy images of case 2. A whitish plaque of 4 mm in diameter in the rectum is seen (a). Narrow-band imaging (b) and indigo-carmine spraying (c) show whitish lesions more clearly. [/fig]
[fig] Figure 3: Pathological image of case 2. Biopsy examination reveals massive deposition of xanthoma cells within the rectal mucosa. [/fig]
|
Measuring oral health during pregnancy: sensitivity and specificity of a maternal oral screening (MOS) tool
Background: Midwives can play a key role in promoting the oral health of pregnant women and assessing their oral health status. A maternal oral assessment tool (MOS) was developed and pilot tested by the study investigators to assist midwives in this role and the results were promising. The aim of this study was to undertake further sensitivity and specificity assessment of the MOS tool using two-comparison approaches-the longer oral health screening tool known as the Oral Health Impact Profile (OHIP-14) and an oral assessment by trained study dentists. Methods: Pregnant women were recruited for this study as part of a larger randomised controlled trial of a Midwifery Initiated Oral Health (MIOH) program. Pregnant women completed the MOS and OHIP-14 as part of their initial assessment undertaken by 38 trained and accredited midwives. A dental assessment was conducted for all women in the intervention group using three trained study dentists with high inter rater reliability.Results: Two hundred and eleven pregnant women participated in the validation of the MOS tool. Results from both approaches found the MOS tool to have high sensitivity, correctly identifying 88-94 % of women at risk of poor dental health, and low specificity (14-21 %).Conclusions:This study has shown that the MOS tool can be successfully implemented by midwives during a woman's first antenatal visit and can identify up to 94 % of women at risk of poor oral health and needing a dental referral. The tool has the potential to be transferable to other antenatal care providers and could be incorporated into hospital obstetric database systems. Trial registration number: ACTRN12612001271897, 6 th Dec 2012, retrospectively registered.
# Background
Population oral health experts within Australian and internationally have developed guidelines [bib_ref] working group 3 of the joint EFPAAPw. Periodontitis and adverse pregnancy outcomes:..., Sanz [/bib_ref] that emphasise the importance of oral health screening for women during pregnancy. Pregnancy increases the risk of oral health problems (periodontal disease and tooth decay) due to physiological changes associated with pregnancy [bib_ref] Oral health during pregnancy, Silk [/bib_ref]. In addition, early screening of women for oral health problems, provides an opportunity for women to learn about how to prevent decay for themselves and also for their future or existing children [bib_ref] Mothers' caries increases odds of children's caries, Weintraub [/bib_ref]. The initial comprehensive review of women's health is conducted at the antenatal clinic by midwives for most Australian women. This provides a unique opportunity to assess and intervene to improve the oral health of women and their children. The potential for midwives and other antenatal care providers to also undertake an assessment of women's health at that initial visit (primary care settings) has been supported in other studies [bib_ref] working group 3 of the joint EFPAAPw. Periodontitis and adverse pregnancy outcomes:..., Sanz [/bib_ref]. The availability of a valid, simple to administer, oral health assessment tool with demonstrated sensitivity and specificity, would facilitate the identification of women with oral health problems and potentiate early dental interventions through referral during the optimal treatment time of the first trimester [bib_ref] Developing and testing of an oral health screening tool for midwives to..., George [/bib_ref] [bib_ref] Piloting of an oral health education programme and knowledge test for midwives, George [/bib_ref].
Concerns raised about oral health during pregnancy go beyond dental caries. Systematic reviews have shown a positive association between adverse pregnancy outcomes such as preterm delivery and periodontal disease, although a cause and effect relationship has not been established [bib_ref] Piloting of an oral health education programme and knowledge test for midwives, George [/bib_ref] [bib_ref] Treatment of periodontal disease during pregnancy a randomized controlled trial, Newnham [/bib_ref]. Several trials have been conducted within Australia to intervene during pregnancy in an attempt to reduce the incidence of adverse birth outcomes such as the SMILE study [bib_ref] Treatment of periodontal disease during pregnancy a randomized controlled trial, Newnham [/bib_ref] and the Midwifery Initiated Oral Health (MIOH) trial [bib_ref] The midwifery initiated oral health dental service protocol: an intervention to improve..., Johnson [/bib_ref]. As part of the MIOH Trial we have included several instruments, and undertaken oral health assessments by dentists to determine the sensitivity and specificity of the Maternal Oral Screening (MOS) Tool.
The MOS tool was developed following a comprehensive literature review of screening items for oral health during pregnancy [bib_ref] Developing and testing of an oral health screening tool for midwives to..., George [/bib_ref]. The result was a preferred twoitem tool: item 1 referring to commonly reported dental problem during pregnancy and those caused by oral diseases [bib_ref] Developing and testing of an oral health screening tool for midwives to..., George [/bib_ref] ; item 2 referring to how often the woman has seen a dentist in the last 12 months [bib_ref] Developing and testing of an oral health screening tool for midwives to..., George [/bib_ref]. A total score of ≥1 (having a dental problem or not seen a dentist in the previous 12 months) indicated that women were at risk of poor oral health and required a referral to a dentist. A visual inspection was optional for confirming the presence of dental problems and was only undertaken if a problem was identified by the pregnant women (See below). An important part of the development process was the measurement of the tools sensitivity and specificity which indicates whether a patient will be screened in for an intervention (in this case dental treatment) or screened out (in this case having good oral health). A tool that allows for both situations to be considered with appropriate proportions is always an aspiration, however, in some clinical situations this may not always be possible. Therefore the emphasis is often placed on whether the tool captures populations at risk of the condition, in this case women with poor oral health. Clinical evaluation of sensitivity and specificity is frequently used as the gold standard in clinical conditions where clinical assessment is a key diagnostic step [bib_ref] Diagnostic validity of self-reported oral health outcomes in population surveys: literature review, Ramos [/bib_ref]. We had previously pilot tested the MOS tool and found that it was promising in a sample of 56 women [bib_ref] Developing and testing of an oral health screening tool for midwives to..., George [/bib_ref]. The pilot results suggested that the tool was acceptable to pregnant women and midwives and was sensitive (98 %) to identifying dental problems and facilitating dental referrals. However, further evaluation of the tool in a larger sample was warranted.
## Aim
This study sought to undertake sensitivity and specificity assessment of the maternal oral health screening tool using two comparison approaches-the 14 item oral health screening tool known as the Oral Health Impact Profile (OHIP-14) and a clinical oral assessment by trained study dentists.
# Methods
This study was undertaken as part of an existing multicenter randomized controlled trial that is evaluating the effectiveness of a midwifery initiated oral health program in improving the uptake of dental services, oral health knowledge, quality of life and oral health status of pregnant women (Trial ID ACTRN12612001271897). At the initial assessment, pregnant women, undergo screening and referral (where appropriate) by appropriately accredited midwives [bib_ref] The evaluation of an oral health education program for midwives in Australia, George [/bib_ref].
## Sample and setting
The pregnant women were recruited from three large antenatal clinics in Sydney. The eligibility criteria for the MIOH trial have been reported elsewhere [bib_ref] The midwifery initiated oral health dental service protocol: an intervention to improve..., Johnson [/bib_ref] and included low risk women with a single pregnancy (more than 12 and less than 20 weeks gestation). These women were screened at their initial antenatal appointment and provided dental referrals by accredited midwives who had completed the MIOH education program [bib_ref] The evaluation of an oral health education program for midwives in Australia, George [/bib_ref] [bib_ref] Piloting of an oral health education program and knowledge test for midwives, George [/bib_ref]. One group of women in the trial was referred to study dentists for a clinical examination regardless of whether they were screened to be at risk of poor oral health. These women formed the study sample for the testing of the MOS tool.
## Characteristics of women attending the three antenatal clinics
Two hundred and eleven participants were recruited for this study. The majority of participants were Australian born (n = 128, 61 %) with a mean age of 29 years (SD = 5.7, range = 18-43 years). Most women were multiparous (65 %), partnered (81 %) and in their second trimester (n = 387, 92 %). The highest level of education recorded was university (18 %) followed by trade school (35 %). Most Maternal Oral Screening tool for antenatal clinics The maternal oral screening tool Item 1. Do you have bleeding gums, swelling, sensitive teeth, loose teeth, holes in your teeth, broken teeth, toothache or any other problems in your mouth?
Yes □ (1) No □ (0) If yes, visual inspection of oral cavity (optional to confirm Item 1) Item 2. Have you seen a dentist in the last 12 months?
Yes
[formula] □ (0) No □ (1) [/formula]
Items 1 and 2 are scored either 0 or 1. Participants with a total score ≥ 1 are referred for a dental check-up.
participants were not working (56 %) or working part time . More than 40 % of participants (n = 83) had a combined annual household income of less than $60,000 and a high proportion (50 %) lived in areas of low socioeconomic advantage (lowest Socio-Economic Index for Area quintile as measured through the Australian Bureau of Statistics).
## Characteristics of midwives undertaking the screening and referral of women
Thirty-eight midwives successfully completed the MIOH education program and undertook the screening and referral of the pregnant women. The midwives had a mean age of 43.4 years (SD 11.5) and an average of 15.8 years of clinical practice experience (SD 11.7). Nearly half the midwives (47.4 %) had postgraduate qualifications.
## Ohip-14
The OHIP-14 is a subjective measure of oral health that has been found to be a precise, valid and reliable instrument (α = 0.88) [bib_ref] Derivation and validation of a short-form oral health impact profile, Slade [/bib_ref]. It has been used previously as a 'gold standard' measure for validating other oral assessment tools [bib_ref] Development and validation of a three-item questionnaire for dietitians to screen for..., Jeganathan [/bib_ref]. OHIP-14 contains 14 questions assessed on a 5-point Likert scale and the total scores range from 0 to 56 with higher scores indicating poorer oral health. This tool was developed based on a conceptual model defined by Locker [bib_ref] Measuring oral health; a conceptual framework, David [/bib_ref] focusing on seven domains-functional limitation, physical pain, psychological discomfort, physical disability, psychological disability, social disability and handicap (see Additional file 1).
## Clinical examination by study dentists
Three experienced dentists were recruited to the study from health services where the study was conducted. Several reliable measures were used to assess the extent of dental problems in pregnant women. These included the decay, missing and filled teeth (DMFT) index [bib_ref] Goals for oral health in the year 2000: cooperation between WHO, FDI..., Aggeryd [/bib_ref] and the periodontal screening and reporting (PSR) index [bib_ref] Comparison of the GOHAI and OHIP-14 as measures of the oral health-related..., Locker [/bib_ref]. The DMFT index identified whether decay was present while the PSR index (scoring 0-4) assessed the health of the gums with a score of two and above indicating gum disease requiring treatment. Pregnant women were determined to have poor oral health if they had any dental decay and a PSR score of greater than two. One of the investigators (SA) undertook to train all dentists on a standard assessment and intervention protocol. The dentists were assessed on five patients for various measures with >80 % inter-rater reliability in the key study measures (DMFT and PSR).
## Procedure
The items for the MOS and OHIP-14 were included in the initial assessment undertaken by the midwife with the consenting woman presenting to the antenatal clinic.
The data collection procedures for the MIOH Trial are more fully described within the protocol [bib_ref] The midwifery initiated oral health dental service protocol: an intervention to improve..., Johnson [/bib_ref]. The dental assessment was conducted for all women in the intervention group that had access to the study dentists [bib_ref] The midwifery initiated oral health dental service protocol: an intervention to improve..., Johnson [/bib_ref]. Ethical approval was obtained from the Human Research and Ethics Committees of Sydney Local Health District (reference no HREC/11/CRGH/28) and Western Sydney University (reference no H9709). Written consent to participate in the trial was obtained from women attending the antenatal clinic at the initial presenting visit.
## Data management and analysis
Statistical analysis was completed using SPSS v22. Descriptive statistics were used to describe the demographic information of the sample population. The relationships between the screening tools were assessed using chisquared analyses after variable dichotomisation. The level of significance (α) was set at 0.05. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were computed using conditional probabilities based on a two-way table. Confidence intervals were determined using the central limit theorem formulation.
# Results
## Mos tool
In total, an average of 67 % (n = 141) of women reported having a current problem or concern with their teeth, gums or mouth, and 39 % reported seeing a dentist in the previous 12 months. Using the screening criteria , 86 % of women were deemed at risk of poor oral health [fig_ref] Table 2: Women's responses to the MOS tool, OHIP-14 and Dental Assessment [/fig_ref].
## Approach 1 -ohip-14
A total of 207 (98 %) participants satisfactorily completed the OHIP-14. The mean OHIP-14 score was 6.8 (SD = 8.6, median = 4.0, range 0-38). As cut-off scores for identifying patients at risk of poor oral health has not been established with the OHIP-14, the total score for each participant was dichotomised using the median split as described by Locker et al. [bib_ref] Comparison of the GOHAI and OHIP-14 as measures of the oral health-related..., Locker [/bib_ref] categorised as either ' At risk' (score greater than 4) or 'Not at risk' (score 0 to 4) [fig_ref] Table 2: Women's responses to the MOS tool, OHIP-14 and Dental Assessment [/fig_ref].
## Approach 2 -dental assessment
Of the 207 participants referred, 131 women completed the dental assessments. Analysis of these data found 90 % (n = 118) of women had a PSR rating of two or more, 62 % (n = 81) had dental decay, and 56 % (n = 74) of women had both of these and were considered to have poor oral health.
## Sensitivity and specificity
The
# Discussion
The focus of this study was to further validate a maternal oral health screening tool for pregnant women. Developing such a tool is important because of the high prevalence of poor oral health among pregnant women and its impact on maternal and infant outcomes. Further, current guidelines advocate the need for all antenatal care providers to undertake oral health education, assessment and referral during early pregnancy. Initial piloting of the MOS tool suggested that it was easy to administer by midwives and had good sensitivity in identifying dental problems in pregnant women and facilitating referrals. However, the pilot study had limited sample size and further testing of the sensitivity and specificity was warranted. This study used a two staged approach using a larger sample size to validate the MOS tool. Current practice during the first antenatal visit involves midwives assessing multiple health areas at one time, thus consideration of time constraints was an important factor in assessing oral health during this visit [bib_ref] Assessing the responsiveness of measures of oral health-related quality of life, Locker [/bib_ref]. This was one of the main reasons why only three items were considered during the development phase of the tool, only two of which were used in this study. The study results reiterate earlier suggestions that the 2-item tool (which is part of the MIOH education program) was easy to administer by midwives and comprehend by pregnant women [bib_ref] Diagnostic validity of self-reported oral health outcomes in population surveys: literature review, Ramos [/bib_ref] [bib_ref] Piloting of an oral health education program and knowledge test for midwives, George [/bib_ref] [bib_ref] Derivation and validation of a short-form oral health impact profile, Slade [/bib_ref].
Validation of the MOS tool (2-item) showed high sensitivity across both gold standards (88-97 %) which is similar to results of the pilot study (75-98 %). This combined with moderated NPV's of 47-80 % confirm that the MOS tool is reliable and able to correctly identify the majority of women in need of a dental referral. The specificity of the tool remained low even with increased sample size (14-19 %), however when interpreting this value consideration must be given to the purpose to the tool. In this situation correct identification of women at risk of poor oral health, compared to identifying those with good oral health, is of paramount importance especially considering that it is recommended that all women should see a dentist early in their pregnancy even if they are unaware of any dental problems [bib_ref] Treatment of periodontal disease during pregnancy a randomized controlled trial, Newnham [/bib_ref]. The positive predictive value of the MOS tool indicates that around 50 % of the women referred for a dental assessment, will in fact be in need of dental care. In addition it should be noted that low specificity values are not uncommon in oral health screening tools with values ranging from 2.9 to 37.5 % observed in studies addressing oral health in early childhood (27) and HIV patients (26). The study reinforces earlier suggestions (15) that the MOS tool can be easily administered by midwives. The fact that the education that accompanies the assessment tool has been successfully implemented in other states in Australia [bib_ref] Derivation and validation of a short-form oral health impact profile, Slade [/bib_ref] indicates a level of tool transference. In the future this tool could be used by other antenatal care providers as an assessment tool. In addition, there is no restriction on this tool being used solely by midwives, therefore other health care professionals who have contact with mothers early in the antenatal period may also be able to successfully undergo the training and administered the MOS tool. Further the tool could also be incorporated into existing obstetric systems to increase the number women captured/screened across the country.
Although the tool has low specificity, in the current Australian system, where we have strict eligibility criteria's for public dental services (28) as opposed to a free universal dental scheme for pregnant women, the impact of over referral will be mostly borne by the individual rather than the government. The potential cost benefits however is not to be disregarded; the encouragement of women to access dental services earlier during pregnancy is likely to have flow on health benefits, improving maternal health and quality of life (29), child oral health (30) and potentially birth outcomes (31) which ultimately could reduce the overall cost burden to society.
Lastly, it should be noted that although the OHIP-14 is frequently used as a gold standard for measures in a variety of populations such as older people [bib_ref] Derivation and validation of a short-form oral health impact profile, Slade [/bib_ref] [bib_ref] Assessing the responsiveness of measures of oral health-related quality of life, Locker [/bib_ref] and HIV patients [bib_ref] Development and validation of a three-item questionnaire for dietitians to screen for..., Jeganathan [/bib_ref] it may not be as appropriate for this population. Only 6 of the 14 OHIP items were scored frequently >0 in the study population. These items were related to the physical pain, psychological discomfort and psychological disability domains of the OHIP-14. As the OHIP tool is derived from the conceptual understanding of Locker et al. [bib_ref] Measuring oral health; a conceptual framework, David [/bib_ref] focusing on disability, the remaining domains and related items may not be as suitable for this sample of healthy women. Further studies should be conducted to develop or modify the OHIP-14 for this population of predominantly young healthy women. Similarly as suggested by Locker et al. [bib_ref] Comparison of the GOHAI and OHIP-14 as measures of the oral health-related..., Locker [/bib_ref] longitudinal studies should be undertaken to identify the ability of the OHIP tool to measure changes over time.
# Limitations
This study was undertaken in South West Sydney and the findings may deliver differing results in other populations of pregnant women from higher socio-economic backgrounds, or indeed from other countries. Similarly this study has focused on only one aspect of psychometric assessment of a tool and other aspects such as construct validity was not undertaken within this sample. We also note that although the OHIP-14 was used as a gold standard, similar results in terms of sensitivity and specificity were also demonstrated with clinical examination by dentists.
# Conclusion
The MOS (2-item) provides a sensitive screening tool that is easy to use by midwives and potentially other antenatal care providers including general practitioners. The items are easily administered and provide an opportunity to raise awareness with women about the importance of oral health to their overall health as well as the health of the unborn fetus. This simple initial screening approach conducted by a non-oral health professional, with specialized training, complements the service provided by dentists where more thorough examination of oral health risk can be undertaken. Further studies should examine the potential for the OHIP-14 or other modifications of the OHIP to more appropriately target items of relevance for young healthy pregnant women.
## Availability of data and materials
The dataset supporting the conclusions of this article is included within the article as an additional file.
Authors' contributions AG and MJ prepared the first draft of the manuscript. AY, EE and AS performed the quantitative data analysis and AG, EE and MJ performed the synthesis and interpretation. AG, HD, AB, SA, SB, SE and MJ conceived and designed the study. All authors (AG, HD, AB, SA, SB, SE, AY, EE, AS and MJ) provided input into versions of the manuscript and read and approved the final manuscript.
## Competing interests
The authors declare that they have no competing interests.
## Consent for publication
Not applicable.
Ethics approval and consent to participate Ethical approval was obtained from two health service Human Research and Ethics Committees with approval number HREC/11/CRGH/28. Written consent to participate in the trial was obtained from women attending the antenatal clinic at the initial presenting visit. Participation was voluntary and privacy and confidentiality of all study information was maintained.
[table] Table 2: Women's responses to the MOS tool, OHIP-14 and Dental Assessment [/table]
[table] Table 3: Sensitivity and specificity of Maternal Oral Screening Tool compared with OHIP-14 and Dental Assessment [/table]
|
Chronic disease prevention policy in British Columbia and Ontario in light of public health renewal: a comparative policy analysis
Background: Public health strategies that focus on legislative and policy change involving chronic disease risk factors such as unhealthy diet and physical inactivity have the potential to prevent chronic diseases and improve quality of life as a whole. However, many public health policies introduced as part of public health reform have not yet been analyzed, such as in British Columbia and Ontario. The purpose of this paper is to present the results of a descriptive, comparative analysis of public health policies related to the Healthy Living Core Program in British Columbia and Chronic Disease Prevention Standard in Ontario that are intended to prevent a range of chronic diseases by promoting healthy eating and physical activity, among other things.Methods: Policy documents were found through Internet search engines and Ministry websites, at the guidance of policy experts. These included government documents as well as documents from non-governmental organizations that were implementing policies and programs at a provincial level. Documents (n = 31) were then analysed using thematic content analysis to classify, describe and compare policies in a systematic fashion, using the software NVivo. Results: Three main categories emerged from the analysis of documents: 1) goals for chronic disease prevention in British Columbia and Ontario, 2) components of chronic disease prevention policies, and 3) expected outputs of chronic disease prevention interventions. Although there were many similarities between the two provinces, they differed somewhat in terms of their approach to issues such as evidence, equity, and policy components. Some expected outputs were adoption of healthy behaviours, use of information, healthy environments and increased public awareness.Conclusions: The two provincial policies present different approaches to support the implementation of related programs. Differences may be related to contextual factors such as program delivery structures and different philosophical approaches underlying the two frameworks. These differences and possible explanations for them are important to understand because they serve to contextualize the differences in health outcomes across the two provinces that might eventually be observed. This analysis informs future public health policy directions as the two provinces can learn from each other.
# Background
Chronic diseases, defined by the World Health Organization as "diseases of long duration and generally slow progression", are becoming a serious problem worldwide. Some major categories of chronic disease that have high morbidity and mortality rates around the world include cardiovascular diseases, cancer, chronic respiratory diseases and diabetes. WHO estimated that chronic diseases accounted for 46% of the global burden of disease in 2001; this proportion is expected to climb to 57% by 2020. Additionally, as the global proportion of deaths from infectious diseases has decreased, the proportion attributed to chronic diseases has increased. Chronic diseases contributed to 63% of deaths around the world in 2008 and are projected to contribute to 75% of deaths by the next decade. This epidemiologic shift is occurring in both developed and developing countries, and Canada is no exception. Forty-two per cent of Canadians over the age of 12 are living with a chronic condition (either a chronic disease or a risk factor for a chronic disease), a figure that grows progressively larger for older age groups. The majority of chronic disease-related deaths are attributed to cardiovascular diseases (CVD), though conditions such as cerebrovascular diseases, diabetes, respiratory illnesses, and cancers are common as well.
The causes of chronic diseases are complex and rooted in social and economic structures, tied to social determinants of health such as income inequality, education, working conditions, food insecurity and housing. However, the WHO has made it clear that it is also possible to prevent some chronic diseases through the reduction of key risk factors. The four main behavioural risk factors are unhealthy diet, physical inactivity, tobacco use and the harmful use of alcohol. Public health strategies such as policy changes are key to preventing chronic diseases, particularly at the population level. Some proven and/or promising strategies include legislation to protect people from tobacco smoke, high taxes on tobacco, and policies to reduce salt content in food and replace trans-fats with polyunsaturated fat. These targeted policy-level solutions are crucial to address the social determinants of health, the unequal distribution of which systematically sustain risk behaviours related to diet, nutrition, tobacco use, and alcohol consumption.
Strategies that focus on legislative and policy change have the potential to alter the lives of Canadians in fundamental ways that not only prevent chronic diseases, but also improve quality of life as a whole. However, many public health policies introduced as part of public health reform have not yet been analyzed, individually or for their potential synergistic effect, because they are still in the initial stages of implementation. This is the case in two Canadian provinces currently undertaking a process of public health system reform in which a major public health policy intervention is being implemented in each province. In British Columbia (BC), the Framework for Core Functions in Public Health, and in Ontario (ON) the new Public Health Standardslaid out policy frameworks for implementing core public health programs for population health assessment; health surveillance; disease and injury prevention; health promotion and health protection. Aspects of the policy related to chronic disease prevention were selected for closer examination in this paper (and the larger research program) as one exemplar to illustrate the implementation of the larger frameworks.
The public health systems in ON and BC are organized very differently (see Additional file 1), and each of these provincial policies takes a somewhat different approach to chronic disease prevention. These naturally occurring variations provide a unique opportunity for a natural experiment of sorts to study the implementation of public health renewal in the two provinces. ON and BC were selected for analysis because, in addition to undergoing simultaneous public health reform processes, these two provinces are quite distinct in terms of geography, population demographics, economics, organizational structures and governance. The analysis described in this paper reports on a first step in a larger study (Renewal of Public Health Systems -RePHS) that explores the implementation and impact of these public health policy interventions in BC and ON, with particular respect to: chronic disease prevention/healthy living and sexually transmitted infection prevention (see http://www.uvic.ca/research/groups/cphfri/ projects/currentprojects/rephs/index.php). Our purpose in this paper is to present the results of a descriptive, comparative analysis of public health policies related to the Healthy Living (HL) Core Program in BC and Chronic Disease Prevention (CDP) Standard in ON that are intended to prevent a range of chronic diseases by promoting healthy eating and physical activity.
Tobacco prevention and control were excluded from this analysis for several reasons: a) their unique long and rich history in both provinces, b) the fact that related legislative and policy measures were more restrictive and focused on risk-mitigating behaviours (e.g. tobacco cessation) and protecting the public from second-hand smoke, rather than health-promoting behaviours (e.g. healthy eating), and c) an analysis of tobacco policy will be reported in a separate paper.
Not only will this analysis illustrate the variations in CDP policy in two provinces but will also provide a baseline against which to judge implementation changes in policy over time. Evaluating the outcomes of policy interventions can be particularly challenging without baseline data, and it is difficult to generate evidence to assess the whether the policy has been successful [bib_ref] Challenges in assessing the implementation and effectiveness of physical activity and nutrition..., Ramanathan [/bib_ref]. As the RePHS project moves forward, further developments in policy documents, program implementation and impacts will be assessed against this baseline analysis.
The Canadian Context: Canada is a country composed of 10 provinces and 3 northern territories. Although on a national level Canada's health-care system is publically funded and guided by the Canada Health Act, jurisdiction over the majority of health care services, funding structure and delivery lies with the provinces/territories. Residents are required to pay into mandatory provincial insurance plans, which then allow them to receive medically necessary physician and hospital services at no cost up front, while health care professionals bill the provincial insurance provider. Provincial plans may cover other services (for example dental care, eye care and drugs), but they are not required to do so, and in many cases private insurance plans supplement provincial insurance to fill this gap. Public health in most provinces in Canada is integrated into regional health authorities that are responsible for providing all health care services. The exception is Ontario in which public health does not fall under the health care system per se. It is its own separate, albeit linked, system (see Additional file 1). However, as noted above, public health services are not funded by federal government transfer payments as are other health care services.
Public health is defined by the Public Health Agency of Canada Act as "population-focused and includ[ing] disease surveillance, disease and injury prevention, health protection, health emergency preparedness and response, health promotion, and relevant research undertakings". There is shared responsibility and jurisdiction over public health between the federal government and the provinces/territories. While protecting Canadians from the spread of diseases (e.g. quarantine) falls within national jurisdiction, the provinces and territories structure, fund, and deliver public health services.
Federal Role: National public health reform became a priority in Canada when public health emergencies such as SARS revealed serious gaps and failings in the existing public health system. In 2004/2005, the federal government provided $400 million in funds specifically to enhance public health capacity, including in the area of chronic disease prevention. While a large portion of these funds went to the provinces for public health revitalization and established important structures such as the Ontario Agency for Health Protection and Promotion (now known as Public Health Ontario), there were also important developments on the national stage. The resulting establishment of PHAC, the Federal/Provincial/ Territorial Health Promotion Expert Committee, and six national public health collaborating centres around the country focused on health promotion issues have significantly impacted research, knowledge and programming around chronic diseases [bib_ref] Health promotion in Canada, Jackson [/bib_ref]. The federal department of health, Health Canada, is also involved in developing policy, promoting research, and funding projects related to health promotion and chronic disease prevention. Some notable initiatives with federal involvement include the Canadian Heart Health Initiative, the Canadian Diabetes Strategy, the Canadian Cardiovascular Disease Action Plan, and ParticipACTION, which demonstrate a policy and program environment that is favourable to chronic disease prevention strategies [bib_ref] Health promotion in Canada, Jackson [/bib_ref]. It should be noted that the extent to which the federal government is involved with chronic disease prevention is debatable. Although it is purportedly committed to health surveillance, high-level policy setting and national leadership, the majority of health promotion and chronic disease prevention programming falls to the provinces. For more information around federal and provincial organizations and structures that impact chronic disease prevention policy and programming, please see Additional file 1.
## The policy interventions bc
The original BC Core Functions of Public Health Framework (CF Framework) that was created in 2005identified twenty-one core public health programs that encompass the areas of health improvement; disease, disability and injury prevention; environmental health; and health emergency management (see [fig_ref] Figure 1: BC Core Functions of Public Health Framework [/fig_ref]. There was a staggered implementation of these core programs by health authorities (HA) beginning in 2007 and continuing until 2010. Although each core public health program in the BC framework was defined originally as an individual and distinct program, most HAs clustered their core programs in some way, often by population, to achieve some economies of scale and to take advantage of synergies in delivery and implementation. HAs are responsible for the implementation and quality of their programs; they are expected to develop programs/services and a performance improvement plan for each core program in line with findings of a provincial evidence review and a model core program paper, a logic model, outcome indicators and regional needs.
The public health strategies employed to deliver core programs include health promotion, health protection, prevention, and health assessment/disease surveillance. A population and an equity lens were expected to be applied to all programs and strategies to address health inequities and to ensure that the needs of specific populations were met, including but not restricted to vulnerable or marginalized groups. Finally, the framework included a Core Public Health Capacity component that identified the health information systems, quality management, research and knowledge development, and staff training This framework outlines the core program categories, the main public health strategies used in the implementation of the programs, the population and inequity lenses that are applied to all programs, and system capacities necessary to carry them out.
## Figure 2
Ontario Public Health Standards. This framework demonstrates elements of the foundational standard and protocol that are used to implement the program standards and protocols (shown in the orange circle). The foundational standard in turn is supported by four principles that are represented by pillars. and capacity development needed to apply public health strategies and implement core programs.
## On
The Ontario Public Health Standards outlined thirteen program standards in five areas: chronic diseases and injuries, family health, infectious diseases, environmental health and emergency preparedness (see . All programs were based on a foundational standard that emphasized the importance of population health assessment, surveillance, research and knowledge exchange, and program evaluation. This foundational standard paralleled, in some ways, the Public Health Capacity component of the BC framework. While the MOHLTC was responsible for the majority of most standards, including the Foundational Standard, (during the period of this study) the Chronic Disease Prevention standard was directed by the former Ministry of Health Promotion (now absorbed into the Ministry of Health and Long-Term Care), and then administered by public health units. The standard had specific goals, desired health and societal outcomes, and requirements attached to it that related to how the Board of Health in each unit would plan and deliver programming around assessment and surveillance, health promotion, policy development, and health protection. There were also more detailed protocols and agreements for provincially funded programs that guided how Boards were meant to implement programming, such as the Nutritious Food Basket Protocol, the Population Health Assessment and Surveillance Protocol and Smokefree Ontario Service Agreements.
It should be noted that in ON, the Chronic Disease Prevention Standard encompassed physical activity and healthy eating. In BC, the policy Framework included two main core programs that address chronic disease prevention activities. The Healthy Living core program included physical activity and healthy eating while the Chronic Disease Prevention (CDP) core program included prevention activities for specific chronic diseases and may not be directly related to these areas, although the expectation was that by addressing physical activity and healthy eating, there will be prevention benefits for several of the chronic diseases that are the focus of the CDP core program (e.g., musculoskeletal disorders, respiratory diseases, and various forms of cancer). For the purpose of this paper we focus on the HL core program in BC and the CDP Standard in ON as they both include physical activity and healthy eating.
# Methods
## Study design
This study was designed as a descriptive comparative analysis of CDP policies in ON and BC using thematic content analysis of key policy documents. Health policy in this case is defined as "courses of action (and inaction) that affect the set of institutions, organizations, services and funding arrangements of the health system", p.6. The research questions were: how do the recent CDP/HL policies compare across BC and ON in terms of their scope and focus, and what are some contextual explanations for these differences? What are the implications of these differences?
## Document search and selection strategy
The search for documents was done in two phases. In the first phase, in 2010, the research team performed a search, using different combinations of the words "healthy eating", "active living", "chronic disease prevention", "policy", "strategy" and "public health" for BC and ON documents using the Internet search engine Google and websites of relevant Ministries in both provinces (the former Ontario Ministry of Health Promotion, the Ontario Ministry of Health and Long-Term Care, the former BC Ministry of Healthy Living and Sport, and the BC Ministry of Health). No other search engines were used because all documents were available in the public domain. The second phase, in 2011, was conducted to ensure that the team had a comprehensive list of documents. In the second phase, in 2011, the research team was expanded to include key policy makers from each province. These individuals provided further guidance on additional government policy documents that would be useful to include in the analysis. The selection criteria also broadened to include documents from key non-governmental organizations (NGOs) that were implementing CDP/HL policies and programs at a provincial level (e.g., the BC Healthy Living Alliance).
Documents for those policies that operate at the provincial level were selected for analysis, with a publication date cut-off of September, 2010. Changes in provincial policy structures, which may be found in documents published after September, 2010, are not reflected in this analysis but will be examined in subsequent follow up analyses. Because the focus of this study was on provincial policy, only documents that identified CDP/HL policies at the provincial level were included; documents that addressed policies and initiatives at the regional health authority (in BC) and local health unit level (in ON) were not used for analysis but will be considered in other analyses being conducted for the larger RePHS study. The larger program of research will also go beyond policy to analyze the implementation and impact of public health programming in both provinces, including the impact of the different organizational, delivery and funding structures for public health in the two provinces. For example, it will examine to what extent in ON local government priorities affect public health programs, given that local governments fund a portion of public health activities.
Documents that provide an overview of public health reform at a national level were used for understanding context but not included in the document analysis. The final list of documents included in the analysis can be found in [fig_ref] Table 1: List of Analyzed Documents for BC and ON• A framework for core... [/fig_ref]. They are categorized into core, supporting and contextual documents. Core documents form the heart of CDP/HL policy as envisioned by BC and ON governments. Beyond the core programs other government documents that related to these policies were seen as supporting documents, and contextual documents have non-governmental authors that provide a more complete picture of the formation and implementation of healthy living policies.
# Document analysis
Documents (n = 31) were analysed using thematic content analysisto classify, describe and compare CDP/HL policies in a systematic fashion. The analysts (AK, DG, MM, DA, GB), who were a mix of researchers and policy makers, initially read through the assigned documents to become familiar with the data. The first author created a preliminary coding framework with a codebook and operational definitions using a key policy document from each province; the framework reflected interest in how the policy tools were constructed, what they might achieve and any evaluation component. The analysts then came together to discuss the framework until a common understanding of the definitions and analytical process was established. A detailed coding protocol was developed and taught to all coders using a short policy document as an example to ensure rigour and inter-coder reliability. Each document was then re-read and coded using the software NVIVO 9 to select sections of text and assign them to an a priori code in the framework. Data that did not fit any of the existing codes were assigned a new code. Only two new codes were created that did not exist in the preliminary framework -"motivators" and "barriers" were added to the category "contextual factors" as a way to capture aspects of the provincial context that acted as facilitators or barriers to CDP/HL policy. Codes were then compared within each document to create higher order categories with shared common characteristics and then the categories were compared across documents to identify broader themes. Repeated team de-briefings were held to question the analysts' assumptions about the data, - OPHS: Requirements*- Public Health Renewal in British Columbia: An overview of core functions in public health- OPHS: Board of Health Outcomes *- OPHS: Goals*- Model Core Program Paper: Healthy Living- OPHS: Societal Outcomes*- Model Core Program Paper for Prevention of Chronic Diseases- Comparison of 2008 OPHS and 1997 MHPSG*Supporting - The nutritious food basket guidance document*- A framework for a provincial chronic disease prevention initiative*- Healthy Eating, Physical Activity and Healthy Weights: guidance document* [26]
- Delivering Effective, Integrated System of Primary and Community Care*- Population Health Assessment and Surveillance Protocol*- Nutritious food basket protocol*Contextual - OPHS: Chronic Disease Prevention Logic Model*- Implementation of healthy living as a core program in public health: final report*Supporting - Leading British Columbia towards a healthy future*- Ontario's action plan for healthy eating and active living*- An environmental scan on primary care and public health in the province of British Columbia*Contextual
- From Vision to Action: A plan for the Ontario Agency for Health Protection and Promotion*- Moving the Healthy Eating and Active Living Strategy forward in Ontario*- Obesity: an overview of the current landscape and prevention-related activities in Ontario*- Informing directions for chronic disease prevention and management in Ontario**Policy documents that were identified in Phase 2 of the document search and selection strategy, as opposed to Phase 1.
and to ensure that the themes were reasonable and plausible. A summary of the results for each code was compiled by DG and discussed by all team members to interpret the findings. Topics most discussed during the team debriefings included the scope and breadth of CDP/HL policies in BC and ON and their respective policy documents. The policy maker team members were able to provide insights into policy legacies and how they shaped the new provincial standards and frameworks.
# Results
Three main categories emerged from the analysis of documents: 1) goals for CDP/HL in BC and ON, 2) components of CDP/HL, and 3) expected outputs of CDP/HL interventions. In the section below we describe these aspects of CDP/HL policy for BC and ON.
## Bc
## Goals for the healthy living core program
The overarching goal for the BC Healthy Living Program was "to optimize health by increasing the adoption of healthy behaviours by British Columbians", p.10, whereas the goal for the BC CDP core program was "to improve the health and well being of British Columbians by preventing and/or reducing the incidence and prevalence of chronic disease among the population", p.20. BC's specific goals focussed on protective factors such as a positive psychosocial environment and community empowerment, which provided a balance between a strengths-based and a risks-based approach. The approach to achieve these goals focused on tackling upstream risk factors, and used an evidence-based population health approach to achieve this. For example, one of BC's priorities was that policies and practices were evidence-based. Evidence related to chronic diseases and strategies for their prevention was drawn from a variety of sources, both formal and experiential, e.g., studies, surveillance statistics, papers on best practices and policy directions, and past experience with other initiatives. The evidence was synthesized in a published formal evidence review that was readily available to the HAs and practitioners. The importance of evidence is also found in the "System Capacity" component of the Core Functions Framework, which stressed that health information systems and research and knowledge development were necessary to implement all core programs.
BC explicitly employed an equity lens as well as a population lens in its public health framework to design appropriate interventions for specific populations. The equity lens is meant to be a perspective which is sensitive to the socioeconomic, political and cultural contexts that create health inequities, and so can be used to identify and track inequitable differences in health status. An equity lens lends itself to working to reduce inequities in health through analysis, community action and advocacy, while the population lens directs attention to particular groups, including but not restricted to vulnerable or marginalized groups. These two lenses were seen as distinct and each was meant to be applied to every intervention or core program. It was recognized in the core BC documents that interventions which were both universal and targeted towards vulnerable groups were necessary to ensure access and maximize equity.
## Components of healthy living
BC documents listed four explicit components of healthy eating and active living strategies (see [fig_ref] Table 2: Explicit components of healthy living core program [/fig_ref]. Two components were advocating for public policy change and increasing awareness of the public and health professionals using social marketing and education. BC also included the community as an integral component, in the context of capacity building (Healthy Communities was another core public health program in BC that addressed the creation of healthy environments and settings for communities. Capacity building is meant to empower individuals and communities to participate in and create sustainable changes to healthy living. For the fourth main component, BC stated the importance of programs and services that involve collaboration between different sectors such as HAs, community organizations and other partners.
In addition to elements specific to the Healthy Living Core Program, BC has implemented other provincial level policies and programs that support chronic disease prevention. Examples are presented in Additional file 2, which are intended to be illustrative rather than exhaustive.
## Anticipated chronic disease prevention outputs
Interventions aimed towards the adoption of healthy behaviours by British Columbians, primarily related to healthy eating, active living and a smoke-free lifestyle, were a major component of CDP/HL in BC. Some initiatives designed to promote adoption of healthy behaviours were previously and concurrently implemented by the BC Healthy Living Alliance or its member organizations (e.g. the Farm to School Salad Bar and Sip Smart! BC), and evaluation of these initiatives were cited to demonstrate that strategies to promote healthy behaviours were successful.
According to BC documents, initiatives that resulted in increased public awareness of chronic diseases, their causes, and ways to prevent them were also seen as essential components of a HL program, i.e., immediate outputs supporting long-term health effects. Tools to increase awareness cited in BC documents included social marketing and public education/awareness campaigns. Increased awareness was seen as building skills-based knowledge, as well as providing the cognitive basis for behaviour change.
Finally, the creation of healthy environments was seen as an important output that is necessary to achieve healthy living and chronic disease prevention. BC's policy documents reflected a multi-pronged settings approachattempting to create healthy environments at home, school and work. Some examples include working with partners to create built environments in communities that encourage physical activity, access to the outdoors, access to healthy food and other factors that contribute to chronic disease prevention. It should be noted that the healthy built environment in BC was explicitly addressed in the Healthy Living core program, whereas healthy social environments and settings were addressed in the Healthy Communities core program.
## On
## Goals for the chronic disease prevention standard
ON's overarching goal for CDP, as stated by the Ontario Public Health Standards (OPHS), was "to reduce the burden of preventable chronic diseases of public health importance" which corresponded with the four main categories of disease stated by WHO: cardiovascular diseases, cancer, respiratory diseases and type II diabetes, p.18. Some of ON's explicit goals focussed more on processes such as the process of championing health promotion and driving partnerships for change. To view BC and ON's goals in more detail, see [fig_ref] Table 3: Goals for healthy eating and active living strategies, BC and ON [/fig_ref].
ON highlighted evidence-based policy and practice and drew from a variety of sources to create an evidence base for chronic disease programming. As well, Health Units were expected to review the evidence themselves to guide their planning and evaluation. In the ON Standards, rather than being integrated into the framework as a whole, program planning, evaluation, research and knowledge exchange were included as part of a separate foundational standard that underlies and directs through situational assessments of all programs and services.
ON did not differentiate between an equity lens and a population lens at the framework level, although both equity and a population focus were acknowledged to be important. An equity lens was used for specific interventions but the province did not state equity as an integral component of their public health framework (readers are directed to Pinto, Pauly, Manson, Thanos, Parks & Cox, 2012 for another RePHS comparison paper providing a detailed analysis of BC and ONs' approach to health equity).
## Components of chronic disease prevention
ON documents, similar to BC, listed four components of healthy eating and active living strategies (see [fig_ref] Table 2: Explicit components of healthy living core program [/fig_ref].
## Programs and services (interventions) grow healthy children and youth
-Interventions may be provided directly by the health authorities or, indirectly through collaboration with partners and community organizations. Intersectoral collaboration is integral to the CF Framework.
-Increase opportunities for physical activity and sport -Support healthy schools -Improve access to healthy food Two components were championing public policy and promoting public awareness of parents, caregivers, professionals and the general public. ON also included the community as an integral component, in the context of building healthy community environments. Creating healthy community environments could involve high-level strategies that impacted communities as well as strategies at a local level. ON's fourth component focused on children and youth, in an effort to improve the environment in which children grow and learn. Other provincial level policies and programs that support chronic disease prevention can be found in Additional file 2.
## Anticipated chronic disease prevention outputs
ON listed several documented initiatives that were aimed at changing population behaviours. A key strategy in prompting a change in attitudes and behaviours in ON documents was social marketing and mass media campaigns; however, there were other complementary strategies in place to encourage adoption of healthy behaviours such as comprehensive program and policy development, e.g., a school food policy that incorporated a skill development component. Another strategy was to ensure that people had the supports they need in order to successfully adopt and maintain healthy behaviours. ON also placed a strong emphasis on raising awarenessin fact, promoting public awareness and engagement was cited as one of the four key strategies of the former Ontario Ministry of Health Promotion to allow for healthier eating and more active living among Ontarians. As with BC, social marketing and media campaigns/interventions were seen as effective tools for raising awareness. It was also recognized that for messages to be as widespread and effective as possible they needed to be coordinated across a broad range of partners: community organizations, public health units, NGOs, and private sector actors. Public awareness included not only awareness of chronic diseases and CDP/HL options, but also awareness of community health status: risk, protective, and resiliency factors, and the importance of creating healthy environments.
ON documents had a stated vision for use of information as an output by a range of users, perhaps owing to knowledge exchange being positioned as a foundational standard for programs and services. Some knowledge translation and exchange initiatives between policy-makers, practitioners, community members and other partners in CDP programs included using the Healthy Eating and Active Living (HEAL) evaluation logic model in local programming, communicating best practices to staff to build their capacity, and disseminating messages about HEAL to the community through the media. Boards of health were also mandated through the OPHS to disseminate surveillance and population health assessment information to government, public health professionals, other boards of health, and across the larger health system.
Finally, an expected output of CDP strategies in ON documents was the creation of healthier, or more supportive, environments. The term "supportive environment" was defined in a broad sense, encompassing physical, social, and economic dimensions of the surroundings that are necessary for healthy living. It could be a reference to the built environment, but it was also meant in a broad context, in which it is a setting in where activities take place -for example neighbourhoods, schools and workplaces. The creation of healthy environments in this case meant creating supports within those contexts for people to be able to make better choices and live healthier lives.
# Discussion
This study presents a descriptive analysis of public health policies related to healthy eating and active living in BC and ON. Possible explanations for provincial differences were drawn from contextual documents and insights provided by policy makers (GB, JS) on the research team; insights were largely related to their - Increased systemic support for healthy living choices, in an integrated manner, at the individual, family, community and regional level.
- Prevention and reduction of high-risk behaviours, including tobacco use, unhealthy eating and physical inactivity, particularly among young people and vulnerable individuals and groups.
- Enhanced surveillance, monitoring and evaluation of healthy living trends and interventions., p.10
## On
- Champion health promotion in Ontario and inspire individuals, organizations, communities and governments to create a culture of health and well-being.
- Provide programs, services and incentives that will enhance health and well-being.
- Make healthy choices easier.
- Harness the energy and commitment of other Government of Ontario ministries, other levels of government, community partners, the private sector, the media and the public to promote health and well-being for all Ontarians.
- Make Ontario a leader in health promotion within Canada and internationally, p.3 *As no specific goals for healthy living were listed in ON core documents, the authors drew on a supporting document's goal.
understanding of the motivations underlying the development of and choices made in the policies. Both provinces are in the midst of a transformational public health renewal process that has, according to our analysis, resulted in slightly different approaches to public health policy for chronic disease prevention as it relates to healthy eating and physical activity. These differences and possible explanations for these differences are important to understand for a number of reasons: 1) they serve to contextualize the differences in health outcomes across the two provinces that might eventually be observed; 2) within each province, the explanations might provide insight with respect to the sustainability of local programs and services; and 3) the analysis informs future public health policy directions as the two provinces, and the country, can learn from them. Even if government structures change, the policies that are put in place at a certain point in time will direct intervention efforts, and it is important to keep track of the policies and immediate outputs in order to trace their influence on health outcomes in the years to come [bib_ref] Why behavioral and environmental interventions are needed to improve health at lower..., Milstein [/bib_ref].
In addition to providing a point of comparison against which subsequent changes in public health policies can be assessed, baseline analysis of reforms being led by ON and BC could be used to inform structural changes that are being spearheaded in other provinces, because public health reform is high on the national agenda. An effective and responsive public health system is an integral part of the larger health care system; likewise an ineffective public health system can increase the stress on other components of the system. This work provides an initial understanding of how reforms might be accomplished through the use of policy instruments that outline direction for public health service delivery. Further, to successfully reduce the burden of chronic disease it is crucial to have sound policies; to date, few studies have examined public health system policies for the prevention of chronic disease. As a first step, this analysis examines what the policies look like, and comments on some anticipated challenges and areas of potential success. This is important because the policy perspective takes a systems look at the future, which can be used to complement a post-hoc look at evaluations of service implementation and delivery.
BC and ONs' foci on cardiovascular disease, cancers, respiratory illnesses and diabetes were almost the same as the WHO's recent disease chronic disease priorities in the areas of heart disease and stroke, cancer, asthma and chronic obstructive pulmonary disease, and diabetes . However, some aspects of chronic disease prevention policies were unique in structure and content due to distinct program framework organization and philosophical approaches underlying the two frameworks and visions for their implementation. We draw on these contextual factors in the discussion below. Despite the challenges, we and others recognize that, "policies need time to be fully implemented and embedded in practice, before judgements about their impacts on distal outcomes…can be made"p.737. This analysis is an important preliminary step to understanding public health policy implementation.
BC and ON organized the scope and breadth of their CDP/HL core programs differently. Each province's unique framework structure might lead one to conclude erroneously that gaps exist in service provision (a challenge we tried to overcome by using supporting documents along with policy documents). For example, although not organized into a specific component in the Healthy Living core program, the BC Framework did address chronic disease prevention needs that went beyond healthy living through a variety of other core public health programs, for example the "Healthy Infant and Early Child Development (0-6 years)," the Healthy Child and Youth Development", "Healthy Communities" (which includes a Healthy Schools focus) and "Chronic Disease Prevention" core programs. All programs were inter-related to allow any one core program to build on others and thus cannot be considered in isolation. As another relevant example, the Healthy Living core program might appear to be focussed primarily on individual lifestyle behaviours within the context of the larger framework, but other core programs in a cluster (e.g., the food security and Healthy Communities core programs) in fact focussed on social and environmental supports for chronic disease prevention. The core programs, as well as the public health strategies, lenses, and system capacity elements, need to be thought of as supporting each other within the overall and integrated framework for core public health services within the health care system at large. In contrast, the ON CDP public health standard, in conjunction with the foundational standard, was self-contained to parallel the delivery of services by health units. That is, it could be used as a stand-alone document that was comprehensive in and of itself.
It may well be that the unique organizational and governance structures for public health in the two provinces will be the most important determinants of the longer term impact of the two provinces' approaches to public health renewal in general, and chronic disease prevention more specifically. In BC, public health is situated within HAs (that are responsible for the totality of health care in the province) and is integrated to a greater or lesser extent in each HA. In keeping with the aim of public health integration into the larger system, BC's framework explicitly stated that the core public health functions are in fact, functions of the entire HA. This means that public health functions and services may be delivered, not just by traditional public health practitioners or by public health departments, but by others working in the system. An integrated framework for public health functions, in which each part supports and contributes to the other parts, is congruent with an organizational philosophy of integrated functions and services as in BC. A framework that comprises self-contained and stand-alone standards is more congruent with a public health system in which each standard is delivered through health units acting autonomously from the larger health care system, as in ON. The evidence in support of public health integration into the larger health care system is limited at present, so only time will tell which approach might offer the best advantages for public health service delivery and improved population health outcomes. Our larger study, we hope, will add to our understanding about the benefits or drawbacks of integrated versus autonomous public health systems; this baseline analysis of policies related to chronic disease prevention will contribute to this understanding over the longer term.
The inclusion of equity considerations in both policies signifies an important move to mainstream a systematic approach to health and health inequities in public health. The WHO has identified the role of public health in reducing inequities through the priority areas of: social investments; increased accountability and outcomes through stakeholder roles and increased community capacity; inter-sectoral action; knowledge improvement and exchange on how socio-economic factors affect health outcomes; and leadership of public health. BC explicitly specified equity and population lenses for all core programs, highlighting both targeted and universal approaches for health gains, while ON focussed on priority populations from an equity perspective (and did not clearly separate out the two concepts).
ON's concept of equity draws from the previous Mandatory Health Programs and Services Guidelines for public health, which also had a focus on priority populations. The Mandatory Guidelines were seen as quite prescriptive. Although the focus on priority populations remained, the intent of the new OPHS was to allow health units to practice their independence regarding priority populations since the determination of such groups varies greatly from unit to unit. Which approachone that explicitly highlights both equity and the needs of specific populations, or one that mostly focuses on priority populations as a proxy for equity [bib_ref] Equity in public health standards: a qualitative document analysis of policies from..., Pinto [/bib_ref] is most effective at improving population health and reducing health inequities for their communities is yet to be seen. Such equity-related health improvements are dependent on changes at the upstream, social determinants of health level, requiring extensive inter-sectoral partnerships and action [bib_ref] Why behavioral and environmental interventions are needed to improve health at lower..., Milstein [/bib_ref] [bib_ref] Building a healthier world by tackling noncommunicable disease, Roses [/bib_ref]. However, as identified in a policy review of the integration of an equity lens in both BC and ON core PH renewal documents [bib_ref] Equity in public health standards: a qualitative document analysis of policies from..., Pinto [/bib_ref] , BC had a more explicit commitment to equity in its model core program papers than in ON's individual public health standards and appears to have resulted in more specific focus on strategies to address health inequities. Follow up on the outcomes of this difference in policy will be important.
Evidence-based decision-making is promoted by governmental and non-governmental bodies worldwide. Both provinces demonstrated differences in the way that health infrastructure related to information and/or evidence (e.g., training, education, evaluation, knowledge exchange) is conceptualized. The BC Framework (core programs and strategies) was accompanied by the system capacity requirements required for success, such as health information systems and quality management. Also important to understand is that the BC core programs were deliberately supported by a detailed evidence review (or best practices in situations in which research was lacking). Thus, the role of evidence is highlighted in the development of the BC Framework and the model core program papers (including Healthy Living and Chronic Disease Prevention). In this document, the BC Core Functions Implementation Process included a number of steps toward implementation that built on the integration of evidence into the model core program papers. Responsibility for implementation lies with the HAs, who were responsible for convening a working group to conduct a gap analysis in which current services were examined in relation to the evidence-informed practices described in the model core program papers. As part of this process, some HAs also conducted epidemiological analyses of the current public health issues affecting particular populations within their region and these data were also taken into account in the gap analysis. From this, the working groups in each HA were to develop performance improvement plans to implement the strategies identified in the model core program papers that would address the gaps in service they had identified. Thus, although the CF resource document did not spell out the role of evidence in CF implementation, it was certainly considered. At the same time, many challenges to implementation in BC were identified by those on the ground responsible for implementing core programs.
In ON, the process of OPHS development also involved province-wide consultations, with the result that Research and Knowledge Exchange were positioned as a foundational standard (i.e., a preliminary step in program planning); this standard signalled a need to provide a rationale for decisions where scarce resources had to be applied to complex health issues such as diabetes prevention for a large population. In other words, the role of evidence is highlighted in the implementation of the OPHS. This resulted in the need to: develop skills among staff related to data analyses and critical appraisal of literature; increase program planning time; introduce change management with staff regarding skills increase and program changes. This has brought about the awareness that an "evergreen" process is required where new data and literature are incorporated into revised standards using a process that includes academics, field/practitioner experts and Ministry staff, as was carried out in the BC example. While ON Health Units are expected to locate, interpret and synthesize evidence for programs, which is a well-documented challenge, they are supported in their capacity to do this by Public Health Ontario, a crown corporation dedicated to providing expert scientific and technical advice around public health issues, as well as professional development services, representing a shared responsibility for evidence-based programming.
Unique policy formulation approaches motivated the development and intent of the new public health functions/ standards, adding another layer of complexity to understanding the policies in the two provinces. Historically, BC did not have a previous public health framework from which to build. BC's approach to developing Core Functions was very consultative, and high-level guidance was provided at the provincial level but details were left to the HAs. The intent was for HAs to address the core public health programs in an integrated way within the overarching framework. Further, the CF framework assumed that it is not just traditional public health practitioners that would be doing "public health" programming. The notion of collaboration within the health sector and between sectors was built into the assumptions of the framework. As well, there exist other community structures, organizations or partnerships that support or have supported Healthy Living and Chronic Disease Prevention programs, e.g., the BC Healthy Living Alliance, and ActNow BC.
In ON, the Foundational standard was designed to identify the steps in public health program or policy development. For example, Step 1 would include gathering data on the health issue; Step 2 would involve reviewing literature on interventions; then in Step 3 staff would determine the most effective strategy given the problem and identify interventions either proven to be effective or, in many cases, interventions that show promise; Step 4 would involve evidence-informed decision making to confirm if staff and resources are available (if not, train staff, obtain resources through grants, partnerships, etc.);
Step 5 related to evaluation and knowledge exchangepublish, present, etc. It is in these steps that issues such as equity, determinants of health, etc., were meant to be given strong consideration in the context of all other influencing factors on the health problemand in the context of how to effectively intervene. Although ON has legislated requirements around the OPHS, these new standards were intended to empower staff to assess local health status and to analyze more data and literature when designing a program that is effective locally. The Foundational standard represents a significant shift from previous Mandatory Health Programs and Services Guidelines that were prescriptive, but it still represents a revision of a previous policy. This contrast in policy developmentinnovative policy reform in BC and policy renewal with incremental changes in ONmight be an important difference when assessing future substantive and procedural successes and failures with respect to implementation and outcomes.
## Next steps
This work leads to obvious next steps that include: assessing policy implementation through embeddedness in practice, measuring anticipated outputs and then measuring long-term outcomes related to these public health policies. Outcomes have been estimated to surface between 5 -25 years after intervention implementation [bib_ref] Why behavioral and environmental interventions are needed to improve health at lower..., Milstein [/bib_ref] , suggesting that both short and immediate term definitions of 'outcomes' (not to mention population-level impacts) ought to be given due consideration. Our larger research program aims to understand the implementation of the policies against the backdrop of the policy content described in this paper. This is important because it reveals whether less-than-expected outcomes are due to the policy itself or failed implementation efforts [bib_ref] Implementing childhood obesity policy in a new educational environment: the cases of..., Amis [/bib_ref].
Findings from this work ought to be considered in light of certain limitations. The scope of our study was limited to policy documents at the provincial level. A different search strategy could have uncovered other documents at the HA or Health Unit level that might have provided further insights about priorities, expected outcomes, etc. Additionally, because this analysis focused on traditional elements of the public health systemprimarily government ministries, HAs, and public health units, it did not allow for inclusion of many other civil society actors that form integral components of the public health system -such as public health agencies and organizations, professional associations, non-profit organizations, and community organizations. While as a result the analysis cannot provide a comprehensive picture of all public health activities, its aim was to compare policies (and subsequent programs) of provincial public health leadership. In subsequent papers, as the RePHS project progresses, it will be possible to make much more confident inferences about how different policy settings affect program implementation within these formal public health structures. However, in order to examine health outcomes at the community level, other actors and systems will have to be taken into account.
As well, our analysis was limited to one influence (policy documents) among many in the policy-making process, such as resources, value and power. Nevertheless, these limitations are compensated by the systematic process of collecting and analysing documents in this study. As well, the authorship team comes with varied expertiseacademics and decision-makers from both provinceswhich brings a more balanced interpretation of findings than might have been presented by a traditional scientistonly research team. This balanced interpretation represents a unique strength of this work.
# Conclusion
Ratzan notes that, "The noncommunicable disease threat will require innovative responses and concerted actionboth public and privateto reverse the troubling trends and turn the tide toward health and well being" [bib_ref] The noncommunicable disease challenge, Ratzan [/bib_ref] , p.2. This analysis describes the different recent approaches in BC and ON to addressing risk factors related to chronic diseases. What becomes clear is that the two provincial policies present different approaches to support the implementation of related programs. While ON initiated the process using historical public health guidelines as a jumping off point, engaging in incremental policy change, BC created an entirely new CF Framework, engaging in policy change that is somewhat experimental. In BC the HAs clustered core programs at the level of service delivery (i.e., in anticipation of synergistic health gains in chronic disease prevention) based on analysis of regional health inequities and gaps in service. ON encouraged health units to develop rationales, local evidence reviews and local health standards to support health improvements. The description of the two policies, and the contextual explanations for the differences, are important because the analysis permits a comparison of reforms associated with different public health structures (regional HAs and public health units), which are common structures for the delivery of public health services in other countries.
[fig] Figure 1: BC Core Functions of Public Health Framework. [/fig]
[table] Table 1: List of Analyzed Documents for BC and ON• A framework for core functions in public health: resource document[6] • Ontario Public Health Standards (OPHS)[7] [/table]
[table] Table 2: Explicit components of healthy living core program (BC) and chronic disease prevention standards (ON) CDP programming [/table]
[table] Table 3: Goals for healthy eating and active living strategies, BC and ON [/table]
|
Salivary Trefoil Factor Family (TFF) Peptides and Their Roles in Oral and Esophageal Protection: Therapeutic Potential
# Introduction
## Saliva
Saliva is a mixed body fluid produced in the oral cavity by different sources, i.e., three pairs of major salivary glands (parotid, sublingual, and submandibular glands) and several minor glands, such as labial and palatal glands. The parotid glands contain only serous acini, whereas all other glands are composed of a mixture of seromucous acini. Saliva is the fluid which has the first contact with ingested food and often the environment. It fulfills important functions for both nutrition (lubrication of the bolus, taste, and first steps of digestion) as well as for protection, particularly of the teeth, the oral epithelial barrier, but also the esophagus (reviews:. For example, saliva has enzymatic, wound healing (cell migratory), and antimicrobial effects. It is also essential for maintaining healthy oral microbiota .
Within the last two decades, the saliva proteome was subjected to a huge number of investigations, which often focused on highly specific aspects, such as certain diseases (also use of saliva as diagnostic fluid) and response to different stimuli, such as psychological stress ("stimulated saliva"). Recently, not only whole saliva was investigated, but also extra-vesicles-enriched saliva, which contains exosomes, i.e., extracellular vesicles, which probably originate from intracellular multivesicular bodies. In normal saliva, more than 3000 proteins were identified with a relative abundance spanning about 14 orders of magnitude. Only about 200 proteins represent about 90% in weight including prolinerich proteins, mucins, amylases, histatins, and statherins . Of note, there are remarkable Int. J. Mol. Sci. 2021, 22, 12221 2 of 12 individual differences and the saliva composition is also subject to hormonal fluctuations and aging .
Typical salivary protein constituents are enzymes (such as amylases, lysozyme), protease inhibitors, growth factors such epidermal growth factor (EGF), antimicrobial peptides (such as histatins, defensins, cathelicidin), immunoglobulins (mainly secretory IgA and IgG), surfactant proteins, the agglutinin Deleted in Malignant Brain Tumor 1/gp340 (DMBT1 gp340 ), IgG Fc binding protein (FCGBP), and secretory mucins (MUC5B, MUC7, MUC19) . Many of these proteins also appear in tears. Furthermore, there are proteins unique to this fluid, such as proline-rich proteins and statherins, which influence calcium phosphate chemistry and initial plaque formation; for example, statherin allows saliva to maintain a state of supersaturation concerning calcium phosphate . Of note, most salivary proteins appear in protein families.
Mucins are glycoconjugates and the primary gel-forming components of mucus. Due to their special rheological properties, they are effective in lubricating the oral cavity including the teeth. They also play a key role for the innate immune defense of the oral cavity (review:. The salivary mucins mainly appear in two molecular entities, the unusual low molecular mass mucin MUC7 (previously termed MG2) and the high-molecular-mass mucin MUC5B (previously MG1). Additionally, MUC19 has been identified on the transcript and protein level. MUC5B, MUC7, and MUC19 are typically expressed in the mucous acini of the major and minor salivary glands. MUC5B and MUC19 are typical gel-forming mucins, which evolved from a common ancestor with von Willebrand factor. In contrast, MUC7 lacks gel-forming properties. Both MUC5B as well as MUC7 form complexes with proline-rich proteins, statherins, and histatin 1. However, MUC5B and MUC7 differ remarkably in their binding characteristics with microbes. MUC7 directly binds Streptococcus strains, but also to Escherichia coli and Staphylococcus aureus. Sialic acid residues from the carbohydrate moiety of MUC7 play a major role for binding of different microbes. In contrast, the binding of MUC5B to oral pathogens is limited and here protein-protein interactions seem to be important, e.g., for binding of Haemophilus parainfluenzae. MUC5B also reduces the virulence of Candida albicans. Furthermore, a mixture of MUC5B and MUC7 inhibits T cells from viral infections, i.e., HIV-1. Generally, there are two potential principles by which salivary mucins could protect the oral cavity; they could agglutinate microbes facilitating their removal or they disperse the microbes, hindering their transition into a virulent state.
Generally, saliva has a fundamental role for the innate immune system of the oral cavity, but also the esophagus and the delicate esophagogastric junction. Here, a wide range of different molecular mechanisms is used, such as adhesions (proline-rich proteins), agglutinins (e.g., mucins, DMBT1 gp340 ), and antimicrobial peptides . On the other hand, saliva is important for taste, digestion, and modulating the pH; it has pronounced rheological properties important for lubrication (mucins, etc.), but it also has to provide the necessary water by aquaporins (water channels) . The importance of a functional salivary flow can be estimated from patients with a catastrophic loss of salivary function, such as patients with radiation therapy because of head and neck cancers, patients with congenital absence of salivary glands, and patients with Sjögren's syndrome. Thus, there is still a need for the development for better formulations of saliva substitutes, particularly with strongly improved antimicrobial properties (see Section 3.2).
## Trefoil factor family (tff) peptides
More than two decades ago, expression of TFF peptides was also demonstrated in human salivary glands, where TFF3 transcripts were most abundant. In saliva, only TFF3 is easily detectable by Western blotting, but not TFF1 or TFF2. The concentration of salivary TFF1 was reported to be about 20% of that of TFF3, whereas the salivary TFF2 concentration was below 1% of that of TFF3. In situ hybridization, laser microdissection, and immunohistochemistry localized TFF expression mainly in mucous acini of both the major and minor salivary glands, TFF3 expression being most abundant. There were remarkable individual differences, also sometimes recognizing TFF3 in serous acini of submandibular glands. TFF3 was also located in parotid gland ducts. Furthermore, TFF3 (and also little TFF1 and TFF2) is synthesized in the oral mucosal epithelium. Expression of TFF2 and TFF3 in oral mucosal tissue is downregulated in patients with oral squamous cell carcinoma and oral lichen planus. Decreased salivary TFF3 levels were also observed in patients with obstructive sleep apnea and rhonchopathy. Salivary TFF1 and TFF3 concentrations are reduced in patients with chronic periodontitis, whereas salivary TFF3 is elevated in children with oral mucositis. TFF expression is increased in salivary gland tumors.
TFF peptides belong to a family of secretory lectins (i.e., sugar-binding proteins), which play different roles for mucosal protection (for recent reviews, see. Thus, they are also considered as a protective shield of the oral cavity. They consist of one (TFF1, TFF3) or two TFF domains (TFF2), each TFF domain being stabilized by three conserved disulfide bridges, i.e., Cys I-V , Cys II-IV , Cys III-VI; for reviews, see. Of note, and highly unusual for secretory peptides, TFF1 and TFF3 contain an odd number of cysteine residues, the seventh unpaired residue (Cys VII ) being C-terminal and outside the TFF domain. The nucleophilicity of Cys VII is modulated by neighboring acidic residues (change of pKa) as well as by steric exposure due to proline residues nearby. This is highly relevant for TFF1, which is directly flanked by four acidic residues and mainly occurs in the stomach as an unusual monomer. TFF2 concentration was below 1% of that of TFF3. In situ hybridization, laser microdissection, and immunohistochemistry localized TFF expression mainly in mucous acini of both the major and minor salivary glands, TFF3 expression being most abundant. There were remarkable individual differences, also sometimes recognizing TFF3 in serous acini of submandibular glands. TFF3 was also located in parotid gland ducts. Furthermore, TFF3 (and also little TFF1 and TFF2) is synthesized in the oral mucosal epithelium. Expression of TFF2 and TFF3 in oral mucosal tissue is downregulated in patients with oral squamous cell carcinoma and oral lichen planus. Decreased salivary TFF3 levels were also observed in patients with obstructive sleep apnea and rhonchopathy. Salivary TFF1 and TFF3 concentrations are reduced in patients with chronic periodontitis, whereas salivary TFF3 is elevated in children with oral mucositis. TFF expression is increased in salivary gland tumors. TFF peptides belong to a family of secretory lectins (i.e., sugar-binding proteins), which play different roles for mucosal protection (for recent reviews, see. Thus, they are also considered as a protective shield of the oral cavity. They consist of one (TFF1, TFF3) or two TFF domains (TFF2), each TFF domain being stabilized by three conserved disulfide bridges, i.e., Cys I-V , Cys II-IV , Cys III-VI; for reviews, see. Of note, and highly unusual for secretory peptides, TFF1 and TFF3 contain an odd number of cysteine residues, the seventh unpaired residue (Cys VII ) being C-terminal and outside the TFF domain. The nucleophilicity of Cys VII is modulated by neighboring acidic residues (change of pKa) as well as by steric exposure due to proline residues nearby. This is highly relevant for TFF1, which is directly flanked by four acidic residues and mainly occurs in the stomach as an unusual monomer.. Schematic structures of the human TFF peptides TFF1, TFF2, and TFF3. Cysteine residues (C; numbering in Roman numerals) as well as the free thiol groups at Cys VII in TFF1 and TFF3 are shown in yellow. TFF2 contains an Nlinked carbohydrate moiety and an additional disulfide bridge between Cys-6 and Cys-104, which creates a circular structure. Additionally outlined are the proline residues (P) at the C-terminal outside the TFF domains. Acidic residues close to the C-terminal cysteine residues are shown in blue.
TFF peptides are characteristically secreted by mucous epithelia and their glands. Here, exocrine secretion occurs, mainly together with different gel-forming mucins. TFF1 is predominantly secreted from gastric surface mucous cells and TFF2 from gastric mucous neck and antral gland cells. In contrast, TFF3 is mainly synthesized in intestinal goblet cells, but also in most other mucous epithelia, such as the respiratory and urogenitary tracts and also the conjunctiva. Furthermore, TFF peptides also undergo endocrine secretion, where minute amounts are released from the central nervous system, the immune system, the endocrine pancreas, and the thyroid. TFF peptides are linked to inflammation (review:and they play different roles in the mucosal innate immune defense (review:. Here, I will discuss the role of salivary TFF peptides for the protection of the oral cavity, the esophagus, and also the delicate esophagogastric junction as well as their therapeutic potential, for example as constituents for improved formulations for artificial saliva. Major emphasis will be put on TFF3, as this is the predominant salivary TFF peptide in human. Schematic structures of the human TFF peptides TFF1, TFF2, and TFF3. Cysteine residues (C; numbering in Roman numerals) as well as the free thiol groups at Cys VII in TFF1 and TFF3 are shown in yellow. TFF2 contains an N-linked carbohydrate moiety and an additional disulfide bridge between Cys-6 and Cys-104, which creates a circular structure. Additionally outlined are the proline residues (P) at the C-terminal outside the TFF domains. Acidic residues close to the C-terminal cysteine residues are shown in blue.
TFF peptides are characteristically secreted by mucous epithelia and their glands. Here, exocrine secretion occurs, mainly together with different gel-forming mucins. TFF1 is predominantly secreted from gastric surface mucous cells and TFF2 from gastric mucous neck and antral gland cells. In contrast, TFF3 is mainly synthesized in intestinal goblet cells, but also in most other mucous epithelia, such as the respiratory and urogenitary tracts and also the conjunctiva. Furthermore, TFF peptides also undergo endocrine secretion, where minute amounts are released from the central nervous system, the immune system, the endocrine pancreas, and the thyroid. TFF peptides are linked to inflammation (review:and they play different roles in the mucosal innate immune defense (review:. Here, I will discuss the role of salivary TFF peptides for the protection of the oral cavity, the esophagus, and also the delicate esophagogastric junction as well as their therapeutic potential, for example as constituents for improved formulations for artificial saliva. Major emphasis will be put on TFF3, as this is the predominant salivary TFF peptide in human.
## Potential roles of salivary tff peptides
## Potential role of salivary tff1
Little TFF1 expression was detectable in mucous acini of submandibular, sublingual, and labial as well as in parotid glands. Due to the minute amounts of TFF1 expected in the saliva, there are no protein data available thus far and it is not clear in which forms salivary TFF1 occurs. However, the situation in the human and murine stomach has been investigated in detail, where large amounts of TFF1 are synthesized. Here, TFF1 mainly occurs as an unusual monomer, but also as a homodimer and as heterodimers with FCGBP and gastrokine 2. By analogy, one might expect that salivary TFF1 might also occur as a monomer, a homodimer, and a TFF1-FCGBP heterodimer, as FCGBP is a constituent of human saliva. The hypothetical existence of a TFF1-FCGBP heterodimer would be also comparable with the TFF3-FCGBP heterodimer present in human saliva.
Monomeric TFF1 with its free and probably highly nucleophilic thiol group at Cys VII (due to flanking acid residues and steric exposure; seecould hypothetically act as a scavenger for reactive oxygen species (ROS) as discussed previously in detail. In short, the free thiol at Cys VII is masked by the four flanking acidic amino acids and thus escapes assembly (dimerization), retention, or degradation in the endoplasmic reticulum, similar to that described for Ig light chains. This might be of biological significance as saliva is a rich source for ROS due to the generation of H 2 O 2 by dual oxidase (DUOX) 2 from salivary glands and secreted lactoperoxidase, which produces microbicidal hypothiocyanite (OSCN − ) anions. Such a protective function as ROS scavenger might also be of special importance for the delicate esophagogastric junction, as reactive nitrogen species (RNS) are also formed there when nitrite from saliva meets the gastric juice. As a prerequisite, salivary nitrate (NO 3 − ), whose concentration is about 10-20 times higher than that in plasma due to the enterosalivary circulation, is reduced to nitrite (NO 2 − ) by the oral microbiome. After acidification in the gastric juice and disproportionation of the instable nitrous acid (HO-NO), the radical nitric oxide (NO) is formed, which is a gasotransmitter and can also react with O 2 − to peroxynitrite (ONOO − ). The latter is the prototype of a toxic RNS. Thus, it could well be that salivary TFF1 might reduce the development of adenocarcinoma particularly at the delicate esophagogastric junction.
Additionally, monomeric TFF1 could be an intracellular chaperone involved in the correct folding of glycoproteins (such as mucins) in the endoplasmic reticulum. In contrast, homodimeric TFF1 is able to interact as a lectin with Helicobacter pylori (for review, see. Homodimeric TFF1 can also bind as a lectin to the gastric mucin MUC6 in vitro, which could also stabilize the inner gastric mucus layer particularly at the delicate esophagogastric junction.
The hypothetical formation of a salivary TFF1-FCGBP heterodimer is most interesting as it could play a role in the innate immune defense of the oral cavity and the esophagus comparable with TFF3-FCGBP (see Section 2.3). Furthermore, by analogy with the situation in the stomach, the formation of additional TFF1 heterodimers is possible.
## Potential role of salivary tff2
Only minute amounts of TFF2 are expressed in mucous acini of major and minor salivary glandsand there are no protein data on TFF2 in the saliva.
Based on studies from the stomach, where TFF2 is a major secretory peptide of mucous neck and antral gland cells together with the mucin MUC6, it is clear that TFF2 is a typical lectin specifically recognizing the GlcNAcα1→4Galβ1→R epitope at the non-reducing terminals of the MUC6 carbohydrate moiety (for review, see. A prerequisite for the biosynthesis of this unusual sugar epitope is α1,4-N-acetylglucosaminyltransferase (A4GNT) and mice lacking this enzyme spontaneously develop antral adenocarcinomas. Gastric TFF2 has probably a role in physically stabilizing the inner insoluble layer of the gastric mucus barrier (crosslinked mucous network) and thus can be considered as part of the gastric innate immune defense. Furthermore, TFF2 has been re-ported to influence inflammatory processes probably via glycosylated basolateral receptors (for reviews, see.
Currently, the biological role of salivary TFF2 is not established and it is not even clear if it is bound to mucins or if it exists in a non-bound form similar to that in the porcine pancreas. There are no positive reports on MUC6 expression in salivary glands; MUC6 transcripts are absent in the esophagus, but they can easily be detected in the stomach starting at the Z-line. There are also no reports on the expression of A4GNT in salivary glands and the esophagus. Thus, there is no indication for an interaction of salivary TFF2 with MUC6 or another mucin in the oral cavity or the esophagus. Possibly salivary TFF2 helps to protect the delicate esophagogastric junction. Furthermore, salivary TFF2 seems to bind as a lectin ligand to the carbohydrate moiety of various transmembrane receptors affecting, e.g., cell migration or an immune response (for reviews, see.
## Potential role of salivary tff3
In contrast to TFF1 and TFF2, TFF3 is easily detectable in human saliva and is mainly expressed in mucous acini of the major and minor salivary glands together with the mucin MUC5B. About 20 to 80% of human salivary TFF3 exist in a high-molecularmass form, which represents a TFF3-FCGBP heterodimer. The lowmolecular-mass fractions mainly represent different homodimeric TFF3 forms. In the latter, a truncated TFF3 form was also characterized missing the C-terminal phenylalanine residue. Degradation of salivary TFF3 might occur due to the presence of pepsin or bacterial proteases from the oral microbiome; of note, in 22% of healthy volunteers, pepsin/pepsinogen was detected in the saliva. the gastric mucus barrier (crosslinked mucous network) and thus can be considered as part of the gastric innate immune defense. Furthermore, TFF2 has been reported to influence inflammatory processes probably via glycosylated basolateral receptors (for reviews, see. Currently, the biological role of salivary TFF2 is not established and it is not even clear if it is bound to mucins or if it exists in a non-bound form similar to that in the porcine pancreas. There are no positive reports on MUC6 expression in salivary glands; MUC6 transcripts are absent in the esophagus, but they can easily be detected in the stomach starting at the Z-line. There are also no reports on the expression of A4GNT in salivary glands and the esophagus. Thus, there is no indication for an interaction of salivary TFF2 with MUC6 or another mucin in the oral cavity or the esophagus. Possibly salivary TFF2 helps to protect the delicate esophagogastric junction. Furthermore, salivary TFF2 seems to bind as a lectin ligand to the carbohydrate moiety of various transmembrane receptors affecting, e.g., cell migration or an immune response (for reviews, see.
## Potential role of salivary tff3
In contrast to TFF1 and TFF2, TFF3 is easily detectable in human saliva and is mainly expressed in mucous acini of the major and minor salivary glands together with the mucin MUC5B. About 20 to 80% of human salivary TFF3 exist in a high-molecular-mass form, which represents a TFF3-FCGBP heterodimer. The low-molecularmass fractions mainly represent different homodimeric TFF3 forms. In the latter, a truncated TFF3 form was also characterized missing the C-terminal phenylalanine residue. Degradation of salivary TFF3 might occur due to the presence of pepsin or bacterial proteases from the oral microbiome; of note, in 22% of healthy volunteers, pepsin/pepsinogen was detected in the saliva. forms a three-leafed structure (TFF domain) by three disulfide bridges between Cys I to Cys VI . The 7th cysteine residue is linked to the high molecular mass glycoprotein FCGBP (see panel B) via a disulfide bridge (not drawn to scale). Cysteine residues are shown in yellow. Additionally represented is a characteristic proline residue (P) separating the TFF domain from Cys VII . (B) Schematic structures of the high-molecular mass glycoproteins FCGBP and DMBT1gp 340 . Outlined are the various modules; some of them are repetitive and cysteine-rich, e.g., R1-R13s, S1-S14 (SRCR domains). Sale bar: 1000 amino acid residues.
The biological function of homodimeric TFF3 in the saliva is not known currently. However, a protective role can be expected for both the oral epithelium as well as the esophagus. Of note, the esophageal epithelium contains few submucosal glands, which also secrete TFF3. Taken together, the synthesis of TFF3 in salivary glands is reminiscent to TFF3 synthesis in glandular structures of the esophagus, the lung, and the cervix uteri, where TFF3 is co-secreted with the mucin MUC5B. A possible protective role of TFF3 can be inferred from its weak motogenic and antiapoptotic activities (for review, see. These cell migratory and survival effects are coordinately regulated in order to ensure synergy, e.g., wound healing (restitution). Recombinant human TFF3 dimer enhances migration of oral keratinocytes. However, the motogenic effect is rather weak and might result from a lectin-triggered activation of basolateral transmembrane glycoproteins, such as CXCR4 and CXCR7(for reviews, see. Of forms a three-leafed structure (TFF domain) by three disulfide bridges between Cys I to Cys VI . The 7th cysteine residue is linked to the high molecular mass glycoprotein FCGBP (see panel B) via a disulfide bridge (not drawn to scale). Cysteine residues are shown in yellow. Additionally represented is a characteristic proline residue (P) separating the TFF domain from Cys VII . (B) Schematic structures of the high-molecular mass glycoproteins FCGBP and DMBT1gp 340 . Outlined are the various modules; some of them are repetitive and cysteine-rich, e.g., R1-R13s, S1-S14 (SRCR domains). Sale bar: 1000 amino acid residues.
The biological function of homodimeric TFF3 in the saliva is not known currently. However, a protective role can be expected for both the oral epithelium as well as the esophagus. Of note, the esophageal epithelium contains few submucosal glands, which also secrete TFF3. Taken together, the synthesis of TFF3 in salivary glands is reminiscent to TFF3 synthesis in glandular structures of the esophagus, the lung, and the cervix uteri, where TFF3 is co-secreted with the mucin MUC5B. A possible protective role of TFF3 can be inferred from its weak motogenic and antiapoptotic activities (for review, see. These cell migratory and survival effects are coordinately regulated in order to ensure synergy, e.g., wound healing (restitution). Recombinant human TFF3 dimer enhances migration of oral keratinocytes. However, the motogenic effect is rather weak and might result from a lectin-triggered activation of basolateral transmembrane glycoproteins, such as CXCR4 and CXCR7(for reviews, see. Of special note, the potential wound healing effect of TFF3 could be even enhanced in vivo by EGF (a typical constituent of human saliva) as synergistic motogenic effects were described with TFF peptides.
There are increasing indications that the high-molecular-mass TFF3-FCGBP heterodimer plays a key role for the mucosal innate immune defense (for review, see. TFF3-FCGBP was originally characterized in the human intestine, where TFF3 is mainly expressed. FCGBP is a secretory, repetitive, cysteine-rich glycoprotein comprising of about 5400 amino acid residues (see, which is auto-catalytically processed at the 11 GD/PH sites with preferential processing at the six WGD/PH sites. However, after processing, the proteolytic fragments are still linked by disulfide bridges. The cysteine-rich repeats show similarity with von Willebrand factor and gel-forming mucins, such as MUC5B. FCGBP is ubiquitous in vertebratesand is typically synthesized by numerous mucous epithelia and their glands, such as salivary glands, and thus is a constituent of many body fluids, such as saliva. From fish to humans, FCGBP is a highly upregulated defense gene after bacterial or viral infections and it regulates pathogen attachment. Thus, it is speculated that FCGBP is involved in the clearing of microorganisms and prevents bacterial infiltration. It has even been suggested to act as a viral trap for HIV-antibody complexes. Generally, FCGBP would be well suited to bind to salivary IgG, which mainly derives from blood through passive leakage. Currently, the role of TFF3 in the TFF3-FCGBP heterodimer is not established, but TFF3 (and maybe also TFF1) could modulate the binding to microorganisms due to their lectin activities.
Of special note, TFF3 could also interact with another constituent of saliva, i.e., the glycoprotein DMBT1 gp340 , a known salivary scavenger and agglutinin (SALSA), or salivary agglutinin (SAG), which has important functions in innate immunity (for reviews, see. DMBT1 appears in many body fluids and its glycosylation seems to be tissue specific. Calcium-dependent binding of DMBT1 gp340 and recombinant homodimeric TFF3 was observed in vitro. In contrast, no binding was observed with monomeric TFF3 or TFF2. DMBT1 gp340 is a repetitive glycoprotein containing mainly scavenger receptor cysteine-rich (SRCR) domains linked by short proline-rich segments. On the one hand, it is able to aggregate Streptococcus mutans and S. sanguis as well as influenza A virus (maybe via its SRCR domains) promoting their clearance from the oral cavity. On the other hand, DMBT1 gp340 probably interacts via its mannose and fucose structures with the C-type lectin receptors DC-SIGN and Langerin, which prevented binding of Candida albicans and Escheria coli to these receptors. Furthermore, DMBT1 gp340 binds to a variety of host proteins, such as surfactant proteins, lactoferrin, MUC5B, galectin 3, and even TFF2. Currently, there are no reports demonstrating an interaction of TFF3 and DMBT1 gp340 also in vivo. However, a lectin interaction might be possible and this would depend on the glycosylation status, which is tissue specific.
Furthermore, an interaction of homodimeric TFF3 (and even TFF3-FCGBP) with salivary mucins cannot be excluded at the moment and has to be considered thoroughly. This could be of importance as it could affect the viscoelastic properties of salivary mucus. Of special note, the TFF3 concentration in the cervical mucus plug was reported to be correlated with the viscoelastic properties. Remarkably, the gel-forming mucin MUC5B is a major constituent in both the cervicovaginal mucus barrier as well as the saliva. Generally, a lectin interaction is most likely as homodimeric TFF3 has documented lectin activities (for reviews, see.
Taken together, all three TFF3, FCGBP and DMBT1 gp340 are synthesized by mucous epithelia and are involved in mucosal innate immune defense mechanisms. Generally, they could form a complex interaction network. Of special note, Dmbt1-deficient (Dmbt1 KO ) mice show the same phenotype as Tff3 KO mice, i.e., they react extremely sensitively in a dextran sulfate sodium (DSS)-induced colitis model. Here, particularly TFF3, which together with FCGBP is mainly secreted by intestinal goblet cells, seems to strengthen the outer colonic mucus barrier by inhibiting microbial attachment, supporting their clearance, and inhibiting penetration of the inner mucus layer (for reviews, see. A similar mechanism could well protect the oral cavity and the esophagus.
## Summary
Taken together, the protective roles of TFF peptides seem to differ at the mechanistic level. TFF3 is the major salivary TFF peptide and large amounts exist as a TFF3-FCGBP heterodimer. Thus, TFF3 and FCGBP are expected to play a key role in oral and esophageal protection. In, the possible roles of salivary TFF peptides are summarized.
## Therapeutic potential and clinical perspectives
## Saliva, esophagus and esophagogastric junction
Saliva contains high amounts of nitrate, about 10-20 times higher than in plasma. This leads to the formation of nitric oxide and peroxynitrite, particularly at the delicate esophagogastric junction as this is the primary site of acidification by the gastric juice (see also Section 2.1). This is probably an important factor for the development of adenocarcinoma at the esophagogastric junction. Of note, in patients with gastro-esophageal reflux disease, the anatomical location where saliva meets the gastric juice is somewhat changed towards the distal esophagus. Remarkably, TFF3 expression is increased at the esophagogastric junction in gastro-esophageal reflux disease. Furthermore, saliva also seems to be a pivotal player in the pathogenesis of oropharyngeal cancer. Here, ROS play a key role and the addition of glutathione with its free thiol group as antioxidant is protective against damages by aldehydes from cigarette smoke. Additionally, monomeric TFF1 may also be protective here (see Section 2.1).
In humans, TFF3 is the predominant TFF peptide in the salivary glands, and saliva and relatively little TFF3 (and no TFF1 and TFF2) is synthesized in the few esophageal submucosal glands. Thus, the human esophagus seems to rely on protection by the saliva. Of special note, and in contrast, the esophagus of the frog Xenopus laevis is protected by massive own synthesis of an ortholog of TFF2, i.e., xP4, by esophageal goblet cells.
## Tff peptides and their use in chemo-and radiotherapy and in artificial saliva
In the past, TFF peptides have been repeatedly used to protect mucous epithelia from damage (for compilation and reviews, see. For example, Tff3 KO mice were more susceptible to chemotherapy-or radiotherapy-induced intestinal damages and oral application of recombinant TFF3 reduced intestinal mucositis. Subsequently, an oral spray of human dimeric recombinant TFF3 was successfully used in a phase II study to treat colorectal cancer patients in order to reduce chemotherapy-induced oral mucositis. In another attempt, all three TFF peptides delivered by genetically modified Lactococcus lactis were shown to prevent DSS-induced colitis in mice. Later, a mouth rinse formulation of L. lactis-secreting TFF1, coded AG013, was applied to reduce radiation-induced oral mucositis in a hamster model. Finally, these positive results were confirmed in a phase Ib study, where patients with locally advanced head and neck cancer (LAHNC) were treated with AG013 during chemotherapy (35% reduction in percentage of days with ulcerative oral mucositis). Taken together, the protective effects from chemotherapy-or radiotherapy-induced oral mucositis might be due to the weak motogenic and antiapoptotic effects of TFF peptides as well as by anti-inflammatory effects, which could be triggered by binding to the carbohydrate moiety of numerous transmembrane glycoproteins (for reviews, see.
Pharmacological inhibition of TFF3 dimerization by a synthetic drug was reported to enhance the sensitivity of colorectal carcinoma to chemotherapy. Thus, on the one hand, TFF peptides (especially homodimeric TFF3) can be expected to protect from chemotherapy-or radiotherapy-induced oral mucositis. On the other hand, the inhibition of TFF3 dimerization may positively support the effect of chemotherapy. Thus, the application particularly of homodimeric TFF3 in oral cancer patients during chemotherapy needs caution and should be investigated in detail as it has the potential to act as a double-edged sword.
TFF3, together with FCGBP, could play a major role in the innate immune defense of the oral cavity and the esophagus (see Section 2.3 and. Thus, the application of TFF3-FCGBP and/or FCGPB seems to be a novel promising strategy to protect the oral cavity from microbial infections, which are typical side effects of radiotherapy and chemotherapy. In addition to radiotherapy or chemotherapy, reduced saliva production may also be caused by certain diseases, medications, or aging, which leads to a chronic sensation of a dry mouth called xerostomia. Currently, there is still a need for the development of better formulations of saliva substitutes, i.e., artificial saliva, and more sophisticated strategies are needed. Besides rheological and lubricating effects, artificial saliva should support wound healing and have antimicrobial properties. Particularly the latter two properties are important for the maintenance of a healthy oral epithelial barrier. A combination of TFF peptides, FCGBP, and DMBT1 gp340 would be promising to support both wound healing and antimicrobial defense. Currently, there is a formulation commercially available from a porcine gastric mucin preparation, which contains relatively large amounts of TFF2, but no detectable levels of TFF1 or TFF3. However, TFF2 is the least abundant TFF peptide in human saliva and thus this formulation does not reflect the natural situation of TFF peptides in human saliva.
Funding: This research received no external funding. |
Limited Systemic Sclerosis Patients with Pulmonary Arterial Hypertension Show Biomarkers of Inflammation and Vascular Injury
Background: Pulmonary arterial hypertension (PAH) is a common complication for individuals with limited systemic sclerosis (lSSc). The identification and characterization of biomarkers for lSSc-PAH should lead to less invasive screening, a better understanding of pathogenesis, and improved treatment.Methods and Findings: Forty-nine PBMC samples were obtained from 21 lSSc subjects without PAH (lSSc-noPAH), 15 lSSc subjects with PAH (lSSc-PAH), and 10 healthy controls; three subjects provided PBMCs one year later. Genome-wide gene expression was measured for each sample. The levels of 89 cytokines were measured in serum from a subset of subjects by Multi-Analyte Profiling (MAP) immunoassays. Gene expression clearly distinguished lSSc samples from healthy controls, and separated lSSc-PAH from lSSc-NoPAH patients. Real-time quantitative PCR confirmed increased expression of 9 genes (ICAM1, IFNGR1, IL1B, IL13Ra1, JAK2, AIF1, CCR1, ALAS2, TIMP2) in lSSc-PAH patients. Increased circulating cytokine levels of inflammatory mediators such as TNF-alpha, IL1-beta, ICAM-1, and IL-6, and markers of vascular injury such as VCAM-1, VEGF, and von Willebrand Factor were found in lSSc-PAH subjects.Conclusions and Significance: The gene expression and cytokine profiles of lSSc-PAH patients suggest the presence of activated monocytes, and show markers of vascular injury and inflammation. These genes and factors could serve as biomarkers of PAH involvement in lSSc.
# Introduction
Pulmonary arterial hypertension (PAH) is a common complication of systemic sclerosis (SSc) associated with high mortality despite modest improvements in survival due to increased screening and treatment. PAH occurs more frequently in limited SSc (lSSc) than in diffuse SSc (dSSc) [bib_ref] Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited..., Steen [/bib_ref]. Early detection and treatment of PAH secondary to SSc (SSc-PAH) might lead to better patient outcome [bib_ref] Screening and therapy of pulmonary hypertension in systemic sclerosis, Bull [/bib_ref]. For example, early treatment of renovascular disease in SSc patients results in improved renal outcomes [bib_ref] Scleroderma renal crisis, Steen [/bib_ref]. In addition, hypoxia from progressive PAH may accelerate vascular injury by stimulating increased ET-1, VEGF, PDGF and endothelial apoptosis [bib_ref] Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis, Carmeliet [/bib_ref] [bib_ref] Endothelial cell responses to hypoxic stress, Faller [/bib_ref]. Current tools used in screening for PAH in SSc patients include echocardiography, pulmonary function testing, and levels of B-type natriuretic peptide (BNP), none of which have demonstrated adequate sensitivity or specificity [bib_ref] Screening and therapy of pulmonary hypertension in systemic sclerosis, Bull [/bib_ref]. Thus, SSc patients could benefit from biomarkers that permit earlier detection of patients at risk for PAH.
SSc patients can have severe disease in several different vascular beds, resulting in digital ischemia, telangiectasias, scleroderma renal crisis and PAH. Similar to other vascular pathology in SSc, SSc-PAH vascular remodeling consists of intimal thickening of pulmonary arterioles and capillaries due to intimal cell proliferation and deposition of extracellular matrix [bib_ref] Fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated..., Dorfmuller [/bib_ref]. Inflammation may play a role in SSc-PAH, as patients have circulating autoantibodies and perivascular inflammatory cell infiltrates such as T-and Blymphocytes, and macrophages [bib_ref] Fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated..., Dorfmuller [/bib_ref]. While there are similarities in the histological appearance of vascular lesions and the presumed pathogenesis between idiopathic PAH (IPAH) and SSc-PAH, the risk of death for SSc-PAH patients is higher [bib_ref] Hemodynamics and survival in patients with pulmonary arterial hypertension related to systemic..., Kawut [/bib_ref] [bib_ref] Clinical differences between idiopathic and scleroderma-related pulmonary hypertension, Fisher [/bib_ref]. There are also indications that SSc-PAH patients have fewer plexiform lesions and more intimal hyperplasia [bib_ref] Fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated..., Dorfmuller [/bib_ref] [bib_ref] Pathogenesis and evolution of plexiform lesions in pulmonary hypertension associated with scleroderma..., Cool [/bib_ref] , as well as differences in the involvement of the pulmonary veins [bib_ref] Fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated..., Dorfmuller [/bib_ref].
Since pulmonary tissue is not readily accessible, it is difficult to perform gene expression studies in SSc-PAH analogous to those of SSc skin biopsies [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref] [bib_ref] Gene profiling of scleroderma skin reveals robust signatures of disease that are..., Gardner [/bib_ref] [bib_ref] Systemic and cell type-specific gene expression patterns in scleroderma skin, Whitfield [/bib_ref]. An alternative is to analyze gene expression of peripheral blood mononuclear cells (PBMCs). Two studies have investigated the gene expression of PBMCs in IPAH and SSc-PAH. Bull et al. examined PBMC samples from 7 patients with IPAH, 3 patients with SSc-PAH (in a total of 8 patients with PAH related to a secondary cause), and 6 healthy controls [bib_ref] Gene microarray analysis of peripheral blood cells in pulmonary arterial hypertension, Bull [/bib_ref]. Genes were identified that discriminated PAH patients from healthy controls. Grigoryev et al. analyzed PBMCs from 9 patients with IPAH, 10 patients with SSc-PAH, and 5 healthy controls [bib_ref] Identification of candidate genes in scleroderma-related pulmonary arterial hypertension, Grigoryev [/bib_ref]. Gene expression concordant between the IPAH and SSc-PAH groups was contrasted with discordant gene expression. Neither study examined the alterations in PBMC gene expression specifically attributable to PAH in SSc. We hypothesized that PBMC gene expression would specifically separate lSSc patients from healthy controls, as well as SSc patients with and without PAH. We report here PBMC gene expression defining lSSc patients with and without PAH, and healthy controls.
# Results
Although PAH can occur in patients with dSSc, in order to provide a more homogeneous population for analysis, we limited this study to patients with lSSc, where it occurs more commonly. Gene expression of PBMC samples was analyzed for 21 lSSc patients without PAH (lSSc-NoPAH), 15 lSSc patients with PAH (lSSc-PAH), and 10 healthy controls. For three patients (two lSSc-NoPAH and one lSSc-PAH), an additional PBMC sample was analyzed one year after the baseline, resulting in 49 total samples. With 5 technical replicates, a total of 54 microarrays were analyzed. LSSc patients with mildly elevated pulmonary capillary wedge pressure (PCWP) [bib_ref] Design of the REVEAL registry for US patients with pulmonary arterial hypertension, Mcgoon [/bib_ref] to #18) were included in our primary analyses consistent with the REVEAL registry with similar rationale [bib_ref] Design of the REVEAL registry for US patients with pulmonary arterial hypertension, Mcgoon [/bib_ref]. Patients that had a mild increase in PCWP included in the primary analysis all had significantly elevated pulmonary vascular resistance (PVR, see [fig_ref] Table 1: Clinical and hemodynamic data on subjects associated with PBMC sample arrays [/fig_ref] , and significant increases in both the pulmonary artery diastolic minus pulmonary capillary wedge pressure (PAd-PCWP) gradient (.10) and the transpulmonary gradient (.15). Thus, each was considered to have PAH by the pulmonary hypertension expert caring for the patient. Further data supported the diagnoses of PAH in these patients, Patient 66 had a right heart catheterization two months prior to the catheterization carried out on the day of study enrollment, showing similar elevated pressures (mPAP = 49) but a normal PCWP [bib_ref] Gene profiling of scleroderma skin reveals robust signatures of disease that are..., Gardner [/bib_ref]. Patient 31 had relatively mild PAH (mPAP = 32) and a relatively mild increase in PVR (207), but no evidence of right (RV) or left ventricular (LV) dysfunction on echocardiogram or by cardiac output. Patient 42 had severely elevated mPAP and PVR, with an echocardiogram showing normal LV function but severely dilated RV consistent with severe PAH. Patient 89 had relatively mild PAH (mPAP = 37, PVR = 205), with mild elevation of the PCWP (PCWP = 16), but a PAd 10 mmHg greater than the PCWP consistent with PAH. A limited number of patients with pulmonary fibrosis, some with extensive pulmonary fibrosis (2 in each group of lSSc-PAH and lSSc-noPAH patients), were also included in our primary analyses because PAH is common in these SSc patients [bib_ref] Prevalence and characteristics of moderate to severe pulmonary hypertension in systemic sclerosis..., Launay [/bib_ref] and it was deemed important to understand whether biomarkers for lSSc-PAH patients with pulmonary fibrosis were similar to biomarkers for lSSc-PAH patients without pulmonary fibrosis. To further support the results of our primary analysis which included these patients, we also carried out a secondary analysis excluding patients with elevated PCWP and/or extensive pulmonary fibrosis, described below.
PBMC gene expression distinguishes lSSc and healthy controls Data were first analyzed for genes that differentiated lSSc from healthy controls. The most significantly differentially expressed genes between lSSc patients, regardless of PAH status, and healthy controls were identified with Significance Analysis of Microarrays (SAM) [bib_ref] Significance analysis of microarrays applied to the ionizing radiation response, Tusher [/bib_ref]. 206 probes were selected with a false discovery rate (FDR) ,0.18% and clustered hierarchically by both sample and probe . The division between the lSSc and healthy control samples was clear, with all healthy controls clustering together . Four out of five technical replicates clustered either immediately adjacent to one another or with a single sample separating them (black bars, . Notably, all three PBMC samples collected one year after baseline demonstrated gene expression nearly identical to the initial samples (yellow bars, . Thus, for these three patients, the gene expression was stable over a period of one year, results remarkably consistent with the longitudinal analysis of gene expression in SSc skin (Pendergrass, Lafyatis, Whitfield, In preparation). The full figure with all probe names is available as ; the complete data file is available as Supplementary Data File S1.
The major dendrogram bifurcation places all healthy controls onto one branch, and divides the lSSc patients into two groups, despite selecting for genes that ideally stratify the groups . Statistically significant dendrogram branches (*, p#0.001) were determined with SigClust [bib_ref] Statistical Significance of Clustering for High-Dimension, Low-Sample Size Data, Liu [/bib_ref]. The left branch is significant at the 0.001 level (red branches, and includes PBMCs from 29 of 36 lSSc patients, while the remaining 7 lSSc samples group with healthy controls (black branches, . As observed for dSSc skin [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref] , most lSSc patients show an expression profile distinct from healthy controls . The 7 lSSc patients that group with healthy controls therefore constitute a 'normal-like' group, similar to that observed in SSc skin [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref].
Genes with increased expression in lSSc PBMCs are associated with inflammation and vascular injury. Those associated with inflammation include interleukin-1, interferon, and TNF-alpha regulated genes such as intracellular adhesion molecule 1 (ICAM1) [bib_ref] Intercellular adhesion molecule-1 deficiency attenuates the development of skin fibrosis in tight-skin..., Matsushita [/bib_ref] , caspase 1 (CASP1) [bib_ref] The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis, Franchi [/bib_ref] , lymphotoxin beta receptor TNFR superfamily, member 3 (LTBR) [bib_ref] Inhibition of the lymphotoxin pathway as a therapy for autoimmune disease, Browning [/bib_ref] , signal transducer and activator of transcription 1 (STAT1), and allograft inflammatory factor (AIF-1) [bib_ref] The role of allograft inflammatory factor 1 in systemic sclerosis, Galdo [/bib_ref]. Genes associated with angiogenesis, proliferation, hypoxia and vascular injury include Pre-B-cell colony enhancing factor (PBEF1/Visfatin) [bib_ref] Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis, Bae [/bib_ref] , Guanylate binding protein 1 (GBP-1) [bib_ref] Guanylate binding protein-1 inhibits spreading and migration of endothelial cells through induction..., Weinlander [/bib_ref] , Tryptophanyl-tRNA synthetase (WARS) [bib_ref] Cytokine-like activities of some aminoacyl-tRNA synthetases and auxiliary p43 cofactor of aminoacylation..., Ivakhno [/bib_ref] , Tissue inhibitor of metalloproteinase 2 (TIMP2), and CyBB cytochrome b-245 beta polpypetide (CYBB/NOX2) [bib_ref] Regulation of NADPH oxidases: the role of Rac proteins, Hordijk [/bib_ref]. Genes with decreased expression in lSSc did not show cohesive biological processes but included protein kinase C eta (PRKCH), which had decreased expression in PBMCs from rheumatoid arthritis patients [bib_ref] Association and expression study of PRKCH gene in a French Caucasian population..., Teixeira [/bib_ref].
## Gene expression in lssc-pah
In addition to the gene expression differences found between the lSSc and healthy control samples, a multi-class SAM analysis identified gene expression that ideally distinguished lSSc-PAH, lSSc-NoPAH, and healthy controls. A total of 305 probes were selected with an FDR,0.14%, and clustered hierarchically in the gene and sample dimensions [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref]. Twelve of 15 lSSc-PAH samples clustered together on a significant branch (Group 1, [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] , and showed the largest differences in gene expression relative to controls. Nine of 21 lSSc-NoPAH samples clustered on the same branch and showed intermediate expression levels. Three lSSc-PAH samples (patients 64, 31, and 2) grouped on the opposite branch (Group 2, [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] with lSSc-NoPAH samples. All technical replicates clustered adjacent to each other, or with only a single sample separating them. Samples collected one year later showed gene expression most similar to the previous sample. The full figure with all probe names is available as [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] ; the data file is available as Supplementary Data File S2. A striking feature of the gene expression is a gradient with the largest differences between the lSSc-PAH and controls, with lSSc-NoPAH showing intermediate expression levels. To validate this finding, the most significant differentially expressed genes were analyzed by quantitative RT-PCR. Nine genes were validated [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref] : intercellular adhesion molecule 1 (ICAM1), associated with vascular injury; Interferon-c receptor 1 (IFNcR1), interleukin 1 beta (IL1B), interleukin 13 receptor a1 (IL13Ra1), janus kinase 2 (JAK2), allograft inflammatory factor 1 (AIF1) and chemokine (C-C motif) receptor 1 (CCR1), all of immunological relevance; Aminolevulinate delta synthase 2 (ALAS2), a possible regulator of the response to hypoxia; and tissue inhibitor of metalloproteinase-2 (TIMP2) a known regulator of fibrosis. All had significantly higher expression in the lSSc-PAH group relative to healthy controls (p#0.05, [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref]. For IL1B, IL13Ra1, and TIMP2 there was a significant difference in expression between lSSc-PAH and lSSc-NoPAH samples (p#0.05, [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref]. The observed differences in gene expression between these groups are maintained even when excluding the four patients with extensive pulmonary fibrosis (two in each of the lSSc-PAH and lSSc-NoPAH groups), or the four patients with mildly elevated PCWP (.15 to #18, all in the lSSc-PAH group), indicating that these biomarkers of PAH are not primarily driven by pulmonary fibrosis or heart failure.
In addition, we carried out a complete secondary analysis excluding all patients with either PCWP .15 or extensive pulmonary fibrosis (both groups indicated in [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref]. 305 probes were again selected using multi-class SAM and clustered hierarchically in the gene and sample dimensions. The dendrogram of patient clustering following this analysis is very similar to that seen when including these patients [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref] with the major groupings maintained. Therefore the inclusion of these patients has little effect on the overall results.
## Gene expression groups are associated with pah severity
We examined the relationship between pulmonary severity metrics and the gene expression groups defined in the three-class analysis. The branch containing most of the lSSc-PAH patients is labeled 'Group 1', and the branch containing all healthy controls is labeled 'Group 2' [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref]. Although SigClust suggested potential subgroups within Group 2, there was high variability at different p-values, suggesting more samples would be needed to define these groups conclusively. We have therefore considered these samples as a single group.
Strikingly, when comparing the mean pulmonary arterial pressure (mPAP) values between Groups 1 and 2, the mPAP was higher for lSSc-PAH patients [fig_ref] Figure 4: Distribution of PAH assessment measures between gene expression groups [/fig_ref] , solid black circles) in Group 1 versus Group 2. The mean PAP of the three patients with lSSc-PAH in Group 2 showed the lowest mPAPs of the patients with PAH, and these patients had a more intermediate gene expression compared to lSSc-PAH patients of Group 1. We also compared the diffusing capacity for carbon monoxide (DLCO) between the two groups, as isolated depression of diffusion capacity is associated with PAH in SSc [bib_ref] Isolated pulmonary hypertension in systemic sclerosis with diffuse cutaneous involvement: association with..., Sacks [/bib_ref]. DLCO was decreased in Group 1 for both SSc-PAH and SSc-NoPAH patients [fig_ref] Figure 4: Distribution of PAH assessment measures between gene expression groups [/fig_ref] , pvalue = 0.000263). In a reciprocal pattern to that seen for the mPAPs, the three lSSc-PAH patients in Group 2 (solid black circles) had more preserved DLCO compared to the lSSc-PAH patients in Group 1. Therefore, lSSc-PAH patients in Group 2 show milder changes from normal in both mPAP and DLCO compared to lSSc-PAH patients of Group 1.
Three patients in Group 1 (2 lSSc-PAH: Pat22 and Pat86, and 1 lSSc-NoPAH: Pat89) and one patient in Group 2 (lSSc-PAH: Pat64) had extensive ILD. The difference in FVC between Group1 and Group 2 was not statistically significant, indicating that ILD was unlikely to be the primary clinical covariate driving gene expression in these groups [fig_ref] Table 1: Clinical and hemodynamic data on subjects associated with PBMC sample arrays [/fig_ref]. Additionally, we were unable to detect clustering related to medications each patient was taking. Fifteen out of 36 patients were not on vasoactive medications at time of the first blood draw [fig_ref] Table 1: Clinical and hemodynamic data on subjects associated with PBMC sample arrays [/fig_ref]. In patients taking vasoactive medications, the most common treatments were: phosphodiesterase type 5 (PDE5) inhibitors, sildenafil (4 lSSc-PAH and 1 lSSc-noPAH patient) and tadenafil (1 lSSc-PAH patient); epoprostenol (3 lSSc-PAH patients and 1 lSSC-NoPAH patient); and nifedipine (9 lSSc-NoPAH patients). Two patients, Pat60 and Pat35, started sildenafil before the second blood draw and showed similar gene expression between the two time points [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] , indicating that this class of medication did not affect the expression analyses.
## Coordinate enrichment of biological processes
To investigate the pathways deregulated in lSSc with and without PAH, we performed a separate multiclass SAM analysis and selected a more inclusive list of 2,313 probes (FDR = 2.67%). This list was analyzed for enriched biological processes using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). [fig_ref] Figure 4: Distribution of PAH assessment measures between gene expression groups [/fig_ref] shows clusters of gene expression analyzed using DAVID. The gene expression signature found in all but three lSSc-PAH patients [fig_ref] Figure 4: Distribution of PAH assessment measures between gene expression groups [/fig_ref] , showed enrichment for GO biological processes associated with proliferation and inflammatory responses (Benjamini-Hochberg corrected, p#0.05), including negative regulation of apoptosis and cell differentiation, I-kappaB kinase/NF-kappaB cascade, myeloid cell differentiation, response to external stimulus, and inflammatory response. Genes included annexin A1 (ANAX1), chemokine ligand 2 (CCL2), BCL2 related protein A1 (BCL2A1), and tumor necrosis factor receptor a1 (TNFRSF1A).
## Gene expression associated with specific cell-types
To identify gene expression associated with specific cell types, as well as to seek out indications of cellular activation and differentiation, we used experimentally derived gene sets from isolated cells. Gene sets were obtained for B-cells, T-cells, macrophages, monocytes, immature and mature dendritic cells (DCs), and myeloid cells (DCs, macrophages, and monocytes) [bib_ref] Cell-type specific gene expression profiles of leukocytes in human peripheral blood, Palmer [/bib_ref] [bib_ref] Cellular genomic maps help dissect pathology in human skin disease, Haider [/bib_ref]. While PBMCs do not contain macrophages, the gene expression profiles for these cells were included in the analysis due to the possibility of monocyte populations showing signs of differen- . Gene expression differentiates lSSc PBMC samples from healthy controls. A. Clustering dendrogram using 206 probes that most differentiated lSSc (red identifiers) and healthy control samples (green identifiers; FDR cutoff,0.18%). Black bars beneath the sample identifiers connect technical replicates. Samples collected one year apart are indicated by yellow bars. Asterisks indicate statistically significant clustering determined by SigClust. The red dendrogram branch contains the majority of lSSc samples, and the black branch contains those lSSc samples most like healthy controls. B. Heat map showing expression of the 206 probes after 2-dimensional hierarchical clustering. Expression values above the mean for each probe are indicated in red and below the mean are indicated in green. The yellow box highlights the gene expression data for the dendrogram branch containing the majority of lSSc samples. A subset of genes is listed next to the heat map. The full figure with all probe names is available as . doi:10.1371/journal.pone.0012106.g001 . The yellow box tiation into macrophages. We used Gene Set Enrichment Analysis (GSEA) to determine gene set signatures significantly overrepresented among the probes ranked by significance of differential expression between lSSc and healthy control samples. Four gene sets were significantly enriched at p#0.1: myeloid cells (p = 0.013, FDR q-value = 0.070); dendritic cells (p = 0.014, FDR q-value = 0.056); macrophages (p = 0.011, FDR q-value = 0.059); and immature dendritic cells (p = 0.079, FDR q-value = 0.237). The monocyte gene set was fifth in the list ordered by significance and although above the p-value cutoff (p = 0.131), was below the suggested FDR cutoff of 0.25 (FDR q-value = 0.239). The enrichment of gene sets for the related cell types of myeloid, dendritic and macrophage cells, coupled with the enrichment of GO biological processes of endocytosis and cell motility, suggests the presence of monocytes undergoing cellular recruitment and differentiation into further cell types such as macrophages, particularly in lSSc-PAH patients. Gene expression and cellular differences in monocytes have been found between SSc and healthy control patients [bib_ref] Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein..., Duan [/bib_ref] [bib_ref] Changes in circulating monocytes in patients with progressive systemic sclerosis, Andrews [/bib_ref] , and the indication here of lSSc-PAH patients in particular having increased gene expression associated with macrophages and dendritic cells suggests an inflammation and injury response with cellular recruitment and differentiation in PAH patients. shows the GSEA enrichment plot and associated gene expression data for the top five gene sets.
## Lssc-pah patients show elevated serum biomarkers of inflammation and vascular injury
Serum samples were analyzed for 18 lSSc-PAH, 19 lSSc-NoPAH, and 6 healthy controls. Of these individuals, 13 lSSc-PAH, 18 lSSc-NoPAH, and 4 healthy controls were in the gene expression analysis. Levels of 89 cytokines were measured using Human Multi-Analyte Profiling (MAP) multiplexed immunoassays. To select cytokines with the greatest differential signal between the three groups, a multi-class SAM analysis was used to compare lSSc-NoPAH, lSSc-PAH, and healthy controls. The protein quantities for 42 cytokines (FDR = 4.93%) were clustered in patient and cytokine dimensions. Significantly stable clusters are indicated (p,0.05, . LSSc-PAH, lSSc-NoPAH, and healthy controls clustered together except for lSSc-PAH-64 and lSSc-NoPAH-91 . Notably, lSSc-PAH-64 also had gene expression similar to the lSSc-NoPAH patients [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] , confirming the gene expression findings.
Markers of vascular injury were increased in the lSSc-PAH patients, including Von Willebrand Factor (vWF) [bib_ref] Cell adhesion mechanisms in platelets, Varga-Szabo [/bib_ref] , C-reactive protein (CRP) [bib_ref] C-reactive protein: a new predictor of adverse outcome in pulmonary arterial hypertension, Quarck [/bib_ref] , and vascular endothelial growth factor (VEGF). VEGF and vWF have been found upregulated in idiopathic pulmonary arterial patients [bib_ref] Reduced number and activity of circulating endothelial progenitor cells in patients with..., Junhui [/bib_ref]. Also found are fatty acid binding protein and myoglobin which are markers of acute myocardial infarction [bib_ref] The value of biochemical markers for risk stratification prior to hospital admission..., Herlitz [/bib_ref].
Found at higher levels in lSSc-PAH are cytokines characteristic of inflammatory response including: intercellular adhesion molecule 1 (ICAM-1), which was also increased in the microarray data and confirmed by qRT-PCR; vascular cell adhesion molecule 1 (VCAM-1); interleukin-8 (IL-8); interleukin-6 (IL-6); interleukin 1beta (IL-1b); and tumor necrosis factor alpha (TNF-alpha). Consistent with the cytokine data, TNF-alpha inducible protein 2 (TNFAIP2) shows increased gene expression in the lSSc-PAH patients. Increased levels of tumor necrosis factor receptor superfamily member 1B (TNFRII/TNFR1B) [bib_ref] Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors, Aderka [/bib_ref] and Beta2microglobulin (b2m) are also observed in the lSSC-PAH patients. High levels of circulating TNFRII and b2m have been reported in SSc [bib_ref] Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein..., Duan [/bib_ref] [bib_ref] An evaluation of some inflammatory, coagulative and immune factors in progressive systemic..., Garcovich [/bib_ref] , however these biomarkers have not previously been investigated specifically for lSSc-PAH patients.
# Discussion
Our results demonstrate gene expression differences between the majority of lSSc patients and healthy controls, as well as differences between lSSc-PAH and lSSc-NoPAH patients. PBMC gene expression divides lSSc patients based on the presence and severity of PAH as assessed by PAP and DLCO. Although some patients with PAH fell into the group without PAH in the hierarchical clustering analysis, these patients uniformly had the mildest PAH in the study. Further studies will be required to determine if the lSSc patients without PAH but with gene expression more similar to patients with PAH, are at higher risk of eventually developing this complication. Our current data show the gene expression of lSSc-PAH and lSSc-NoPAH samples are stable over the course of approximately one year. Further analyses over an extended period are needed to rigorously test whether gene expression can predict the onset of PAH, and how gene expression in lSSc patients changes over time.
In addition to gene expression differences between lSSc-PAH and lSSc-NoPAH patients, we found differences in cytokines between the two groups. The cytokine and gene expression profiles suggest activated and differentiating monocytes. Notably, IL1beta, produced primarily by stimulated monocytes [bib_ref] Monocyte expression of the human prointerleukin 1 beta gene (IL1B) is dependent..., Kominato [/bib_ref] , and Caspase 1, an important enzyme for cleaving the IL-1beta precursor [bib_ref] Differential requirement for the activation of the inflammasome for processing and release..., Netea [/bib_ref] were both increased in lSSc-PAH. Gene expression of TLR4, a key receptor for monocyte activation and IL1 upregulation, and MYD88, a TLR signaling molecule, was also upregulated. Consistent with TLR activation of NF-kappa B [bib_ref] Regulation of lipopolysaccharide-inducible genes by MyD88 and Toll/IL-1 domain containing adaptor inducing..., Hirotani [/bib_ref] , GO biological processes enriched in lSSc-PAH include the I-kappaB kinase/NF-kappaB cascade. Collectively these observations suggest that activated monocytes play an important role in the inflammatory response in lSSc-PAH patients.
The combined gene expression and cytokine data indicate the involvement of other myeloid cell types beyond monocytes in lSSc-PAH patients. The most significant GSEA results were for gene sets associated with myeloid cells, dendritic cells, and macrophages. GO biological processes of myeloid cell differentiation, endocytosis, cell motility, and cell projection biogenesis were also enriched. Dendritic cells have been implicated in the immunopathology of IPAH with increases in DCs found in vasculopathy rat models of PAH, and increased DC infiltrates in affected vessels in human IPAH [bib_ref] Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension, Perros [/bib_ref]. lSSc-PAH patients showed increased IL-6, which signals monocytes to differentiate into macrophages, has previously been shown upregulated in PAH patients and can induce PAH in transgenic mice [bib_ref] IL-6 switches the differentiation of monocytes from dendritic cells to macrophages, Chomarat [/bib_ref] [bib_ref] Increased plasma monocyte chemoattractant protein-1 level in idiopathic pulmonary arterial hypertension, Itoh [/bib_ref] [bib_ref] Interleukin-6 overexpression induces pulmonary hypertension, Steiner [/bib_ref]. Cytokines ICAM-1 and VCAM-1 are increased in lSSc-PAH, along with increased expression of the ICAM-1 gene. These cytokines are induced through IL1B and TNF-alpha (both increased in lSSc-PAH patients on the cytokine array) and initiate the binding of monocytes to the endothelium. Through a combined gene expression and cytokine analysis approach, these data support a role for activated DCs and macrophages in lSSc-PAH. LSSc-PAH patients also showed upregulated circulating highlights the gene expression of Group 1, which contains most of the lSSc-PAH PBMC samples. A subset of genes from the 305 probes are listed. The full figure with all probe names is available as Supplementary [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref]. Patients with PCWP .15 (mm Hg) are indicated with a purple asterisk (*) and those with extensive ILD are indicated with a blue asterisk (*). The analysis was repeated without these patients and nearly identical groupings were obtained [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref]. doi:10.1371/journal.pone.0012106.g002 levels of other inflammatory cytokines, IL1B and TNF-alpha and downstream targets of these mediators, notably the adhesion molecules, ICAM-1 and VCAM-1 These data support a role for activated DCs and macrophages in lSSc-PAH.
## Grigoryev et al. found genes upregulated in both ipah and
SSc-PAH compared to healthy controls, as well as a relationship between decreasing gene expression and increasing right atrium pressure [bib_ref] Identification of candidate genes in scleroderma-related pulmonary arterial hypertension, Grigoryev [/bib_ref]. In this study we found upregulation of some of the same genes including adrenomedulin (ADM) and pentraxinrelated gene (PTX3), but we found increased expression of these genes in some of the lSSc-NoPAH patients as well.
We found increases in some of the same cytokines, including ICAM-1, TIMP1, and vWF, previously noted by Duan et al. to be increased in lSSc patients compared to healthy controls [bib_ref] Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein..., Duan [/bib_ref]. We found levels of these cytokines were higher in lSSC patients with PAH compared to those without PAH, indicating the importance of these as biomarkers of lSSc-PAH.
Increased expression of inflammatory cytokines in the serum of SSc patients with PAH suggests the possibility that these cytokines might play a role in pathogenesis. Notably, TNF was found to be increased in the serum of lSSc-PAH patients in this study and TNF increases pulmonary vascular resistance [bib_ref] Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils, Horvath [/bib_ref] , stimulates endothelin-1 [bib_ref] Synergistic induction of endothelin-1 by tumor necrosis factor alpha and interferon gamma..., Wort [/bib_ref] and leads to PAH in TNF transgenic mice [bib_ref] Pulmonary hypertension in TNF-alpha-overexpressing mice is associated with decreased VEGF gene expression, Fujita [/bib_ref] TNF and TNF-regulated genes are increased in patients with rheumatoid arthritis, where TNF inhibition provides a clear therapeutic benefit [bib_ref] Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis, Zhang [/bib_ref]. In SSc, elevated TNF has been described in diffuse cutaneous patients with pulmonary fibrosis [bib_ref] Elevated serum tumor necrosis factor-alpha levels in patients with systemic sclerosis: association..., Hasegawa [/bib_ref]. One recent open label study has suggested that TNF inhibition may ameliorate skin disease [bib_ref] An openlabel pilot study of infliximab therapy in diffuse cutaneous systemic sclerosis, Denton [/bib_ref] and a case report has suggested some possible value for PAH [bib_ref] Infliximab treatment in a patient with systemic sclerosis associated with lung fibrosis..., Bargagli [/bib_ref]. IL6 was found to be increased in lSSc-PAH patients on the cytokine array, and IL6 is also associated with PAH in the context of chronic obstructive pulmonary disease (COPD) [bib_ref] Role for interleukin-6 in COPD-related pulmonary hypertension, Chaouat [/bib_ref]. Further supporting a possible role in SSc-associated PAH, IL6 transgenic mice develop PAH [bib_ref] Interleukin-6 overexpression induces pulmonary hypertension, Steiner [/bib_ref] , and hypoxia-induced PAH in mice is ameliorated in IL6-deleted mice [bib_ref] Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice, Savale [/bib_ref]. In our study, although IL6 protein was elevated in the serum, IL6 mRNA levels were not elevated in PBMC samples, suggesting that IL6 might be secreted primarily from other cell types, such as endothelial cells, fibroblasts or neutrophils. Thus these inflammatory mediators, possibly through activation of an innate immune response, may play a role in SSc-associated PAH.
Our results provide potential biomarkers that identify patients with lSSc, showing a specific subset of lSSc patients with PAH that can be identified through cellular and circulating biomarkers, and suggest pathogenic cellular and immunologic pathways that are upregulated in these patients. The gene expression profiles in PBMCs in our study may also provide biomarkers to predict the risk of lSSc patients for developing PAH.
# Methods
# Ethics statement
This study was approved by the Boston University Medical Center Institutional Review Board, and the Committee for the Protection of Human Subjects at Dartmouth College. All patients signed informed written consent forms approved by the Boston University Medical Center Institutional Review Board.
## Patient selection
Subjects included limited cutaneous systemic sclerosis (lSSc) patients according to criteria in LeRoy et al. [bib_ref] Scleroderma (systemic sclerosis): classification, subsets and pathogenesis, Leroy [/bib_ref] and healthy controls. Subjects with lSSc were stratified into those with or without PAH on the basis of echocardiogram and pulmonary artery catheterization according to consensus criteria with exceptions noted below. LSSc subjects with echocardiogram showing a systolic pulmonary arterial pressure (PAP) ,35 mm Hg and no clinical features suggesting PAH were considered to not have PAH. Subjects showing evidence of PAH by echocardiogram or other clinical features who underwent right heart catheterization and showed a mean PAP#25 mm Hg were also considered to not have PAH (4 patients). Subjects showing evidence of PAH by echocardiogram or other clinical criteria who underwent catheterization and showed mean PAP .25 mm Hg and pulmonary capillary wedge pressure (PCWP) #15 were considered to have PAH, or with PCWP .15, but #18 considered to have PAH if adjudicated by the attending . A subset of cytokines shows association with lSSc and lSSc-PAH. A MAP panel was used to profile the cytokines in plasma from lSSc-PAH, lSSc-NoPAH, and healthy control samples. Cytokines with significantly different levels between the three groups were selected using a multiclass SAM analysis (FDR,4.93%). 42 cytokines were selected and organized by 2 dimensional hierarchical clustering. The dendrogram shows lSSc-PAH (red), lSSc-NoPAH (blue), and healthy controls (black) group distinctly. Asterisks indicate stable groups as determined by SigClust. Increasing brightness of red indicates relative fold change increase in cytokine levels. Increasing brightness for green pixels indicates decreasing cytokine levels. Yellow boxes highlight the cytokines with increased levels in the three major groups, lSSc-PAH, lSSc regardless of PAH status, and healthy controls. doi:10.1371/journal.pone.0012106.g005 pulmonologist on the basis of PVR, PAd-PCWP gradient and transpulmonary gradient (see further details in results section for individual patients meeting this criteria). Most subjects entered into the study had no or minimal interstitial lung disease. The extent of disease in those with interstitial lung disease (9 subjects) was stratified as either mild (5 subjects) or extensive (4 subjects) according to previously described methodology using highresolution chest computerized tomography (HRCT) and forced vital capacity (FVC) criteria [bib_ref] Interstitial lung disease in systemic sclerosis: a simple staging system, Goh [/bib_ref]. Blood was collected from patients on the day of catheterization for catheterized subjects, or within three months of the date of echocardiogram for subjects who were not catheterized.
## Peripheral blood mononuclear cell isolation
PBMCs were collected in Becton Dickinson vacutainer CPT tubes and processed within 30 minutes after collection. Tubes were centrifuged at 18006g for 30 min at room temperature . The PBMC cell layer was then transferred to a 15 mL tube, and the PBMCs washed twice with PBS and lysed in RNeasy RLT buffer (Qiagen, Valencia, CA).
## Rna isolation and microarray hybridization
Total RNA was prepared from PBMCs using the RNeasy Mini Kit (Qiagen). 250 ng of RNA was converted to cDNA and amplified as labeled cRNA using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Patient and healthy control RNA were labeled with Cy3 fluorescent dye, and Universal Human Reference RNA (Stratagene) was labeled with Cy5 fluorescent dye. Labeled cRNA was hybridized to Agilent 4644,000 element DNA microarrays in a reference-based design as previously described [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref] , with the following changes. Patient or healthy control cRNA was co-hybridized with UHR cRNA to microarrays for 17 hours at 65uC. Arrays were washed for 1 minute each in 66 SSPE, 0.005% N-Lauroylsarcosine at room temperature and then in 0.066 SSPE, 0.005% N-Lauroylsarcosine at 37uC. This was followed by an acetonitrile wash for 1 minute at room temperature and Stabilization and Drying Solution for 30 seconds at room temperature. Microarrays were scanned, processed, and data normalized and filtered as previously described [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref].
## Data access
All microarray data from this study has been deposited to NCBI's Gene Expression Omnibus (GEO; http://www.ncbi.nlm. nih.gov/geo/; Accession Number GSE19617) and is MIAME compliant.
## Gene selection and hierarchical clustering
Gene selection was performed using Significance Analysis of Microarrays (SAM) [bib_ref] Significance analysis of microarrays applied to the ionizing radiation response, Tusher [/bib_ref]. For the two class unpaired t-test, arrays were grouped by lSSc vs. healthy controls and probes selected with a false discovery rate (FDR) ,0.18%. The gene expression levels of the X (inactive)-specific transcript (XIST) gene were removed from the analysis because it caused one healthy control sample to group by gender rather than disease [fig_ref] Figure S6: Disease vs [/fig_ref].
For the multiclass analysis, samples were divided by lSSc-PAH, lSSc-NoPAH, and healthy controls. We performed a stringent analysis that identified 305 probes (FDR,0.14%) and a less stringent analysis that identified 2,313 probes (FDR,2.67%) . Average linkage hierarchical clustering was performed as previously described [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref].
# Pathway analysis
The Database for Annotation, Visualization, and Integrated Discovery tool (DAVID) [bib_ref] The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to..., Huang Da [/bib_ref] was used to analyze coordinately regulated groups of genes for enriched GO biological processes.
Gene Set Enrichment Analysis (GSEA) [bib_ref] Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles, Subramanian [/bib_ref] , was used to determine enrichment of cell-type specific gene sets. Genes were ranked by significance of differential expression between lSSc and healthy controls. For each gene set, GSEA determined if the set was found at the top or bottom of the ranked list. Significance was determined by permuting the data. Gene sets for B and T cells were obtained from Palmer et al. [bib_ref] Cell-type specific gene expression profiles of leukocytes in human peripheral blood, Palmer [/bib_ref]. Gene sets for macrophages, monocytes, immature and mature dendritic cells, CD3/CD28 activated T cells, total PBMCs and myeloid cells (DCs, macrophages, monocytes) were obtained from Haider et al. [bib_ref] Cellular genomic maps help dissect pathology in human skin disease, Haider [/bib_ref].
## Quantitative rt-pcr
RT-PCR was carried out as previously described [bib_ref] Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal..., Farina [/bib_ref] , using ABI primers for the following genes: ICAM1 (Hs00164932_m1), IFNcR1 (Hs00166223_m1), IL1B (Hs01555410_m1), IL13Ra1 (Hs00609817_m1), JAK2 (Hs01078136_m1), AIF1 (Hs00357551_ g1), CCR1 (Hs00174298_m1), ALAS2 (Hs00163601_m1), TIMP2 (Hs00234278_m1). Relative RNA quantity was determined using the delta-delta CT method [bib_ref] Analysis of relative gene expression data using real-time quantitative PCR and the..., Livak [/bib_ref].
## Cytokine panel
Serum samples were collected from 43 patients, and sent to Rules-Based Medicine (http://www.rulesbasedmedicine.com) for the Human Multi-Analyte Profiling (MAP) multiplexed immuno assay. MAP assays were carried out two different times including approximately equal numbers of samples from all three subject groups. The first assay obtained analyte measurements for 89 cytokines, the second for 90 cytokines. Results that were not detectable were replaced with the reported least detectable amount for that cytokine. To control for bias between the two MAP assays, the data in the second MAP assay were normalized for each cytokine to the proportion of the average values in the first MAP assay divided by the average values in the second MAP assay. Multiclass SAM analysis was used to compare cytokines detected between LSSc-NoPAH, LSSc-PAH, and healthy controls, and the resultant data at an FDR cutoff of 4.93% was clustered in both the patient and cytokine dimension.
# Statistical analysis
The R statistical package was used for box plots, two-group ttest analysis of FVC, DLCO, and PAP, as well as log transformation, ANOVA, and Tukey H.S.D. analysis of the qRT-PCR data.
## Sigclust
An iterative implementation in R of SigClust [bib_ref] Statistical Significance of Clustering for High-Dimension, Low-Sample Size Data, Liu [/bib_ref] was used to determine the number of stable clusters of arrays found after the hierarchical clustering of the gene expression data as described in [bib_ref] Molecular subsets in the gene expression signatures of scleroderma skin, Milano [/bib_ref]. Three P-value cutoffs of p#0.05, p#0.01, and p#0.001 were used for SigClust analysis for each dataset. All gene names for . This file is intended to be viewed digitally, as the text is small and requires the ability to zoom in and out. Found at: doi:10.1371/journal.pone.0012106.s001 (0.80 MB PDF) [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] All gene names for [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref]. This file is intended to be viewed digitally, as the text is small and requires the ability to zoom in and out. Found at: doi:10.1371/journal.pone.0012106.s002 (1.07 MB PDF) [fig_ref] Figure 3: Quantitative Real Time PCR validation of gene expression [/fig_ref] Comparison of three group clustering in the presence and absence of a patients with elevated wedge pressure or extensive ILD. A. Patients with PCWP .15 (mm Hg) and those with extensive ILD were removed and the analysis shown in [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] repeated. The hierarchical clustering dendrogram was generated using 305 probes selected by a multi-group SAM analysis. Even without these patients, the majority of the arrays that fell within ''group 1'' (purple) in the previous analysis, still were in group 1, and likewise for those arrays that fell within ''group 2'' (black) in the previous analysis. B. This shows the same dendrogram as that of S3A, in this case the arrays are indicated in color according to diagnosis, lSSc-PAH (red), lSSc-NoPAH (blue), healthy controls (green). Found at: doi:10.1371/journal.pone.0012106.s003 (0.17 MB PDF) [fig_ref] Figure 4: Distribution of PAH assessment measures between gene expression groups [/fig_ref] Gene expression differentiating lSSc-PAH, LSSc-noPAH and healthy controls, less stringent cutoff. Supplemental figures 3A and B show the clustering dendrogram and resultant heatmap after hierarchically clustering in the array and gene dimension the resultant 2313 probes that passed a multiclass SAM analysis with an FDR of 2.67%. [fig_ref] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls [/fig_ref] shows the clustering dendrogram with the sample identifiers. A black bar beneath the sample identifiers connects technical replicates, and samples collected approximately one year later samples are connected to the baseline samples by a yellow line. To seek out pathways associated with coordinate gene expression utilizing David analysis, gene lists were created from the regions marked in red, green, purple, and blue. The bottom panel shows the gene expression plot from the PBMC dataset for genes/probes that matched each respective gene list (the gene names associated with each probe are available in supplemental data). Gene expression in blue represents decreased gene expression, while red represents increased gene expression. The Normalized Enrichment Score (NES) is shown for each gene set along with the FDR q-value. An FDR q-value,0.25 is considered to be significant. The lSSc-PAH samples consistently show increased expression (boxed, red cells) relative to the lSSc-noPAH samples and healthy controls (highlighted in yellow) for each gene set. This suggests increased expression of genes associated with these cell types in the lSSc-PAH samples. The most significant enrichment was found in the myeloid cell gene set, whereas the least significant enrichment was found in the monocyte gene set. Data File S1 This file list the 206 probes shown in along with the gene expression matrix. Genes were selected with a false discovery rate (FDR) ,0.18% hierarchically clustered by both sample and probe, for the genes most consistently and significantly differentially expressed between lSSc patients (lSSc-NoPAH and lSSc-PAH) and healthy control.
## Supporting information
[fig] Figure 2: Gene expression differentiating lSSc-PAH, lSSc-noPAH and healthy controls. A. Hierarchical clustering dendrogram generated using 305 probes selected by a multi-group SAM analysis (FDR,0.14%). LSSc-PAH sample identifiers are indicated in red, lSSc-NoPAH in blue, and healthy controls in green. Black bars connect technical replicates and yellow bars connect samples collected one year apart. Statistically significant branches determined by SigClust are indicated. The major bifurcation in the dendrogram divides Group 1 and Group 2. B. Heat map showing the expression values of the 305 probes after 2-dimensional hierarchical clustering. Gene expression ratios are colored as in [/fig]
[fig] Figure 3: Quantitative Real Time PCR validation of gene expression. RT-PCR validation was performed for nine genes (ICAM1, IFNcR1, IL1B, IL13Ra1, JAK2, AIF1, CCR1, ALAS2, TIMP2) in healthy control, lSSc-NoPAH, and lSSc-PAH samples. Bars indicate comparisons with statistically significant differential expression. Symbols indicate the level of significance between groups (asterisks p#0.05, open circles p#0.001). doi:10.1371/journal.pone.0012106.g003 [/fig]
[fig] Figure 4: Distribution of PAH assessment measures between gene expression groups. 'Group 1' and 'Group 2' were defined by the major bifurcation in the clustering dendrogram of figure 2, with Group 1 containing all lSSc-PAH samples but three, and Group 2 containing all of the healthy control samples. Solid circles indicate PAH measures from lSSc-PAH patients, open circles indicate those from lSSc-NoPAH patients. A. mPAP measurements compared between patients in Group1 versus those in Group 2. B. DLCO values compared between Group 1 and Group 2. doi:10.1371/journal.pone.0012106.g004 [/fig]
[fig] Figure S6: Disease vs. No Disease SAM Analysis with Additional Probe. A. Clustering dendrogram for the hierarchical clustering in the array and gene dimension of the 54 arrays and the 207 probes that most distinguished via gene expression between lSSc and healthy control samples (FDR cutoff,0.18%), including X (inactive)-specific transcript (XIST), a gene expressed exclusively from the X inactivation center of the inactive X chromosome. The sample identifiers are marked with lSSc in red and healthy control in black. A black bar beneath the sample identifiers connects technical replicates. Samples collected approximately one year apart are connected to baseline samples by a yellow line. The dendrogram tree is marked to indicate if samples were from lSSc patients, or healthy control. B. Heat map showing gene expression after hierarchical clustering in the array and probe dimension. Red is upregulation of gene expression, green is down regulation of gene expression. Found at: doi:10.1371/journal.pone.0012106.s006 (0.75 MB TIF) [/fig]
[table] Table 1: Clinical and hemodynamic data on subjects associated with PBMC sample arrays. [/table]
|
Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.
T yrosine hydroxylase (TH; EC. 1.14.16.2) catalyzes the hydroxylation of L-tyrosine (L-Tyr) to L-3,4-dihydroxyphenylalanine (L-Dopa), the first and rate-limiting step in the synthesis of the catecholamines (CAs) dopamine (DA), noradrenaline and adrenaline [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref]. In the brain, CAs are essential neurotransmitters and neuromodulators involved in processes such as motor control, emotion, reward, biorhythms and learning 2 . Mutations in the TH gene are associated with congenital TH deficiency (THD, OMIM #605407), with phenotypes ranging from L-Dopa responsive dystonia (DRD) and infantile parkinsonism to severe, complex encephalopathy with neonatal onset [bib_ref] Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular..., Fossbakk [/bib_ref] [bib_ref] Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis, Willemsen [/bib_ref]. Furthermore, a deficiency in striatal TH is a hallmark of Parkinson's disease [bib_ref] Selective neuronal vulnerability in Parkinson disease, Surmeier [/bib_ref].
TH belongs to the non-heme iron-and tetrahydrobiopterin (BH4)-dependent aromatic amino acid hydroxylase (AAAH) family, which also includes phenylalanine hydroxylase (PAH) and the tryptophan hydroxylases (TPH1 and TPH2). PAH catalyzes the hydroxylation of L-Phe to L-Tyr, the first and committed step in L-Phe catabolism, and the TPHs hydroxylate L-Trp to 5-hydroxy-Trp, the rate-limiting step in the biosynthesis of the neurotransmitter serotonin. Mammalian AAAHs mainly present as homotetramers with a three-domain subunit structure (Supplementary Figs. 1a, 2a): an N-terminal regulatory domain (RD) that consists of a structured ACT (aspartate kinase-chorismate mutase-TyrA) domain preceded by a less structured N-terminal tail of varying length; a central catalytic domain (CD) that contains the active site iron and binding-sites for substrate and cofactor; and a C-terminal oligomerization domain (OD) responsible for dimerization and/or tetramerization [bib_ref] Structural insights into the regulation of aromatic amino acid hydroxylation, Fitzpatrick [/bib_ref] [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref]. Structures of rat and human PAH that include all three domains have recently been solved by X-ray crystallography [bib_ref] First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an..., Arturo [/bib_ref] [bib_ref] Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled..., Meisburger [/bib_ref] [bib_ref] Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin, Flydal [/bib_ref] [bib_ref] Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of..., Arturo [/bib_ref] and Cryo-EM [bib_ref] Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin, Flydal [/bib_ref]. For human and rat TH and human TPH, crystal structures encompassing CD + OD are available [bib_ref] Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for..., Goodwill [/bib_ref] [bib_ref] Threedimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis..., Wang [/bib_ref]. For rat TH, an NMR structure of the isolated dimeric ACT [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref] is also available (Supplementary [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] , which allowed preparation of composite models of full-length TH based on SAXS data [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref] [bib_ref] The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants..., Szigetvari [/bib_ref]. These TH models present a very dynamic structure, notably at the N-terminal tail and the long loops between the RD and CD.
In humans, a single TH gene gives rise by alternative splicing to four very similar TH isoforms (TH1-4), with the only difference being a variable length of the flexible N-terminus [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref]. In this work, we have used TH1 (named just TH from this point onward) as it is the most studied human isoform and most similar to the only TH in non-primate mammals [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref]. Although it has the shortest N-terminus, its RD still includes~70 residues preceding the folded ACT (residues . ACT domains are typical in many allosteric enzymes, including PAH. However, since TH is not known to be allosterically regulated, it has been suggested that this domain has lost its original function in TH while maintaining its structure throughout evolution [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref] [bib_ref] The ACT domain: a small molecule binding domain and its role as..., Grant [/bib_ref]. The N-terminal tail (residues 1-70) is considered to be largely disordered and variable across species, however residues 40-49 are highly conserved and followed by a poly-alanine segment of variable length (residues 51-59 in humans) that seems to have appeared in evolution after the jawless fish [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref]. This alanine-rich region has been predicted to have high helical propensity [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref].
In order to maintain CA homeostasis, the activity and conformational stability of TH are regulated at the transcriptional and translational levels, and also posttranslationally through feedback inhibition by CAs and phosphorylation at serine/ threonine residues in the N-terminal tail [bib_ref] Complex molecular regulation of tyrosine hydroxylase, Tekin [/bib_ref] [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref]. TH is phosphorylated on T8, S19, S31 and S40 by several protein kinases with different site specificities [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref] [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref]. The different phosphorylation sites regulate TH through binding to 14-3-3 proteins (S19), cellular localization to the Golgi and synaptic vesicles (S31), and activity (S40) [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref] [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref] [bib_ref] Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along..., Jorge-Finnigan [/bib_ref]. Early preparations of TH from the adrenal medulla revealed co-purified CAs in the active site, forming a strong bidentate catecholate-Fe(III) complex [bib_ref] Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase)...., Andersson [/bib_ref]. TH inhibition by CAs is competitive with respect to BH4, and CAs are released from the active site either by incubation with BH4 or by phosphorylation at S40, which is performed by several kinases, including cAMPdependent protein kinases (PKAs) [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref] [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref] [bib_ref] Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at..., Haycock [/bib_ref]. Indeed, all four human TH isoforms are inhibited by DA and phosphorylated by PKA at the corresponding phosphosites to S40 in TH1 (S44 for TH2, S67 for TH3 and S71 for TH4), which decreases the affinity by DA and reactivates enzyme activity [bib_ref] Effects of phosphorylation by protein kinase A on binding of catecholamines to..., Sura [/bib_ref] [bib_ref] Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation, Lehmann [/bib_ref]. Thus, the strength of feedback inhibition by DA can be modulated by S40-targeting signalling pathways, e.g., enforced by inhibitory auto-receptors that lower PKA activity [bib_ref] Dopamine D(2) receptors regulate tyrosine hydroxylase activity and phosphorylation at Ser40 in..., Lindgren [/bib_ref].
DA acts not only as a feedback inhibitor, but its binding also stabilizes TH, as seen as an increased thermal stability [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref] [bib_ref] Brain catecholamine depletion and motor impairment in a Th knock-in mouse with..., Korner [/bib_ref] [bib_ref] Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect..., Martinez [/bib_ref] and decreased susceptibility to proteolysis [bib_ref] Brain catecholamine depletion and motor impairment in a Th knock-in mouse with..., Korner [/bib_ref] [bib_ref] Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect..., Martinez [/bib_ref] [bib_ref] Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase, Thony [/bib_ref] , as well as increased lifetime of TH activity in crude extracts from rat striatum [bib_ref] Conversion of tyrosine hydroxylase to stable and inactive form by the end..., Okuno [/bib_ref]. This stabilizing effect is important for maintaining TH levels in vivo, particularly in the axo-terminal compartment, as has been shown in mouse models of DA deficiency [bib_ref] Brain catecholamine depletion and motor impairment in a Th knock-in mouse with..., Korner [/bib_ref] [bib_ref] The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine..., Homma [/bib_ref]. In addition to the direct interaction of DA with the active site iron [bib_ref] Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase)...., Andersson [/bib_ref] , mutagenesis and truncation studies have revealed the importance of the N-terminal tail on the high-affinity binding of DA and other CAs [bib_ref] Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in..., Ota [/bib_ref] [bib_ref] Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines, Nakashima [/bib_ref]. The N-terminal region is also where CA-feedback inhibition is modulated by signalling mediated S40-phosphorylation. Still, due to a lack of detailed structural information for full-length TH, a deep understanding of the molecular determinants of the interaction between TH, in particular the RD, and DA is missing. In this work, we have used state-of-the art Cryo-EM to obtain the structure of full-length human TH, both in the absence and presence of its feedback inhibitor DA, and that of S40 phosphorylated TH (THS40p), which reactivates TH. These structures provide us with a better understanding of the inhibitory and stabilizing role of DA in the regulation of TH activity and TH protein turnover.
# Results
Three-dimensional reconstruction of the ligand-free TH (apo-TH). The high flexibility of the N-terminal RD and of the RD-CD linker has likely been a major hurdle for structural and functional analysis of the full-length TH; to date there have only been crystal structures of truncated CD + OD domains (PDBs 1TOH and 2XSN) and the NMR structure of part of the RD domain (PDB 2MDA) [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. We set out to optimize conditions for structural determination of the full-length human TH, this time using Cryo-EM [bib_ref] Cryo-EM: A unique tool for the visualization of macromolecular complexity, Nogales [/bib_ref]. Recombinant human TH was expressed and purified as in [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref] [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. When analyzed by gel filtration on a Superdex 200 Increase 3.2/300, the profile of purified TH showed a main peak containing tetrameric TH preceded by a smaller octamer peak 15 [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. The tetrameric population was used for optimization using a 200 keV FEI Talos Arctica located at the Centro Nacional de Biotecnología (CNB-CSIC; Madrid), and the best grid was used for data acquisition on a 300 kV Titan Krios at the DLS-eBIC facility (Oxford) (Supplementary [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref] ; EPU 1.12 was used for data collection with parameters described in . The image processing and subsequent 3D reconstruction procedures are described in detail in the Methods section and in Supplementary Figs. 4 and 5. The final map, with an estimated resolution of 3.9 Å (FSC = 0.143) [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] , shows a central tetrameric structure formed by the CD and OD domains that is 110 Å long, 86 Å wide and 38 Å high. The four subunits of this central structure have an asymmetric arrangement that could be explained by the tetramer being formed by a dimer of dimers. The two small masses of the RDs, separated 15 Å from the main body, have a dimeric structure and a parallelepiped-like shape of 40 × 40 × 22 Å. The two masses were placed on opposite sides of the central part of the TH structure, allowing full access to the active sites of the enzyme. In the 3D reconstruction, many structural features could be assigned to α-helices and loops in the central structure formed by CD and OD domains, in particular the α-helices involved in tetramerization. In the RD domains, four α-helices were clearly visible, two of them involved in dimer formation. The same views with the flexible fitting with iMODfit 1.51 of the atomic model in the 3D map for further sharpening steps. c Atomic model of the CD + OD and RD domains built from the Cryo-EM 3D reconstruction. The connections between the RD and CD are highlighted with red arrows on one side of the structure. In the left figure, the RD has been made slightly transparent for the CD + OD domains to be better observed. However, there were areas of lower resolution, so we sought to determine the local resolution of each area of the map using MonoRes [bib_ref] MonoRes: Automatic and accurate estimation of local resolution for electron microscopy maps, Vilas [/bib_ref] [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] , which showed a nonisotropic resolution distribution of the map, between 2 Å at the central part of the tetrameric structure and 10 Å at the RDs. An attempt to improve the local resolution of the latter by treating them as single particles did not result in a noticeable increase to resolution (7.1 Å), suggesting that this dimeric structure has intrinsic flexibility [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. The central structure was also subjected to a further refinement by masking out the RDs, and the final resolution reached 3.4 Å (Supplementary [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. Sharpening programs were used to improve the interpretability of the obtained map and to start the model building, as described in the Methods section and in [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] -e. The final atomic model generated (residues 163-497) [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] ; PDB 6ZZU) was very similar to that described in the crystallographic structure of the CD + OD domains (PDBs 1TOH and 2XSN), showing the presence of iron in its active site with the coordinating residues H330, H335 and E375 [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] [bib_ref] Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for..., Goodwill [/bib_ref] [bib_ref] Selectivity and affinity determinants for ligand binding to the aromatic amino acid..., Teigen [/bib_ref]. Surrounding residues G292, L293, A296, F299, E331, S367 and Y370 form the BH4 binding site, and R315, S323, W371, S394 and D424 form the substratebinding site, where the latter residue is a specificity determinant for L-Tyr hydroxylation to L-Dopa in TH (Supplementary [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref] [bib_ref] Selectivity and affinity determinants for ligand binding to the aromatic amino acid..., Teigen [/bib_ref] [bib_ref] Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of..., Daubner [/bib_ref]. There was also an extra density where one ironcoordinating water molecule could be placed, which was previously observed in the structure of truncated rat TH 12 . In this model, residues 160-170 corresponding to the linker between the RD and CD were also present (marked with red arrows in [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. The final PDB file for the apo-TH tetramer included coordinates for residues 78-497 of the four subunits (PDB 7A2G), but not for the 77 N-terminal residues, which are known to be important in TH regulation.
Structural characterization of dopamine-bound TH (TH(DA)). As TH plays a pivotal role in DA synthesis and homeostasis, it is important to understand its regulation 2,37 . The RD is essential here as it conveys communication between feedback inhibition by DA and activation by S40 phosphorylation 1 . It was, therefore, essential to characterize the RD domain structurally at the highest possible resolution and its positioning in the full-length protein.
We, therefore, set out to investigate the structural differences between apo-TH and TH in the presence of DA (TH(DA)).
The formation of stable TH(DA) was carried out as described in the Methods and used for vitrification following the same conditions as for apo-TH. The cryogrids were first analyzed in our 200 keV FEI Talos Arctica, and the best one was used to record a total of 4422 movies on a 300 kV Titan Krios at the ESRF Grenoble facility (data collection parameters are described in Supplementary . The image processing and subsequent 3D reconstruction procedures followed are described in detail in the Methods section and in [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref]. The final 3D reconstruction yielded a map at 4.1 Å resolution [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref] and [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] , although analysis of the local resolution using MonoRes revealed the same differences in resolution described for apo-TH [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref].
The reconstruction obtained showed similar structural features and dimensions to those described for apo-TH (compare Figs. 1a and 2a), except for the presence of an extra cylindrical structure, strongly suggestive of an α-helix protruding from the central mass and contacting the RDs (red arrow in [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref]. These new densities together with the connections between the RD and the CD + OD were masked, and subsequent refinement and sharpening of this part of the enzyme resulted in an increase of the resolution up to 3.8 Å [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref].
We next carried out an atomic model building and refinement with COOT and PHENIX. Validation statistics showed reasonable correlation values for the attained resolution. We were only able to rigid-body fit a goodquality homology model (82% identity) into the lower resolution RD density. This explains the lower quality of the RD fitting in comparison with the atomic resolution modeling of the CD. For this reason, we built a model in which the side chains were omitted from the RD structure. In this map, the density of the extra cylindrical volume presented an even clearer helical shape, revealing a new α-helical region in the N-terminus (the first 70 residues) not visible in apo-TH. Unfortunately, the lower resolution of this helical region only allows to build a backbone tentative model (residues 40-497; PDB 6ZVP). Earlier secondary structure predictions and modelling by replica exchange molecular dynamics (REMD) have shown the α-helix propensity of the 45-59 alanine-rich region [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref] [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref]. The presence of an α-helix in the 39-58 region is biochemically supported 1,31,38 and, as described in the Methods section, the N-terminal α-helix configurations with lowest energy computed with KORP (knowledge-based orientational potential) [bib_ref] KORP: knowledge-based 6D potential for fast protein and loop modeling, Lopez-Blanco [/bib_ref] corresponded to the 39-50 region. We nevertheless performed secondary structure predictions using PSIPRED 4.0 40 and I-TASSER 5.1 41 , which actually pointed to a longer region of highest α-helical propensity at residues 39-58, and shorter regions in segment 13-29 with lower propensity . Independently, we modeled a 20-residue α-helix from residues 39-58 based on the corresponding density of the Cryo-EM map (see "Methods" for details). A model for the TH(DA) tetramer comprising coordinates for residues 40-57 and 163-497 for the four subunits is also included [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref] ; PDB 6ZN2) .
Structural differences between apo-TH and TH(DA). Comparison of the apo-TH and TH(DA) of the CD + OD structures indicates that no large rearrangements take place upon DA binding [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref] and Supplementary Movie 1). However, there were some interesting changes, all related to the active site. When a difference map between TH(DA) and apo-TH was calculated [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref] , the major differences found were the α-helix penetrating the active site and a mass located where DA should be placed (arrow in [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. Since the current resolution did not allow accurate fitting of DA, its atomic structure in the PAH(DA) complex (PDB 5PAH) was used to place it in the TH active site, bound to the iron with a bidentate coordination [bib_ref] Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol..., Erlandsen [/bib_ref]. Based on how DA was placed into TH as guided by the PAH(DA) structure, DA could form hydrogen bonds with the iron-coordinating residues H330, H335, E375 and Y370 and by hydrophobic interactions with P326 [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. All these DA-interacting residues are conserved in PAH where they are also involved in complex formation [bib_ref] Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol..., Erlandsen [/bib_ref]. The presence of DA was also accompanied by a rearrangement of the loop between residues C176 and D196 and a shift of the side chain of W371 towards the active-site iron [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. We evaluated whether the observed closure of the active site affects L-Tyr binding by alignment of the structure of substrate and BH4-bound PAH-CD (PDB 1KW0) [bib_ref] Crystal structure of the ternary complex of the catalytic domain of human..., Andersen [/bib_ref] with the TH(DA) structure [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. The docked structure revealed a steric clash of DA with the cofactor, but we did not observe clashes between DA and either L-Tyr or substratebinding residues [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref] , in agreement with the DA inhibition being competitive only towards BH4 [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref] [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref]. Insertion of the α-helix into the active site would leave ample space for the disordered residues 1-39 to thread out of active site into the exterior [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref].
The major difference found between apo-TH and TH(DA) involves the above-mentioned presence of the N-terminal α-helix (residues 39-58) inserted into and blocking the active centre. To further confirm the presence of an α-helix in the 39-58 region of the N-terminal regulatory domain, we performed the 3D reconstruction of a mutant with the first 35 residues deleted (THNΔ35). The mutant was purified, incubated with DA (THΔN35(DA)) and vitrified as described above for apo-TH and TH(DA). A total of 13,213 movies were acquired at the Diamond Light Source (DLS) electron Bio-Imaging Centre (eBIC), and 1,626,575 particles were automatically selected and subjected to 2D and 3D classification as described for the two other 3D reconstructions. The final 3D reconstruction of THΔN35(DA)) (4.6 Å resolution) showed the presence, as in the case of TH(DA), of an α-helix fixed at the iron-coordinated DA in the active site, with no residues resolved upstream (arrows in [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref]. The similarity of both 3D reconstructions (although at different levels of resolution), which reveals the presence of the α-helix in the same place as in the case of TH(DA) [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] , reinforces the notion that the αhelix is formed by residues of the 39-58 region, and that its stabilization in the active site is associated with tight DA binding and strong TH inhibition.
The binding of the N-terminal α-helix (residues [bib_ref] KORP: knowledge-based 6D potential for fast protein and loop modeling, Lopez-Blanco [/bib_ref] [bib_ref] The PSIPRED Protein Analysis Workbench: 20 years on, Buchan [/bib_ref] [bib_ref] I-TASSER server: new development for protein structure and function predictions, Yang [/bib_ref] [bib_ref] Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol..., Erlandsen [/bib_ref] [bib_ref] Crystal structure of the ternary complex of the catalytic domain of human..., Andersen [/bib_ref] [bib_ref] pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation..., Haavik [/bib_ref] [bib_ref] Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of..., Ramsey [/bib_ref] [bib_ref] Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease, Waløen [/bib_ref] [bib_ref] A flexible loop in tyrosine hydroxylase controls coupling of amino acid hydroxylation..., Daubner [/bib_ref] [bib_ref] Substituting Tyr(138) in the active site loop of human phenylalanine hydroxylase affects..., Leandro [/bib_ref] [bib_ref] Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain, Patel [/bib_ref] [bib_ref] Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase, Briggs [/bib_ref] [bib_ref] Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and..., Ramsey [/bib_ref] [bib_ref] Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine..., Wang [/bib_ref] [bib_ref] Striatal synaptosomal dopamine synthesis: evidence against direct regulation by an autoreceptor mechanism, Compton [/bib_ref] [bib_ref] Dopamine concentration in the cytoplasmic compartment of single neurons determined by capillary..., Olefirowicz [/bib_ref] [bib_ref] Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref] [bib_ref] Dopamine oxidation products as mitochondrial endotoxins, a potential molecular mechanism for preferential..., Biosa [/bib_ref] [bib_ref] Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the..., Calvo [/bib_ref] [bib_ref] Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent..., Vie [/bib_ref] to DA at the active site enhances the inhibition and thermal stability of TH. The α-helix observed in the TH(DA) and THNΔ35(DA) structures could also be present in apo-TH despite not being visualised due to its great flexibility, or it could be formed upon DA binding. We, therefore, measured the secondary structure content of the two TH conformations by synchrotron circular dichroism spectroscopy (SRCD) [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. The far-UV spectra were completely overlapping, and calculation of the secondary structure showed no increase in helix content upon incubation with DA , supporting the presence of the N-terminal α-helix in apo-TH. In order to better define the helical content of the N-terminus, we generated two deletion mutants in addition to THΝΔ35: one lacking the N-terminal 43 residues (THΝΔ43), and another with the entire N-terminal removed (THΝΔ70). Far-UV scans [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] and secondary structure calculations (Supplementary showed that whereas THNΔ35 and THΝΔ43 were almost identical to apo-TH and their secondary structure was not affected by DA binding, THΝΔ70 presented a significantly lower α-helical content. These results strengthen the notion that the N-terminal, alanine-rich α-helix is preformed and not in stable contact with any structured domain of apo-TH but gets locked into the active site groove upon DA binding, allowing its visualization by Cryo-EM.
Next, we analyzed apo-TH and TH(DA) by crosslinking mass spectrometry (XL-MS), which can provide information about nearby regions and possibly help to elucidate whether the presence of DA stabilizes the protein and produces any detectable rearrangement that would account for the presence of the α-helix. Both complexes were incubated in the presence of the crosslinker BS3 prior to trypsin digestion and LC-MS/MS analysis. The MS/ MS data were searched with StavroX and the results were filtered at a false discovery rate (FDR) < 5%. We identified 300 (apo-TH) and 340 (TH(DA)) peptide-spectrum matches (PSMs) corresponding to, respectively, 128 and 120 unique crosslinked peptides. We then focused our analysis on the crosslinks detected in the flexible, N-terminal region and for that, we queried for crosslinks that mapped the first 20 N-terminal residues of TH, the most flexible region of the enzyme. The number of crosslinked peptides encompassing this region of the ligand-free complex (19 peptides, 55 PSMs) almost doubled that of the dopamine-bound one (10 peptides, 24 PSMs) . Moreover, the crosslinked peptides observed in TH(DA) were a subset of those found in apo-TH, as no TH(DA)-specific crosslinks were identified . Finally, two of the apo-THspecific peptides reflected an interaction between the N-terminus of the protein and K204, a residue found in an external α-helix that surrounds the active centre. The larger number of crosslinks involving the N-terminus in apo-TH compared with TH(DA) speaks of a higher flexibility of the former, which is consistent with the presence of an immobile αhelix in the latter that would limit the movement of the entire N-terminal tail (bottom images in , indicative of increased protein stability.
We then analyzed a possible effect of DA on the thermal stability of TH and the three deletion mutants by differential scanning calorimetry (DSC) measurements, which provides the temperatures for onset of thermal denaturation (T onset ) and the transition maximum (T max ). The stabilization by DA, seen by delayed T onset and increased T max , was highest for WT and T m decreased with the length of the deletion, as shown in the showing the extra density connecting the RD with the CD active sites. b The same, detailed view of the active sites of (left) apo-TH, (middle) a difference map of TH(DA) minus apo-TH (red) superimposed into the apo-TH map. The red cylinder shows the internalized α-helix and the mass representing DA (red arrow). Other small masses come from changes between both conformations. The contour level is set to 3σ. The right panel shows the fitting of the atomic structure of DA into the corresponding density. c The same, detailed view of the atomic model of the apo-TH active site (left) highlighting the segment encompassing residues 176-196 (dark green) and those involved in iron coordination (H330, H335 and E375). In the middle, the same view of the atomic model of TH(DA) with the 176-196 segment highlighted in red. Y370, which is in the BH4 binding pocket and forms an H-bond with the bound DA, is also modelled, and the 39-58 α-helix has been removed for convenience. The right image is a superposition of the two previous images, revealing the different arrangement surrounding the active site.
representative scans for WT [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] and THΝΔ70 [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] and the parameters summarized in . These results support the role of the α-helix sustaining DA-dependent stabilization.
We also performed activity assays to analyze the inhibitory effect of DA on full-length TH and the three truncated forms while THNΔ35 shows a slightly lower IC50 (0.22 ± 0.02 µM) than TH [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. Altogether, our results indicate that residues at the N-terminal part of the α-helix are important for the high-affinity binding of DA.
Modelling the structural response to S40 phosphorylation. The feedback inhibition of TH by DA is alleviated by PKA phosphorylation of TH at S40, both in vitro and in vivo [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref] [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref] [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref]. We prepared S40 phosphorylated TH (THS40p), which, as expected [bib_ref] Effects of phosphorylation by protein kinase A on binding of catecholamines to..., Sura [/bib_ref] [bib_ref] Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation, Lehmann [/bib_ref] , presented an increased IC50 for DA (12.4 ± 2.3 µM), 25-fold higher than unphosphorylated TH [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] , and not significantly different from the IC50-values measured for truncated forms THNΔ43 and THNΔ70. SRCD analysis of THS40p revealed a spectrum very similar to that of the unphosphorylated sample and comparable content of secondary structure elements [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] and Supplementary , indicating that the decreased affinity for DA may arise from a separation of the helix from the ironcoordinated DA in the active site rather than from disruption of the helical structure. This separation is expected to result in loss of visualization of the helix in the Cryo-EM structure, as in the apo-TH structure, and to understand the structural causes for the observed effect of this phosphorylation we generated by Cryo-EM a 3D reconstruction of THS40p. Aliquots of this sample were vitrified and 9241 movies from the best cryogrid were acquired at the Diamond Light Source (DLS) electron Bio-Imaging Centre (eBIC). From these, a total of 1,610,418 particles were automatically selected and 2D and 3D classified as described in the "Methods" section. The 3D reconstruction of THS40p [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] shows a similar overall three-dimensional structure to that of apo-TH, TH(DA) and THNΔ35(DA) with the main point being that, as in the case of apo-TH, no α-helix is visualized blocking the active centre (asterisks in [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref].
The low affinity of THS40p for DA binding [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] , f) hampered the preparation of homogeneous samples of DA bound to S40-phosphorylated TH (THS40p(DA)) for Cryo-EM investigation, and in order to model the initial conformation of this complex we used the 3D reconstruction of TH(DA) and modelled a phosphate group at S40 [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. The added phosphate did not seem a priori to clash with any other residue in the surroundings, though it was close to E325, E375 and D424, which may exert some electrostatic repulsion over the phosphate and contribute to a displacement of the helix. More insights on the possible interactions between the phosphate, DA and TH were obtained from molecular dynamics (MD) simulations on four structures (the apo forms of TH and THS40p, in addition to TH(DA) and THS40p(DA)) left it run for 0.5 µs. The four systems seemed to equilibrate within the first 200 ns and then remained relatively stable for the rest of the 0.5 µs simulations . The ACT domain (residues 71-165) showed higher average mobility when compared to the CD, as shown by calculated backbone atom fluctuations . After initial equilibration we monitored some selected distances, averaged for all subunits, reflecting initial changes affecting the N-terminal part of the helix and around iron and DA (Supplementary . S40-phosphorylation of TH caused slightly longer distances between the N-terminal part of the helix (D44 and S40) and both Fe and DA (notably seen in the comparison of TH(DA) with THS40p(DA)) (Supplementary . The elimination of DA (comparison of TH(DA) with TH and of THS40p(DA) with THS40p) also results in longer interatomic distances between both D44 and S40 and Fe, which together with the DA-inhibition results [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] and the Cryo-EM structure of THS40p, supports that the TH activation induced upon S40 phosphorylation is associated with the release of the α-helix that was blocking the TH active site in TH(DA).
# Discussion
The AAAHs have important functions in the synthesis of biogenic monoamines that are essential for many physiological processes, and disruption of these functions (e.g., through mutations) can lead to severe disorders [bib_ref] Selectivity and affinity determinants for ligand binding to the aromatic amino acid..., Teigen [/bib_ref] [bib_ref] Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease, Waløen [/bib_ref]. The dynamic behaviour of the RDs, which is probably closely linked to their important regulatory roles, has until now hindered detailed structural studies. The structure of full-length TH had been elusive until now, herein solved thanks to the latest advances in Cryo-EM. The structure of human apo-TH (3.9 Å resolution) [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] shows a planar, tetrameric core that comprises the CD and OD domains, and two smaller densities on opposite sides and 15 Å apart from the central structure. This great separation explains not only the difficulty of crystallization, but also in obtaining homogeneous preparations for Cryo-EM, which is reflected in the lower resolution obtained for the RDs (Supplementary [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref].
The high resolution reached in the TH core of the apo structure allowed building of its atomic model [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] and enabled comparison of this area with a previous crystallographic structure (PDB 2XSN). The comparative analysis showed differences in the region encompassing residues 176-196 [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] : whereas the Cryo-EM model revealed a helical and loop structure arrangement, the crystallographic structure shows only a loop that is shifted from the former. This difference could be ascribed to contacts established by this flexible loop in the crystallographic arrangement [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. The presence of DA in the DA-bound TH was also accompanied by a rearrangement of the loop between residues C176 and D196 and a shift of the side chain of W371 towards the active-site iron [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref]. Nevertheless, residues 176-190 centred around F183 have indeed been shown to have an important catalytic role in TH, controlling the coupling of amino acid hydroxylation to tetrahydropterin cofactor oxidation [bib_ref] A flexible loop in tyrosine hydroxylase controls coupling of amino acid hydroxylation..., Daubner [/bib_ref]. In the equivalent Y138-centred active site loop in PAH, which is involved in enzyme activation and catalysis [bib_ref] Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin, Flydal [/bib_ref] [bib_ref] Substituting Tyr(138) in the active site loop of human phenylalanine hydroxylase affects..., Leandro [/bib_ref] , large conformational changes effected by ligand binding have also been observed.
Similar to what was found in the recent Cryo-EM PAH structure [bib_ref] Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin, Flydal [/bib_ref] , there was anisotropy in the resolution, with the RDs showing less resolution both because of their inherent flexibility and the dynamic linkers that connect them to the central structure. However, while the resolution in most of the TH core ranges from 2 to 4 Å, it was only about 5 Å in PAH. Whereas tetramers of TH (and also TPHs) form by leucine-zipper interactions, the many polar residues in the PAH OD [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref] may allow for different conformations and a dimer-tetramer equilibrium that would limit the possible resolution. There were also differences in the CD that have to do with its arrangement relative to the central OD, with a 12.7°outward tilt of the CD in TH compared with that of PAH [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. By comparing the maps for TH [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] and PAH 10 , you can clearly see: (1) RDs in TH are arranged as dimers and the ACTs are separated~15 Å from the central part of the core structure (~30 Å from the active site); and (2) ACTs in resting-state PAH are arranged as monomers, in physical contact with the core structure [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] , and shifted 88°compared to the TH monomer [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. In agreement with this arrangement, isolated TH RDs form a dimer [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref] [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] , whereas the PAH RDs are monomeric in the absence of L-Phe 49 . Allosteric substrate activation of PAH has been proposed to lead to dimerization of adjacent dimers, reaching a conformation resembling that seen in TH [bib_ref] First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an..., Arturo [/bib_ref] [bib_ref] Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled..., Meisburger [/bib_ref] [bib_ref] Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of..., Arturo [/bib_ref].
The ingress and stabilization of α-helix 39-58 into the active site largely explains the tight binding of DA, since in addition to the bidentate interactions of the catechol moiety with the Fe(III) and active site residues H330, H335 and P326 [bib_ref] Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for..., Goodwill [/bib_ref] [bib_ref] Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase)...., Andersson [/bib_ref] , in our atomic model of TH(DA) the catecholamine establishes interactions with L41 and D44, and with Y370 in the D360-E375 α-helix [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. The increased thermal stabilization of TH upon DA binding as well as the low k off for DA, which is the main rate constant affected by S40 phosphorylation [bib_ref] Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of..., Ramsey [/bib_ref] , are largely explained by the immobilization of the 39-58 α-helix through the interactions with DA and additional CD residues. Together with S40, DA interacting residues L41 and D44 are located at the beginning of the 39-58 α-helix, which appears as a pertinent region for TH regulation by DA. The regulatory α-helix runs parallel to and establishes contacts with the D360-E375 helix and the V290-R297 loop (A45 with A296), and with the central region of the Q420-S429 loop (I42 with Y422) [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. It is important to note that this model is reinforced by mutational and MS-monitored hydrogen/deuterium exchange studies that show the involvement of several of these residues in DA binding [bib_ref] Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in..., Ota [/bib_ref] [bib_ref] Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with..., Wang [/bib_ref] [bib_ref] Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase, Briggs [/bib_ref]. Moreover, truncation studies of the N-terminal residues have shown that absolute removal of DA inhibition requires deletion of residues ≥ 39 [bib_ref] Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in..., Ota [/bib_ref]. It is however important to note that in the TH(DA) structure no residues were resolved upstream of Q39 in either TH(DA) or THNΔ35(DA). This unresolved part of the N-terminal hosts important residues for determining the conformational changes effected by phosphorylation and DA binding affinity, such as R37 and R38, which form the recognition site for PKA [bib_ref] Tyrosine hydroxylase and regulation of dopamine synthesis, Daubner [/bib_ref] [bib_ref] Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines, Nakashima [/bib_ref]. We cannot then disregard that these unobserved residues may establish interactions with TH, despite not being stabilized enough to be resolved by Cryo-EM. Furthermore, the structural fixation of the N-terminal α-helix could explain the reported inhibitory effect of DA on S40 phosphorylation rate [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref].
The structural stabilization of the N-terminal residues 39-77 in the DA-bound state, both in full-length TH and THNΔ35, was necessary for their visualization, as they were not observed in the apo-TH state (whose structural model starts at the ACT domain) or in the THS40p conformation. Moreover, the detailed structural information on the N-terminus provided by the TH(DA) and THNΔ35(DA) Cryo-EM structures permitted us to perform MD simulations to obtain additional structural insights on the feedback inhibition by DA and its release by S40 phosphorylation. Physiologically, TH is activated when S31 or S40 are phosphorylated, but only S40 phosphorylation affects CA binding, leaving this site as the main target for signal-mediated activation of TH [bib_ref] Tyrosine hydroxylase phosphorylation in vivo, Dunkley [/bib_ref]. In our structural model of TH(DA), S40 was placed on the N-terminal side of the α-helix, at the base of the active site. The MD-simulated phosphorylated structures pointed to an electrostatic repulsion from the nearby E325, E375 and D424 as a possible mechanism for the separation of the helix [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. This offers a structural explanation to the functional result since the release of the α-helix and a lower affinity for DA would result in higher TH activity [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref].
All these results allowed us to propose a model in which TH is in an active conformation when the N-terminal region (which includes the α-helix) is free and detached from the main structure [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref]. DA coordination to the TH iron [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] , I → I′) favours the interaction of the α-helix with DA and the active site [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] , I′ → II and [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref]. Phosphorylation of S40 [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] , II → III) would initiate, through electrostatic and steric repulsion, the egress of the helix from the active centre [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref] , III → IV′). Subsequent release of DA would result in an active TH [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] , IV′ → IV). Thus, based on data presented here and, in the literature, we expect that state I′ is only transiently populated during DA binding as state II will be more stable. Similarly, for THS40p(DA) we expect that state III is destabilized and less populated than state IV′. This model is supported by kinetic studies where S40 phosphorylation mainly increases the dissociation rate constant (k off ) of DA 51 and the mobility around the phosphorylation site [bib_ref] Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of..., Ramsey [/bib_ref] [bib_ref] Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine..., Wang [/bib_ref]. DA binding to TH inhibits PKA-mediated S40 phosphorylation, mainly by increasing the K M for TH, suggesting that S40 is less available in state II than in state I 21 . However, as the inhibition of TH phosphorylation by DA is not as strong as the inhibition of TH activity, we cannot exclude that S40 can be accessed in the closed conformation [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref] [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref] [bib_ref] Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and..., Ramsey [/bib_ref].
Our results also highlight the importance of the α-helix in the regulation of neuronal DA homeostasis for unphosphorylated TH, as an IC50 ≈ 0.5 µM fits well with IC50-values obtained in physiologically relevant systems, such as rat striatal synaptosomes [bib_ref] Striatal synaptosomal dopamine synthesis: evidence against direct regulation by an autoreceptor mechanism, Compton [/bib_ref]. Cytoplasmic DA concentrations sufficient for a functional feedback inhibitory effect (low µM) are in agreement with the free cytoplasmic concentration of DA in neurons (~2 µM) [bib_ref] Dopamine concentration in the cytoplasmic compartment of single neurons determined by capillary..., Olefirowicz [/bib_ref]. As is the case for the deletion of α-helix 39-58(THNΔ70) or only its N-terminal part (THNΔ43), S40 phosphorylation leads to a great increase (approx. 25-fold) of the IC50 value for DA inhibition, in agreement with values obtained in previous studies [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref] [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. Thus, the DA-mediated TH inhibition was not totally eliminated upon S40 phosphorylation, but rather seems to adjust the set point of cytosolic DA to a higher level depending on the signalling strength. Previous studies have shown similar mechanisms for inhibition by different CAs and their release by Ser40 phosphorylation [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref] [bib_ref] Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of..., Ramsey [/bib_ref] , with a small difference in affinity among DA, epinephrine and norepinephrine, which might be explained by the similar location of the catechol group and the primary amine, but a slight difference in charge distribution at this amine.
DA signalling in the brain occurs mainly through volume transmission, meaning that diffusion vs. reuptake kinetics control the spread of released DA [bib_ref] Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway, Rice [/bib_ref]. Reuptake by the DA transporter also provides neurotransmitter that can be stored in vesicular pools and reused. In the absence of feedback inhibition, the DA synthesis flux would become unaffected by the accumulating cytosolic DA at full vesicular pools, and the steady-state level of cytosolic DA would be determined by the balance between the unconstrained synthesis rate and DA metabolization by high-K M monoamine oxidase. However, DA and other CAs are redoxactive and considered neurotoxic at increased levels [bib_ref] Dopamine oxidation products as mitochondrial endotoxins, a potential molecular mechanism for preferential..., Biosa [/bib_ref] , which may be a reason for the evolution of a strong feedback inhibition of TH, not found for serotonin and TPH. The presence of a flexible N-terminal region in TH is important for the stabilization of DA feedback inhibition and allows for an additional level of regulation, as the S40 site enables signalling pathways to modulate the feedback inhibitory strength of CAs. The CD-interacting α-helix 39-58, containing the S40 site, seems to be a key structural determinant for this regulatory crosstalk and it is unique to TH among the AAAHs [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref]. Despite the low TH sequence identity among different organisms, the helix is likely to be highly conserved from fishes to primates with only small variations in the poly-alanine region [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref] , but not in non-vertebrates where the feedback regulation by DA is different or absent [bib_ref] Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the..., Calvo [/bib_ref] [bib_ref] Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent..., Vie [/bib_ref] [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref]. The S40 PKA site is however older, but has an unresolved function in C. elegans 57 , whereas in D.
melanogaster PKA phosphorylation activates the brain TH isoform, but the epidermal isoform is only activated in the presence of DA [bib_ref] Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent..., Vie [/bib_ref]. The fully functional DA ↔ S40p structural motif seems to have had a step-wise evolution; first establishing an activating S40-phosphosite, then co-evolving higher DA binding affinity that may rely on a stable helical structure to minimize entropy loss. In vertebrates, as a result of the structural N-terminal regulatory cross-talk motif, phosphorylation-mediated modulation of feedback inhibition would allow rapid anticipatory adjustment of TH activity to changes in neuronal activity, either through stimulatory or inhibitory receptors. Thus, dopaminergic autoreceptors inhibit cellular PKA activity and increase the strength of DA feedback according to levels of extracellular transmitters 25 , allowing TH activity to adapt to the anticipated lower need of DA as a result of high extracellular DA.
THD is an autosomal recessive parkinsonian disorder caused by mutations in the TH gene, which are registered in the PND database (www.biopku.org/pnddb/). The protein numbering in the PND database is for the longest human isoform, TH4, which is 31-residue longer than TH1. The effects of the mutations on TH activity, stability and oligomerization have been previously PKs and protein phosphatase(s) (PP) control the transition between THS40p and unphosphorylated TH for both DA bound (I′ ↔ IV′ and II ↔ III) and apo-TH (I ↔ IV). States I′ and III are expected to be only transiently populated during DA binding as state II and IV' will be more stable [bib_ref] Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation...., Almas [/bib_ref] [bib_ref] Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and..., Ramsey [/bib_ref] (see main text). To indicate that our data do not support the presence of stable or well populated states I′ and III, these are faded in the figure. S40 is also expected to be less accessible in state II than in state I (see main text), which is indicated by stippled lines for phosphorylation of TH in state II. The case for dephosphorylation is not known, but it could be expected that state III is a poorer substrate for PP than the open states IV′ and IV. The dephosphorylation reaction III → II is therefore also stippled. The states where we provide structural details in this work (I, II and IV) are marked with circles. studied for a large number of mutants [bib_ref] Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular..., Fossbakk [/bib_ref] [bib_ref] The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants..., Szigetvari [/bib_ref]. Analyses based on prokaryotic expression and characterization of mutationassociated effect on thermal stability, protein solubility and residual TH activity have provided a good correlation to phenotype severity for a large number of type A (L-Dopa-responsive dystonia) mutations, but less so for some of the severe type B (nonresponsive infantile parkinsonism) mutations [bib_ref] Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular..., Fossbakk [/bib_ref] [bib_ref] Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis, Willemsen [/bib_ref]. As seen in [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] , where the mutations are mapped on the TH(DA) subunit structure and coloured according to their presence in type A or B patients [bib_ref] Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular..., Fossbakk [/bib_ref] [bib_ref] Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis, Willemsen [/bib_ref] , the mutations are spread over the CD and OD, and the more severe type B appear located closer to the active site cavity. As DA-mediated TH stabilization largely determines the steady-state levels of TH and DA in vivo [bib_ref] Brain catecholamine depletion and motor impairment in a Th knock-in mouse with..., Korner [/bib_ref] [bib_ref] The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine..., Homma [/bib_ref] , the full-length TH(DA) structure obtained in this work improves our understanding of the structure-based pathogenic mechanism in THD. In particular, the interactions of helix 360-375 with the N-terminal helix 39-58 and loop 290-297 around the active site [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref] appear relevant to understand the deleterious type B THD mutations R297W and T368M (R328W and T399M in TH4). R297 is at the centre of a crucial H-bonding and electrostatic network, and T368 interacts with I42 in the N-terminal helix [fig_ref] Figure 6: Modelling of the TH active site [/fig_ref] and [fig_ref] Figure 1: Structure of the human tyrosine hydroxylase [/fig_ref]. Mutations in these residues are expected to affect the proper localization of the N-terminal helix upon DA binding, further affecting TH stabilization.
This work constitutes a significant step forward in the knowledge of the structure, regulation and stabilization of TH through feedback inhibition by DA and its reversal by PKAmediated S40 phosphorylation. Determining the full-length structure of apo-TH also allowed us to corroborate the differences in oligomeric organization of the RDs in resting-state AAAHs. The dimeric assembly of the RDs in TH is consistent with the stable tetrameric structure of the protein, and for setting the stage for the far N-terminal tail (residues 1-70) as a main player in TH regulation. A major path in future studies remains the region preceding the 39-58 α-helix. The first 40 residues have not been visualised in this work and yet some of them (e.g., T8, S19 and S31) are important players in TH regulation.
In any case, the new structural knowledge allows for a more detailed mechanistic understanding of key physiological regulation of TH and contributes to improve the genotype-phenotype correlations in THD. A better structural understanding may also pave the way to the development of novel stabilizing/chaperoning therapies to address the deleterious neurological manifestations, very much needed by patients with L-Dopa-unresponsive THD type B. Pharmacological chaperones, which are target-specific small molecules that stabilize functional states and/or facilitate folding of non-native intermediates, have shown therapeutic potential by recovering function in mutant proteins, including phenylketonuria-associated PAH mutants, and are often discovered through high-throughput target-based experimental and/ or virtual screenings [bib_ref] Pharmacoperones as novel therapeutics for diverse protein conformational diseases, Tao [/bib_ref] [bib_ref] Phenylalanine hydroxylase misfolding and pharmacological chaperones, Underhaug [/bib_ref]. Virtual screening and structure-based drug design are preferably carried out with 3D-structures with a resolution ≤ 3 Å for the target protein [bib_ref] Cryo-EM for small molecules discovery, design, understanding, and application, Scapin [/bib_ref]. In the case of cryo-EM structures with near-atomic resolution , as is the case for our TH structures, a combination of MD simulations and ensemble docking protocols appears to provide a promising strategy for their successful application in structure-based drug discovery [bib_ref] Revealing molecular determinants of hERG blocker and activator binding, Dickson [/bib_ref].
# Methods
Expression and purification of the TH variants. TH with an N-terminal His-MBP tag containing a TEV protease-site was expressed from the pETMBP1a/TH plasmid 7 , and purified with amylose resin affinity chromatography. The HisMBP-TH fusion protein was eluted with 10 mM maltose, followed by cutting with TEV protease (1:50 w/w for 1 h at 4°C). Subsequent isolation of the tetrameric TH by size-exclusion chromatography using a HiLoad™ Superdex™ 200 prep grade column (1.6 cm × 60 cm) in 20 mM Na-Hepes pH 7, 200 mM NaCl. Constructs for THNΔ35, THNΔ43 and THNΔ70 were derived from the pETMBP1a/TH construct (Genscript) and also expressed and purified as tetramers essentially as TH [bib_ref] Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length..., Bezem [/bib_ref]. For synchrotron circular dichroism (SRCD), the SEC was performed in 50 mM Na-Phosphate pH 7. Protein concentrations were determined using a Nanodrop UV-Vis spectrophotometer and the theoretical extinction coefficients.
Preparation of stable TH(DA) complex. Once TH was purified and its functionality assessed by determining the enzyme activity at standard conditions and 37°C (see section 'Assay of TH activity and inhibition by DA', below), providing a specific activity of 1630 ± 184 nmol L-Dopa/min/mg (mean ± SD, n = 5 independent samples), the protein was incubated with iron and DA to obtain the stable Fe(III)-catecholate complex [bib_ref] Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase)...., Andersson [/bib_ref] [bib_ref] Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics..., Frantom [/bib_ref]. Essentially, ferrous ammonium sulfate (FAS) was dissolved in degassed water, added to TH (at a molar ratio 1:1 FAS:TH subunit) and incubated for 2 min at room temperature (RT) before DA addition (2:1 DA:TH subunit) with subsequent incubation for 3 min.
Phoshorylation of TH on S40. TH was phosphorylated on S40 (control without kinase) 3 , using the catalytic subunit of cAMP-dependent protein kinase (CatPKA, kind gift from S.O. Døskeland 64 ). TH (4 mg/ml) was assayed in 50 mM Na-βglycerophosphate pH 7.5, 1 mM DTT, 0.1 mM EGTA, 1x protease inhibitor cocktail (without EDTA, Pierce), 0.5 mM ATP, 5 mM MgCl 2 and incubated with the catalytic subunit of CatPKA (200 nM, diluted from 20 µM in 60% glycerol, 0.5 mg/ml soya bean trypsin inhibitor) for 30 min at 25°C. Phosphorylation stoichiometry (0.98 ± 0.03; mean ± SD, n = 3 independent samples) was measured by radioactive [ 32 P]-γ-ATP 3 . After phosphorylation, H89 (Sigma) (5 µM) was added and the samples were exchanged into 20 mM Hepes pH 7.0, 200 mM NaCl.
Synchrotron circular dichroism (SRCD) spectroscopy. Small aliquots of concentrated TH (apo-TH, THNΔ35, THNΔ43, THNΔ70, and THS40p) were diluted to 0.5 mg/ml in 50 mM Na-phosphate pH 7 with a two-fold molar ratio of iron and with two-fold molar ratio of DA when appropriate. SRCD spectra were collected as from 280 to 170 nm (3 scans averaged) at 25°C on the AU-UV beamline at ASTRID2, part of the Institute for Storage Rings (ISA) facility at the University of Aarhus, Denmark. All spectra for baselines and samples were measured in the same cuvette, a 121-type closed cylindrical cell with 100 µm pathlength. The path length of the cell was confirmed using an interferometry method and found to be 100.1 µm. The protein concentration was measured using Abs 280nm prior to the measurements and more accurately by A 205nm from the absorption spectra measured simultaneously with the CD spectra, using the respective theoretical extinction coefficients [bib_ref] Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm, Anthis [/bib_ref]. The A 205nm measurements were used in the conversion of raw CD units to Δε(M −1 cm −1 ) values. Secondary structure content was determined in the 250-180 nm range using the BeStSel algorithm [bib_ref] BeStSel: a web server for accurate protein secondary structure prediction and fold..., Micsonai [/bib_ref].
Differential scanning calorimetry (DSC). A PEAQ-DSC automated differential scanning calorimeter (Malvern Panalytical) was used to obtain the melting profile of the TH proteins with and without DA. In all experiments, 9 μM TH in 20 mM Na-Hepes pH 7, 200 mM NaCl was used, with the same buffer as reference, heating from 25 to 75°C at a scan rate of 200°C/h. A two-fold molar ratio of iron was added to all the samples and a two-fold molar ratio of DA or water when appropriate. DA was added after 2 min incubation with FAS at room temperature. The DSC thermograms were buffer-subtracted and normalized on scan rate and concentration and analyzed in the PEAQ-DSC 1.61 analysis software. T max was taken as the temperature with maximum Cp-value and T onset at 5% of T max pre-transition.
Assay of TH activity and inhibition by DA. The activity of purified TH and truncated forms was assayed at 37°C 7 . The purified enzymes were kept on ice and diluted with 0.5% (w/v) bovine serum albumin in 20 mM Na-Hepes pH 7, 200 mM NaCl and centrifuged at 10,000 × g for 5 min at 4°C. The standard assay mixture contained 20 mM Na-Hepes, pH 7, 0.1 mg/ml catalase, 10 µM FAS, 50 µM L-Tyr and 0-800 µM DA. TH was added to a final concentration of 1 µg/ml (17 nM subunit) and preincubated at 37°C for 1 min. The reaction was started by adding 200 µM BH4 and 5 mM DTT and stopped by adding one volume of 1% (v/v) acetic acid in ethanol after 5 min for TH, THNΔ35, THNΔ43, and THS40p and after 2 min for THNΔ70. Protein was removed by precipitation at −20°C for 90 min followed by centrifugation at 20,000 × g for 14 min at 4°C. The amount of L-Dopa in the supernatant was determined using a 1200 series high performance liquid chromatography (HPLC) system (Agilent technologies). The chromatographic separation was obtained using an Agilent Zorbax 300-SCX column with 20 mM HAc pH 3.5, 2% (v/v) propanol as mobile phase at a flow rate of 3 ml/min. The fluorescence detector was set to λ ex = 280 nm and λ em = 314 nm.
Crosslinking experiments and mass spectrometry analysis. Apo-TH and TH(DA) were subjected to chemical crosslinking coupled with mass spectrometry (XL-MS). Approximately 10 μg of apo-TH and TH(DA) were incubated with 3 mM of the crosslinker Bis(sulfosuccinimidyl suberate) (BS3) in 50 mM Na-Hepes pH 7, 200 mM NaCl for 40 min at RT. The reaction was quenched for 15 min at RT by adding 50 mM Tris-HCl pH 7.0. Purified BS3-crosslinked proteins were incubated in Laemmli sample buffer (0.02% [w/v] bromophenol blue, 2% [w/v] SDS, 10% [v/ v] glycerol, 60 mM Tris-HCl pH 6.8) for 5 min at 96°C and loaded onto a 12% polyacrylamide gel. Electrophoresis was stopped as soon as the protein sample reached the top region of the resolving gel. The gel was then stained with Quick Coomassie (Generon) and the coomassie-stained band was excised and subjected to automated reduction, alkylation with iodoacetamide and trypsin digestion in a Proteineer DP robot (Bruker Daltonics). The resulting peptide mixture was speedvac dried and re-dissolved in 0.1% (v/v) formic acid.
The LC-MS/MS analysis was carried out using a nano-LC Ultra HPLC (Eksigent, Framingham, MA) coupled online to a 5600 triple TOF mass spectrometer (AB Sciex, Framingham, MA) through a nanospray III ion source (AB Sciex) equipped with a fused silica PicoTip emitter (10 µm × 12 cm; New Objective, Woburn, MA). Chromatography was performed at a flow-rate of 250 nl/ min at 50°C under the following gradient elution conditions: 2% B for 1 min, a linear increase to 30% B in 109 min, a linear increase to 40% B in 10 min, a linear increase to 90% B in 5 min and 90% B for 5 min. The ion source was operated in positive ionization mode at 150°C with a potential difference of 2300 V. Each DDA cycle included a survey scan (350-1250 m/z) of 250 ms in high resolution mode and a maximum of 25 MS2 spectra (100-1500 m/z) in high sensitivity mode.
For peptide identification, raw MS data were searched against a custom-made database containing the sequences of the proteins in each sample. The MS/MS ion search was performed with Stavrox 3.6.6 [bib_ref] StavroX-a software for analyzing crosslinked products in protein interaction studies, Gotze [/bib_ref]. Search parameters were set as follows: trypsin as enzyme allowing 2 and 3 missed cleavages for R and K, respectively, BS3 as crosslinker, MS tolerance of 20 ppm and MS/MS tolerance of 40 ppm, carbamidomethylation of cysteine as fixed modification and oxidation of M as variable modification. Peptide identifications were filtered at a FDR < 5%.
Sample preparation for Cryo-EM. Purified apo-TH was subjected to a second size exclusion chromatography step to ensure protein homogeneity and stability. The sample was loaded onto a Superdex 200 Increase 3.2/300 (GE Healthcare) equilibrated with the same buffer at 4°C. Fractions of 30 μl were collected in an ÄKTAmicro (GE Healthcare) device. Cryo-EM grids were immediately vitrified using a Vitrobot Mark IV (FEI). Quantifoil UltraAufoil 1.2/1.3 300 mesh grids were previously glow-discharged for 30 s at 15 mA. Aliquots of 3 μl of the different samples were incubated onto the grids, blotted for 1 s at 4°C and 95% humidity and plunged into liquid ethane. TH(DA), THNΔ35(DA) and THS40p were directly vitrified under the same conditions with no extra size exclusion chromatography step.
Cryo-EM data acquisition. All the samples were first checked using a 200 kV FEI Talos Arctica equipped with a Falcon III direct electron detector at the Centro Nacional de Biotecnología (CNB) cryo-EM facility. Data acquisition for apo-TH was performed at the Diamond Light Source (DLS) electron Bio-Imaging Centre (eBIC) using a Titan Krios electron microscope operated at 300 kV, equipped with a Gatan Quantum K2 Summit direct electron detector operated in counting mode. A total of 3867 movies were acquired at a nominal magnification of 130,000× (corresponding to a pixel size of 1.047 Å/pixel), with a defocus range of −1.6 to −2.2 μm. Movies were fractionated to 36 frames with a total exposure of 9 s. The electron dose was 39.6 e − /Å 2 for a total dose of 1.1 e − /Å 2 on the specimen (Supplementary .
Movies of TH(DA) were collected on a FEI Titan Krios electron microscope (Krios 1) operated at 300 kV, equipped with a Gatan Quantum K2 Summit direct electron detector mounted on a Gatan Bioquatum LS/967 energy filter at the European Synchrotron Radiation Facility (ESRF) in Grenoble. Data collection was carried out with a 130,000× nominal magnification (yielding a pixel size of 1.053 Å/ pixel), with a defocus range of −1.8 to −3.2 μm. A total of 4422 movies were recorded and each movie was fractionated to 40 frames with a total exposure of 6 s. The electron dose was 37 e − /Å 2 on the specimen (Supplementary .
Data acquisitions for THNΔ35(DA) and THS40p were performed at the Diamond Light Source (DLS) electron Bio-Imaging Centre (eBIC) using a Titan Krios electron microscope operated at 300 kV, equipped with a Gatan Quantum K3 detector operated in counting mode with 0.5X-binning (super-resolution mode). A total of 9241 and 13213 movies were acquired for THΔ35(DA) and THS40p respectively, at a nominal magnification of 81,000× with a defocus range of −1.6 to −3.4 μm. Movies were fractionated to 40 frames with a total exposure of 2 s for both samples. The electron dose was 30 e − /Å 2 (Supplementary . Motion-corrected movies were extracted and binned 2× to the physical pixel size of 1.07 Å/pixel.
Image processing and three-dimensional reconstruction. Image processing of apo-TH, TH(DA), THΔ35(DA) and THS40p samples was performed following a similar workflow. All programs used to obtain the different 3D models are implemented in the Scipion 2.0 software platform [bib_ref] Scipion: A software framework toward integration, reproducibility and validation in 3D electron..., De La Rosa-Trevin [/bib_ref]. First, the beam-induced motion of the movies was corrected using the MotionCorr2 software [bib_ref] MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy, Zheng [/bib_ref]. After movies alignment, the contrast transfer function (CTF) was calculated and corrected by Gctf [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] [bib_ref] Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled..., Meisburger [/bib_ref]. Particles were automatically picked with Xmipp3 -auto-picking software [bib_ref] A pattern matching approach to the automatic selection of particles from low-contrast..., Abrishami [/bib_ref]. To save computational time during the first processing steps and to increase the signal to noise ratio, particles were downsampled by a factor of 4, and extracted with Xmipp3extract particles [bib_ref] A pattern matching approach to the automatic selection of particles from low-contrast..., Abrishami [/bib_ref]. The extracted particles were subjected to a first 2D classification using Relion 2.0 [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] [bib_ref] RELION: implementation of a Bayesian approach to cryo-EM structure determination, Scheres [/bib_ref] and the best classes were subjected to several further rounds of 2D classification, allowing a much better detection of bad particles such as aggregates or particles that were very close to each other.
For apo-TH and TH(DA), different de novo initial models (Supplementary Figs. 4c and 7c) were obtained using both EMAN2 72 and RANSAC 73 . Another initial model was obtained by low resolution (60 Å) filtering of the atomic structure of the CD of the human TH (PDB 2XSN). In the case of apo-TH, the first rounds of 3D classification were performed using Relion 2.0 without any symmetry imposition and using the different initial models [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref]. No significant differences were found among the best class obtained from the low-pass filtered atomic structure and the de novo initial model. Particles belonging to that class were subjected to refinement using Relion 2.0-3D auto-refine software. Since clear symmetric features were observed in this class, we sought to determine whether C2 or D2 symmetry was applicable and could contribute to better define our 3D models. The application of C2 symmetry resulted in different classes showing good arrangement in the CD and OD, but the mass corresponding to the RD was distorted showing artefactual densities. On the other hand, the D2 symmetry maintained the RD size and shape as expected according to its atomic structure (PDB 2MDA) [bib_ref] The solution structure of the regulatory domain of tyrosine hydroxylase, Zhang [/bib_ref] and to the best volume obtained before symmetry imposition [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref]. In the case of TH(DA), D2 symmetry was directly applied for 3D classification [fig_ref] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by... [/fig_ref]. The particles selected after refinement (250,712 particles for apo-TH and 125,033 for TH(DA)) were reextracted at the original pixel size to continue the image processing. Auto-refine with the original particles [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref] rendered a final 3D map at 3.9 and 4.1 Å resolution for apo-TH and TH(DA), respectively, as estimated by the Fourier shell correlation (FSC) method, with a cut-off of 0.143 (Supplementary Figs. 4f and 7f) [bib_ref] RELION: implementation of a Bayesian approach to cryo-EM structure determination, Scheres [/bib_ref]. This approach calculates the cross-correlation between two halves of the 3D map in the spatial frequency shells to give the resolution, but it does not contemplate the flexibility of the protein. For each 3D map, local resolution was then calculated using Xmipp3-MonoRes 34 (Supplementary Figs. 4g and 7g). The same sets of particles were also subjected to Relion Bayesian particle polishing, however, no improvement in resolution was observed. To improve the low resolution found in the RD, these domains were extracted and processed as single particles to generate a localized reconstruction of the RD 74 [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. The final refined map at 3.9 Å resolution was used for subtracting the region of interest. First, a mask surrounding the selected part of the map (subparticles from now on) was generated and used to extract the subparticles. An initial volume was generated with Relion-reconstruct [bib_ref] RELION: implementation of a Bayesian approach to cryo-EM structure determination, Scheres [/bib_ref] and used for 3Dclassification of the subparticles. The best class was auto-refined to improve the quality of the data and the resolution. The RD final map resolution obtained reached 7.1 Å. This approach was not successful for TH(DA), probably due to the extra density affecting the proper alignment of the subparticles. Another way of improving the different domains in a protein complex is by masking the density of interest. This approach was used to increase the resolution in the CD and OD. Starting from the best auto-refined volume, a mask was generated that excluded the RD. The density inside the mask was subjected to a 3D classification, auto-refine and further postprocessing to obtain a good density map for next model building steps [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref]. The maps were further subjected to sharpening using Relion post-processing 71 , LocScale [bib_ref] Model-based local density sharpening of cryo-EM maps, Jakobi [/bib_ref] and LocalDeblur 76 [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref]. The combination of all of them provided the best interpretability of the 3D reconstructed maps. For the difference map carried out between TH(DA) and apo-TH, the two maps were first normalized and filtered to the same resolution (that of the lowest one) and subtracted with the "vop subtract" option in the Chimera package.
For THNΔ35(DA) and THS40p, image processing was performed following similar steps and using Relion-3 77 and cryoSPARC [bib_ref] cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination, Punjani [/bib_ref]. The 3D reconstruction process was carried out with D2 symmetry imposition. After several rounds of 2D and 3D classification, 152,128 and 148,453 particles were re-extracted at the original pixel size and used to generate the final 3D maps at 4.6 and 4.5 Å resolution, respectively.
Model building, refinement and validation. First, the CD and OD domain structures from the human homotetrameric structure (PDB 2XSN) were fitted rigidly using Chimera [bib_ref] UCSF Chimera-a visualization system for exploratory research and analysis, Pettersen [/bib_ref]. Although a good fit in the Cryo-EM density map was obtained, a further flexible fitting step with iMODFIT 80 was performed to optimize the arrangement of some flexible segments. The majority of the RD was modelled from the NMR structure of the rat homodimer (PDB 2MDA) with whom the human version shares 82% amino acid identity [fig_ref] Figure 2: Structure of human TH in complex with dopamine [/fig_ref] , using the SWISS-MODEL homology-modelling server [bib_ref] SWISS-MODEL: homology modelling of protein structures and complexes, Waterhouse [/bib_ref]. The resolution of the RD precluded accurate model building, but was sufficient to rigid-body fit the domain in a position compatible with its connection with the CD. Unfortunately, no homologous structure was found for the first 70 N-terminal amino acids of the RD. However, an un-modelled density was observed initially in the DA-bound structure that could accommodate a long α-helix structure. The secondary structure predictions obtained from I-TASSER [bib_ref] I-TASSER server: new development for protein structure and function predictions, Yang [/bib_ref] and PSIPRED 40 servers agreed in the existence of an approximately 20-residue long α-helix in this region . Based on these observations, a generic 20-residue helix was modelled and fit into the corresponding density with Chimera. Then, we exhaustively scanned all possible orientations and sequence shifts for the first 70 residues of TH within this NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27657-y ARTICLE NATURE COMMUNICATIONS | (2022) 13:74 | https://doi.org/10.1038/s41467-021-27657-y | www.nature.com/naturecommunications generic helix. The scanning included all 50 possible sequence shifts within a 20residue window, as well as translations of the helix axis from −2 to +2 Å in 0.5 Å steps. The N-terminal α-helix configurations with lowest energy computed with KORP (knowledge-based orientational potential) [bib_ref] KORP: knowledge-based 6D potential for fast protein and loop modeling, Lopez-Blanco [/bib_ref] corresponded to the 39-50 region, or to very close poses (i.e., slightly rotated and translated versions). Interestingly, the identified region corresponded to the fragment with highest α-helical propensity . The minimum energy helix arrangement is proposed as the tentative backbone model. Supporting this model, the reconstruction lacking the first 35 residues (THNΔ35) in the presence of DA maintains an equivalent helix in the active site [fig_ref] Figure 4: Three-dimensional reconstructions of different TH variants [/fig_ref]. Finally, the loops connecting the RD with the CD and the N-terminal helix were predicted with the RCD + server [bib_ref] RCD+: Fast loop modeling server, Lopez-Blanco [/bib_ref]. This online tool efficiently applies the RCD loop-closure algorithm [bib_ref] Random coordinate descent with spinor-matrices and geometric filters for efficient loop closure, Chys [/bib_ref] to generate feasible loop conformations that are refined and ranked in PyRosetta 3.0 [bib_ref] PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta, Chaudhury [/bib_ref]. The best loop conformations were selected to illustrate the feasibility of the connection.
The final TH models [fig_ref] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity... [/fig_ref] were subjected to a double realspace refinement, first manually using COOT [bib_ref] Features and development of Coot, Emsley [/bib_ref] and then, an automatic procedure with PHENIX 1-19-4092-000 86 or REFMAC 5.8.0258 [bib_ref] Refinement of macromolecular structures by the maximum-likelihood method, Murshudov [/bib_ref] implemented in the CCP-EM software platform [bib_ref] Recent developments in the CCP-EM software suite, Burnley [/bib_ref]. The restraints used in the real-space refinement were both standard (bond, angle, planarity, chirality, dihedral, and non-bonded repulsion), and with some additional restraints (Ramachandran plot, C-beta deviations, rotamer, and secondary structure). A local grid search-based fit was included in the refinement strategy to resolve side-chain outliers (rotamers or poor map fitting). Several rounds of real-space refinement were performed until a stable final model was obtained and subsequently validated. For both apo-TH and TH(DA), several comparable Cryo-EM maps were used to combine information and refine the tracing of the whole atomic structure. Validation of the final models was done using the phenix_validation_Cryo-EM module in PHENIX. The figures with maps and structures have been rendered with PyMOL 2.2.0 and Chimera 1.14.
Molecular dynamics (MD) simulations. A total of 8 all-atom unbiased MD simulations of tetrameric TH were carried out for 0.5 µs each. Simulations were performed with and without DA coordinated to the active site iron, as well as with and without phosphorylation of S40 (THS40p), for a total of four individual systems. The atomic tetramer models were based on the Cryo-EM structure of TH(DA) by inserting DA with the same orientation and iron-oxygen bonding distances as that of human PAH (PDB 5PAH), and by adding a phosphate group to S40. To enhance sampling and statistics, each state was simulated in parallel, differing in their generated random initial velocities. All atomic models were prepared with Amber 18 and the corresponding Amber14SB forcefield [bib_ref] ff14SB: Improving the accuracy of protein side chain and backbone parameters from..., Maier [/bib_ref]. Parameters for DA as well as iron with coordinating residues were prepared with Antechamber 70 and the general Amber forcefield 90 using a semi-empirical model. Protonation states of side chains were assigned based on the 3D-structure using PROPKA at pH 7.0 86 . For each of the simulations, the system was neutralized using a mixture of Cl − and Na + counter ions, and the protein was solvated in a periodic truncated octahedron box with TIP3 water molecules 91 , providing 16 Å of water between the protein surface and the periodic box edge. The solute was minimized for 10,000 steps, followed by 10,000 steps of minimization of the whole system with restraints on the protein backbone, and finally 20,000 steps of minimization of all atoms. The protein was then heated to 100 K with weak restraints for 20 ps, and to 300 K for 1 ns. Equilibration with constant pressure and temperature (NPT) of the system was performed for a total of 20 ns prior to the production with reduced restraints on the solute. The production runs lasted 500 ns and were performed at a constant volume and energy. All simulations were run with a 2 fs time step, using SHAKE constraints on bonds involving hydrogens. All simulations were run with GPU acceleration 92 on Nvidia RTX 2080Ti cards, producing on average 48 ns of molecular dynamics per day of the systems simulated. The simulations were analyzed using cpptraj 93 (AmberTools20). Distances, clustering and fluctuation profiles were shown at the monomer level, averaged over the 4 subunits from two simulations. The K-means algorithm was used for clustering and the conformation representing the largest cluster over the last 50 ns was selected to represent each system.
Statistics and reproducibility. Statistical analyses were performed with Graphpad Prism software version 8.3.0. For SRCD, DSC and TH activity measurements, three enzyme samples from independent protein purifications were measured and the mean ± SD (standard deviation) was calculated. The homogeneity of variances was confirmed by Brown-Forsythe tests and multiple comparisons were made using one-way analysis of variance (ANOVA) followed by a post hoc HSD Tukey test. Differences in secondary structure content, thermal onset (T onset ) and temperature maximum (T max ) and half-maximal inhibitory concentration (IC50) of the individual TH forms when compared to themselves with/without DA and to full-length TH in the same state were considered significant when p < 0.05. The individual curves were fitted to a four-parameter logistic curve by the [Inhibitor] vs. response -Variable slope equation (y = min + ((max − min)/(1 + IC50/X)^Hill Slope) in Graphpad and the mean IC50 ± SD reported.
The results in the representative SDS-gel (Supplemental [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref] and Cryo-EM micrograph (Supplemental [fig_ref] Figure 3: Structural comparison of apo-TH and TH [/fig_ref] for purified apo-TH have been observed with at least three protein preparations in independent experiments.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
## Data availability
The data that support this study are available from the corresponding authors upon reasonable request. Cryo-EM data have been deposited in the Electron Microscopy Data Bank under accession codes EMD-11624 for full length apo-TH, EMD-11467 for full length TH(DA), EMD-11587 for CD + OD domains of apo-TH, EMD-11309 for CD + OD domains of TH(DA) and EMD-13442 for THNΔ35. The associated atomic models have been deposited in the Protein Data Bank under accession codes 7A2G for full length apo-TH, 6ZVP for full length TH(DA), 6ZZU for apo-TH, CD + OD domains, 6ZN2 for TH(DA), CD + OD domains and 7PIM for THNΔ35. Previously reported structures used in this work are 1TOH, 2XSN, 2MDA, 5PAH, 1KW0, 6HYC. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024519. Source data are provided with this paper.
Received: 6 September 2020; Accepted: 3 December 2021;
[fig] Figure 1: Structure of the human tyrosine hydroxylase (apo-TH). a Three orthogonal views of the Cryo-EM map showing the main features of the full-length apo-TH at 3.9Åresolution. The scale bar indicates 50 Å. b [/fig]
[fig] Figure 2: Structure of human TH in complex with dopamine (TH(DA)). a Three orthogonal views of the TH(DA) Cryo-EM map (4.1 Åresolution) including the RDs and masses connecting them with the CDs. The red arrow points to one of the new, helical densities connecting with the CD. b Atomic model of the CD + OD domains of TH(DA) docked in the 3.8 ÅCryo-EM map of the same region. c Predicted model of TH(DA) that includes the proposed α-helix. Each monomer is drawn in a different colour. | (2022) 13:74 | https://doi.org/10.1038/s41467-021-27657-y | www.nature.com/naturecommunications [/fig]
[fig] Figure 3: Structural comparison of apo-TH and TH(DA). a Three orthogonal views of the Cryo-EM maps of apo-TH (grey) and TH(DA) (yellow, mesh) [/fig]
[fig] Figure 4: Three-dimensional reconstructions of different TH variants. a Three orthogonal views of the three-dimensional reconstruction of THNΔ35 in the presence of DA (THNΔ35(DA)). The black arrows point to a new mass visualized in TH(DA), which is not present in apo-TH. b The same views with the atomic model of CD + OD domains of THNΔ35 (the RDs have been removed) docked into the corresponding mass of the THNΔ35 three-dimensional reconstruction. The black arrows point to the α-helix entering into the active site. c Comparison of the atomic model of the TH active site of TH(DA) (left) and THNΔ35(DA) (right) showing the similar location of the α-helix inside the active site. d Three orthogonal views of the three-dimensional reconstruction of phosphorylated on S40 (THS40p). The asterisks point to the position where the N-terminal α-helix is located in both TH(DA) and THNΔ35(DA) reconstructions, but absent in this structure. | (2022) 13:74 | https://doi.org/10.1038/s41467-021-27657-y | www.nature.com/naturecommunications [/fig]
[fig] Figure 5: Biophysical characterization of different TH samples and inhibition of their enzyme activity by DA. Representative synchrotron circular dichroism spectroscopy (SRCD) profiles of a apo-TH (broken line) and TH(DA) (continuous line), and b apo-TH (black line), the deletion mutants THNΔ35 (pink line), THNΔ43 (green line) and THNΔ70 (orange line), and TH with phosphorylated S40 (THS40p; blue line). c, d Representative differential scanning calorimetry (DSC) profiles, in the absence (broken line) and presence (continuous line) of DA, of TH (c) and THNΔ70 (d). T onset and T max are indicated by arrows in (c). e Relative TH activity versus DA concentration for apo-TH (black dots), the deletion mutants THNΔ35 (pink dots), THNΔ43 (green dots), THNΔ70 (orange dots), and THS40p (blue dots). Dots represent the mean + SD (n = 3) and solid lines are fittings to the four-parameter logistic nonlinear regression model. f The bars represent the IC50 values obtained from the fitting the four-parameter logistic nonlinear regression model to triplicate curves, presented as mean ± SD, with the individual values as dots. Asterisks indicate significant changes compared to TH using one-way ANOVA followed by Tukey's multiple comparisons test; p = 0.0001 (***) for TH vs. THNΔ43, p > 0.0001 (****) for TH vs. THNΔ70, p = 0.0026 (**) for TH vs. THS40p. Source data are provided as Source Data File. [/fig]
[fig] Figure 6: Modelling of the TH active site. a Modelling the effect of S40 phosphorylation on the interaction of the N-terminal α-helix with DA. Representative conformations from the last 50 ns of the 500 ns MD simulations for TH(DA) (grey ribbon) and pS40-TH(DA) (light blue ribbon). See "Methods" for preparation of the representative conformation. The resulting structures show a slight shift of the N-terminal α-helix upon phosphorylation, most probably due to electrostatic repulsion between the phosphate and E325, E375 and D424. b A detailed view of the atomic model of the TH(DA) active site. (left) The N-terminal α-helix (orange), which establishes connections with the adjacent helix D360-E375 and with residues of the 290-297 and 420-429 loops (blue, right). c A cartoon depicting the interactions established between residues of the N-terminal α-helix that enters the active site, and residues of adjacent regions. | (2022) 13:74 | https://doi.org/10.1038/s41467-021-27657-y | www.nature.com/naturecommunications [/fig]
[fig] Figure 7: Cartoon model of DA-mediated feedback inhibition of TH and its regulation by S40 phosphorylation. In the active, apo and non-phospho state, the 39−58 α-helix of TH is detached from the main structure (I, apo-TH). The feedback inhibitor DA binds to the TH active site, most likely in the open conformation (I′, TH(DA)). DA-binding favours interaction of the N-terminal α-helix with the same binding site, which blocks DA exit and contributes to the high-affinity binding and strong inhibition of TH activity (II, TH(DA)). Protein Kinase (PK) phosphorylation of S40 in TH(DA), leads to state III (THS40p(DA)), prompting the detachment of the α-helix from the TH active site (IV′), which opens up for DA-dissociation and activation (IV, THS40p). [/fig]
|
A systematic review of barriers and motivators to physical activity in elderly adults in Iran and worldwide
On one hand, physical activity (PA) helps elderly people to improve their strength and flexibility, although the most effective type of activity is still unclear[1]. On the other hand, the elderlydue to health problems and insufficient facilities-are more likely to face barriers to access than others[2].For adults aged 60 and over, important motivations for PA have been reported to include social support, health benefits, and enjoyment, while the main barriers have been found to be insufficient guidance and a lack of role models [3]. For people aged over 80 years, the most important motivators and barriers have been reported to be the health benefits of PA, various types of fears, individual preferences, and social support[4]. In general, due to the increasing percentage of the older population in developing (lowand middle-income) countries and the burden of health care and treatment costs, reducing the prevalence of physical inactivity has received attention as an important goal[5]. In addition, PA develops as a behavior through complex and dynamic interrelations among individual, social, and environmental factors, underscoring the usefulness of using multidimensional models to study PA[6,7]. As an example of this approach, the socio-ecological model of McLeroy et al. [8] was used to study the barriers and motivations to PA in the elderly. The aim of this study was to systematically review the motivators and barriers to PA in people aged 60 years and older in Iran and other countries.OBJECTIVES:This study was conducted to identify and characterize the barriers and motivations to physical activity (PA) for elderly adults in Iran and other countries.METHODS:We searched 6 databases (PubMed, Embase, Scopus, Web of Science, Magiran, and the Scientific Information Database) from 2000 to the November 2017, using "aged 60 and over, " "physical activity" or "exercise, " and "motivator" and "barrier" as keywords. Two reviewers independently performed the search, screening, and quality assessment of the studies.RESULTS:In total, 34 papers were finally included in the study. The most important barriers, based on the frequency of factors, included physical problems, having no companions, and physical barriers to walking. The motivators included improving one's physical condition, being social, and suitability of the physical environment.CONCLUSIONS: Important motivators and barriers to PA were more closely related to intrapersonal factors than to the interpersonal and environmental domains. The barriers and motivators to PA in the elderly were not markedly different between Iran and other countries. Therefore, a general strategy could be designed to improve PA in the elderly.
# Introduction
November 2017. The exclusion criteria were non-original articles (i.e., letters to the editor, case reports, interventional studies, reviews, meta-analyses, and articles presented at seminars and conferences were excluded), articles with no specific definition of age, those that were conducted before 2000 but were accepted in 2000 or later, articles analyzing elderly individuals living in nursing homes, and articles with a very low quality score. In addition, a backward search (checking bibliographic mining of identified papers for any additional studies) was conducted to identify any studies that were not retrieved using the main search strategy. All quantitative and qualitative designs were included. In total, 1,981 articles were retrieved, of which a total of 34 articles remained after the review process shown in. Finally, 2 authors carefully examined 34 full-text articles.
In this study, 2 types of studies were investigated: (1) quantitative studies (5 from Iran and 15 from other countries); (2) qualitative studies (1 from Iran and 13 from other countries).
There were 6 studies in Persian and 28 studies in English.
# Materials and methods
## Search strategy
To collect the data, a comprehensive search was performed of several electronic databases (PubMed [MeSH terms], Web of Science, Scopus, Embase, Scientific Information Database, Magiran) to identify all potentially relevant publications in the Persian and English languages from 2000 to November 2017. The following keywords were used: "aged (or age 60 and over), " "physical activity (or exercise), " and "motivator and barrier" (Supplementary Material 1). The detailed search strategy implementation in PubMed was as follows: ([elderly OR Aged OR "60 over aged"] AND ["Physical activity" OR "Exercise" OR "Physical exercise" OR "Motor activity"] AND [Motivation OR Motivat* OR Barrier*]). The inclusion criteria were (1) articles published in the Persian and English languages;original research examining the barriers and motivators to PA in the elderly; (3) studies that examined outcomes in terms of physical and mental illness among the elderly (aged 60 and over); and (4) studies conducted from from 2000 to
## Evaluating the quality of articles
The quality of qualitative studies was assessed using the qualitative methodological checklist of the National Institute of Clinical Nursing (NICE). In general, according to the NICE checklist, ++ means that all or most of the checklist criteria have been fulfilled, + means that some of the checklist criteria have been fulfilled, and -means that few or no checklist criteria have been fulfilled. Quantitative studies were assessed using the Newcastle-Ottawa Quality Assessment Scale (NOS) adapted for cross-sectional studies. The NOS is based on 3 domains, including the selection of study groups, the comparability of groups, and the description of exposures and outcomes. This scale, which includes 8 items is scored in terms of stars, assesses the quality of each study in each domain. All items except the comparability domain have 1 star, while the maximum score for the comparability domain is 2 stars. The total number of earned stars is calculated as the total quality score for each study, which ranges from 1 star (very poor) to 10 stars (high quality). Studies were classified as high-quality, medium-quality (6-7), or low-quality ( < 6). Two review authors (SY and HMS) completed the quality assessment independently. In cases of disagreement or items that remained unclear, a third review author (AR or MG) was consulted.
## Data extraction
We used a structured form to extract the data. The extracted data included study and participant characteristics (e.g., gender, location, country, population, age, type of instrument, type of study, year of study), as well as motivators and barriers to PA. Two authors (SY and HMS), who conducted the study selection independently, performed the data extraction. Any disagreements were discussed with a third review author (AR or MG) if necessary. The data were classified using the socio-ecological model that McLeroy et al.developed in 1988 as a theoretical framework involving interpersonal and intrapersonal factors, organizational and social factors, and environmental factors. The identified factors were prioritized based on the frequency of participants' responses.
# Ethics statement
This study is a systematic review and does not deal with human participants.
# Results
## Quality of qualitative and quantitative studies
Fortunately, almost all the qualitative studies followed the items in the NICE checklist for qualitative studies. Two of the 14 qualitative articles were high-quality, 12 were good-quality, and 5 were poor-quality, as indicated in . The quantitative studies were evaluated using the NOS scale, and 18 articles were found to be moderate-to high-quality, while 2 were moderate-quality.
## Description of reviewed studies
The articles were published between 2000 and November 2017, and the sample sizes ranged from 9 (in a qualitative study) to 4,227 participants (in a quantitative study). The samples consisted of both men and women subjects in 30 of the 34 studies, exclusively of women subjects in 4 studies, and exclusively of men subjects in 1 study. The most common type of PA was walking, followed by swimming and fitness, and a description of the type of PA was not available in 17 studies.
In, the characteristics and major findings of the studies are presented.
## Motivators and barriers to the physical activity among the elderly
In the framework of the McLeroy model, we examined the motivators and barriers to PA among the elderly in the intrapersonal, interpersonal, and environmental domains as follows. According to the study population-people aged 60 years and older-the items from the organizational domain of the Mc-Leroy model were excluded. For quantitative studies, only significant variables were evaluated. Due to the heterogeneity across studies and input variables, it was not possible to conduct a metaanalysis of the results. Below, the most important factors for all dimensions of the model, based on the frequency of participants' responses, are presented.
## Intrapersonal factors
Through the literature review, 23 barriers and 16 motivators were identified as intrapersonal factors. In several articles, physical problems-such as difficulty in walking, physical health problems, physical weakness, respiratory problems, and lack of energy-were mentioned as key barriers [. Time limits were the second most important barrier related to intrapersonal factors. The third most important intrapersonal barrier was fear of falling.
The most important intrapersonal set of motivators that resulted in PA was improving one's physical condition, which included improving one' s balance and walking ability, reducing muscle pain, improving sleep, and strengthening one' s muscles. Enjoyment, addressing psychological issues (which involved relief from stress, feeling more efficient, having positive perceptions of PA, having a positive self-image, being less depressed, AgeIssues related to individual beliefsHousehold choresSecurity concernsSingle and widower statusBeing active enoughSexLack of energyInsufficient understanding of physical activityAn unpleasant experienceLack of self-disciplineLow level of education, retirement, life problemsHeavy weightFeeling self-awarenessImproving one's physical conditionEnjoymentUnderstanding psychological issuesHaving motivation and access to physical activity resourcesLack of knowledgeHealth concernsBeing economicalFeeling securityHaving a long lifeFear of falling and illnessPainLonelinessSocioeconomic status, having sports skills, trainingHaving enough timeJoining physical activity to daily lifeSpending free timeInterpersonal Having no companionFamily responsibilitiesHaving no professional guidance, inadequate informationSocial pressure, having less time to spend with friends and familyExercise clubs devoted to young adults and the lack of planning in the at clubsWorking with others, different views of othersBeing socialSpecialist health careAvailability of facilitiesHaving a companion for exercise, fear of dependencyAssessment of exercise program by a professional instructorSocial pressureEnvironmental Physical barriers to walkingTemperature, season, and weatherLack of facilities for exerciseTrafficInappropriateness of the timing of sports classesLack of personal safetyCommuting and distance from home to sports facilitiesSuitability of the physical environmentEnvironmental securityAccess to public transportationAccess to sports facilitiesSocial network of neighbors, air quality, living in an apartment, proximity to sports facilitiesEconomic and financial agents, holding walking meetingsand enhancing sleep, and increasing motivation and access to PA resourcescomprised the second most important set of intrapersonal motivators.
The third most important set of intrapersonal motivators included a lack of knowledge, health concerns, and being economical.
## Interpersonal factors
In the interpersonal domain, 6 barriers and 7 motivations were identified. The most important interpersonal barrier to PA was having no companion. Family responsibilities (taking care of grandchildren, children, and sick people at home) were the second most important interpersonal barrier to PA.
The most important interpersonal motivator was being social, which included communication with friends, peer support, communication with others, exercise with friends, social coherence, moderate and high local dependency, an abundance of companions for walking, and support from others. Supervision of health professionals was the second interpersonal motivator. In addition, 3 articles identified specialist health careas a motivator for PA. Another important interpersonal motivator for PA was the availability of sports facilities.
## Environmental factors
Overall, 7 barriers and 6 motivators were identified at the community level. The most important barrier was physical barriers to walking, which included problems related to safety, parked motorcycles next to the street, potted plants, rubber tiles in playgrounds, food retailers, paved streets, broken sidewalks, scaffolds, snow accumulation along the street in winter, devoted seats in parks for children, the lack of facilities such as benches for resting, poor locations, unsafe roads, stray dogs, and hills. The second most important set of barriers were related to temperature, season, and weather. More intense PA among the elderly was observed in the spring (40.1%), in sunny weather (76.8%), and at moderate temperatures (56.2%). The third most important barrier was a lack of facilities for exercise.
The most important environmental motivator was the suitability of the physical environment. This factor included pleasant landscapes, streetlights, sidewalks, bike riding routes, walking paths, the neighborhood's suitability for walking, interconnections between streets and an attractive environment, an environment free from non-cultural social activities (e.g., smoking, drinking alcohol, gambling), green space, attractive architecture, benches for resting, a place for dog parks, a smooth surface for hiking, and food availability in urban centers. Environmental security was the second environmental motivator.
# Discussion
In the present study, information on PA among the elderly was reviewed from 2000 to November 2017. We aimed to identify the motivators and barriers to PA among individuals aged over 60 in Iran and worldwide; therefore, some studies were excluded because they did not analyze participants under or over 60 years of age as 2 different groups. In general, the ecological model is a comprehensive multilevel framework that includes contributors to active behavior at all levels: individual (interpersonal and in-trapersonal), social, environmental, and policy.
A systematic review by Baert et al.on adults aged over 79 confirmed that quantitative research has a greater focus on the interpersonal and community levels, while qualitative research tends to focus more on the interpersonal level. We found that more research is needed into barriers and motivators at the organizational level, while Baert et al.showed that community-based barriers and motivators need more research because policy-makers may be able to exert influence on these factors.
## Intrapersonal factors
Health status was highlighted in most articles, either as a barrier (18 times) or as a motivator (5 times) for PA. To summarize, in the literature review, 23 articles reported that poor health was a relevant factor for PA among those aged 60 and over. Moreover, the beneficial effects of PA on health status (such as improving balance, improving walking ability, reducing muscle pain, improving sleep, and strengthening) are well established. In addition, Baert et al.reported that health status was both a barrier and motivator. Nonetheless, health improvement has been reported as an important motivator, and research has highlighted that health benefits can be a major factor for promoting PA. In this review, most studies were conducted in Iran and the USA. In these countries, special consideration should be given to the proportion of the young population in light of current barriers. For example, since Iran is a country with a young population, the proportion of the elderly in Iran is expected to peak in the next 50 years, and Iran will face similar challenges to Europe and the USA between 2040 and 2050.
Fear emerged as a special barrier. Fear is a complex phenomenon that can occur in different situations (e.g., fear of walking at night in order to exercise outside the house). It can be related to health status, such as fear of injury or pain, fear of falling, and fear of being dependent on others. Lim & Taylorreported that fear of falling was associated with inadequate levels of PA. Moreover, fear of falling was identified as a barrier to PA in different races, including African-Americans, Whites, and American Indians. In particular, American Indians were worried about falling when there was nobody to help them. Furthermore, anxiety and fear of injury were mentioned as a barrier. Health care providers should recognize this type of fear, and should consider it as an important barrier to be dealt with appropriately, if necessary.
In our study, time limits or lack of time was identified as a barrier. This barrier has also been described in other studies. In a focus group study, lack of time among people aged over 65 was found to be a barrier to PA. In many countries and cultures, the responsibilities of taking care of children and the home take up many hours in the day, with consequent negative effects on health behavior.
Enjoying PA was reported as a motivator, as in other studies. Factors related to enjoyment increase the pleasure experienced during PA, which depends on individual preferences (doing physical exercise in a group or enjoying the landscape). Fortunately, health care providers can provide support and guidance in this respect.
## Interpersonal factors
As has been found for other age groups, being social was identified as a motivator. In general, social support was also reported as a motivational factor. However, elderly individuals need more social support than younger adults. Unfortunately, elderly people are often single, causing them to be socially isolated. Supervision by health professionals has an important impact on PA in the elderly; in particular, health care providers can encourage elderly people to participate in group exercises. It has been suggested that by providing information and raising awareness about PA, health care providers can augment the self-confidence of elderly individuals to begin exercise regimens.
## Environmental factors
A lack of sports facilities was considered to be an especially important barrier. In this regard, construction of playgrounds, sidewalks, parks, or other fitness facilities could motivate individuals to participate in exercise, such as walking. Governments play an important role in providing subsidies and funding for health facilities, such as health centers and walking paths. It was found that a lack of adequate facilities in organizational settings led to a decrease in enthusiasm for PA. Likewise, in Iran, a study confirmed that the presence of exercise facilities, parks, and walking or cycling routes increased elderly individuals' motivation to engage in PA.
Some studies have shown that the availability of a resting place, such as benches along walking paths, may facilitate walking among the elderly. In this regard, for the elderly, it is very important to ensure easy access to safe, beautiful, and interesting places for walking. For these reasons, the elderly were found to prefer routes with places for them to rest.
The weather, season, and temperature were identified as potential barriers. Nadri et al., in Iran, reported that participants considered an inappropriate environment to be a barrier. In addition, other studies have shown relationships between natural changes (season, weather, and temperature) and the intensity of PA. In general, more intense PA was observed in the spring (40.1%), in sunny weather (76.8%), and at moderate temperatures (56.2%). Elderly individuals were found to engage in more frequent walking in sunny weather than in rainy weather, and their walking rate was higher at temperatures below 60°F than at high temperatures (81°F). Schmidt et al.reported that "unpleasant weather" such as cold, snow, and extreme heat was a barrier. In addition, a study confirmed that the weather was a potential barrier for the oldest old people.
It is worth mentioning that we found some similar barriers and motivators. In some reviewed studies, a link was found between the benefits of prayer and PA. The energy cost of Muslim daily prayers was about 80 calories per day, implying that daily prayers could be considered a form of PA.
There are two important implications of these findings. First, the factors identified as important herein should be analyzed with regard to gender. Second, it is necessary to consider geographical areas and the accessibility of facilities. Additionally, the type of intervention program (community-or individual-based) is an especially important factor for encouraging the elderly to participate in exercise. This systematic review has the following limitations. First, no information was extracted on differences between men and women. Therefore, further research is needed to identify any such potential differences. Second, it is possible that differences existed among participants and authors in the definition of exercise and PA. Third, most studies were conducted in developed countries, which may have yielded country-specific results and decreased the generalizability of the findings. As a final limitation, due to inconsistencies and heterogeneity in the data gathering and analysis methods used in the quantitative articles, it was not feasible to conduct a meta-analysis.
# Conclusion
This study presents a comprehensive literature review and analysis on the barriers and motivators to PA in the elderly. Most barriers involved the intrapersonal and interpersonal domains. According to the population composition of the countries that were analyzed-especially Iran-interventions to address this issue are essential. In addition, the elderly may have different perceptions of barriers and motivators that need to be considered.
# Supplementary materials
Supplementary material is available at http://www.e-epih.org/.
## Orcid
|
The m6A/m5C/m1A Regulated Gene Signature Predicts the Prognosis and Correlates With the Immune Status of Hepatocellular Carcinoma
RNA modification of m6A/m5C/m1A contributes to the occurrence and development of cancer. Consequently, this study aimed to investigate the functions of m6A/m5C/m1A regulated genes in the prognosis and immune microenvironment of hepatocellular carcinoma (HCC). The expression levels of 45 m6A/m5C/m1A regulated genes in HCC tissues were determined. The functional mechanisms and protein-protein interaction network of m6A/m5C/m1A regulated genes were investigated. The Cancer Genome Atlas (TCGA) HCC gene set was categorized based on 45 m6A/m5C/m1A regulated genes, and survival analysis was used to determine the relationship between the overall survival of HCC patients in subgroups. Cox and least absolute shrinkage and selection operator (LASSO) regression analyses were used to construct the risk model and nomogram for m6A/m5C/m1A regulated genes. The relationships between m6A/m5C/m1A regulated gene subsets and risk model and immune cell infiltration were analyzed using CIBERSORT. m6A/m5C/m1A regulated genes were involved in mRNA and RNA modifications, mRNA and RNA methylation, mRNA and RNA stability, and other processes. There was a statistically significant difference between cluster1 and cluster2 groups of genes regulated by m6A/m5C/m1A. The prognosis of cluster1 patients was significantly better than that of cluster2 patients. There were statistically significant differences between the two cluster groups in terms of fustat status, grade, clinical stage, and T stage of HCC patients. The risk model comprised the overexpression of YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3, which contributed to the poor prognosis of HCC patients. The high-risk score was associated with prognosis, fustat status, grade, clinical stage, T stage, and M stage and was an independent risk factor for poor prognosis in HCC patients. High-risk score mechanisms included spliceosome, RNA degradation, and DNA replication, among others, and high-risk was closely related to stromal score, CD4 memory resting T cells, M0 macrophages, M1 macrophages, resting mast cells, CD4 memory activated T cells, and follicular helper T cells. In conclusion, the cluster subgroup and risk model of m6A/ m5C/m1A regulated genes were associated with the poor prognosis and immune microenvironment in HCC and are expected to be the new tools for assessing the prognosis of HCC patients.
# Introduction
Liver cancer is one of the most prevalent cancers worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancer [bib_ref] MTFR2Upregulated Expression of as a Novel Biomarker Predicts Poor Prognosis in Hepatocellular..., Li [/bib_ref]. Recent studies have demonstrated that targeted therapy and immunotherapy significantly affect HCC patient survival [bib_ref] First-Line Sorafenib Sequential Therapy and Liver Disease Etiology for Unresectable Hepatocellular Carcinoma..., Shimose [/bib_ref] [bib_ref] Nivolumab + Ipilimumab for Patients With Hepatocellular Carcinoma Previously Treated With Sorafenib, Tsang [/bib_ref] [bib_ref] Clinical Significance of Radiotherapy Before and/or During Nivolumab Treatment in Hepatocellular Carcinoma, Yu [/bib_ref]. For example, Shimose et al. reported that sorafenib (SORA) improves overall survival (OS) in HCC patients. SORA can improve the prognosis of patients with unresectable HCC as first-line therapy [bib_ref] First-Line Sorafenib Sequential Therapy and Liver Disease Etiology for Unresectable Hepatocellular Carcinoma..., Shimose [/bib_ref]. Nonetheless, the prognosis for HCC patients remains unsatisfactory. Therefore, it is extremely important to identify new targets or immunotherapies to improve the prognosis of HCC patients.
In eukaryotic messenger RNA (mRNA) regulation, N 6methyladenosine (m6A), N 1 -methyladenosine (m1A), and 5methylcytosine (m5C) modifications exist. Several studies have confirmed that m6A, m1A, and m5C regulated genes play important roles in m6A, m1A, and m5C modifications [bib_ref] The Prognostic Value and Immune Landscapes of a M6a/M5c/M1a-Related LncRNAs Signature in..., Wang [/bib_ref] [bib_ref] RNA Modifications and Epigenetics in Modulation of Lung Cancer and Pulmonary Diseases, Teng [/bib_ref] [bib_ref] Differential Analysis of RNA Methylation Regulators in Gastric Cancer Based on TCGA..., Li [/bib_ref] [bib_ref] METTL3 Regulates M6a in Endometrioid Epithelial Ovarian Cancer Independently of METTl14 and..., Ma [/bib_ref]. Recent research has found that m6A, m1A, and m5C regulated gene expression levels are associated with tumor progression [bib_ref] METTL3 Regulates M6a in Endometrioid Epithelial Ovarian Cancer Independently of METTl14 and..., Ma [/bib_ref] [bib_ref] RNA N6-Methyladenosine Reader IGF2BP3 Regulates Cell Cycle and Angiogenesis in Colon Cancer, Yang [/bib_ref] [bib_ref] piRNA-14633 Promotes Cervical Cancer Cell Malignancy in a METTL14-Dependent M6a RNA Methylation..., Xie [/bib_ref] [bib_ref] M1a Regulated Genes Modulate PI3K/AKT/mTOR and ErbB Pathways in Gastrointestinal Cancer, Zhao [/bib_ref] [bib_ref] NSUN2 Modified by SUMO-2/3 Promotes Gastric Cancer Progression and Regulates mRNA M5c..., Hu [/bib_ref] [bib_ref] FMRP Promotes Transcription-Coupled Homologous Recombination via Facilitating TET1-Mediated M5c RNA Modification Demethylation, Yang [/bib_ref]. For example, the m6A-regulated gene methyltransferase 3 (METTL3), METTL14, and WTAP are involved in the initiation of the m6A modification process. METTL3 expression is elevated in endometrioid epithelial ovarian cancer (EEOC) tissues. The METTL3 overexpression levels correlate with the degree of malignancy and the OS of EEOC patients. Inhibiting METTL3 expression in TOV-112D and CRL-11731D cells attenuates cancer cell proliferation and migration and promotes apoptosis. METTL3 overexpression promotes EEOC progression by regulating m6A methylation [bib_ref] METTL3 Regulates M6a in Endometrioid Epithelial Ovarian Cancer Independently of METTl14 and..., Ma [/bib_ref]. The m5C methyltransferase NSUN2 is significantly upregulated in gastric cancer and is predictive of a poor prognosis in gastric cancer patients. In vitro, NSUN2 promotes the proliferation, migration, and invasion of gastric cancer cells. Small ubiquitin-like modifier (SUMO)-2/3 promotes the oncogenic activity of NSUN2 by stabilizing NSUN2 [bib_ref] NSUN2 Modified by SUMO-2/3 Promotes Gastric Cancer Progression and Regulates mRNA M5c..., Hu [/bib_ref]. The risk model has been employed as a tool for determining the prognosis of cancer patients [bib_ref] Clinical Roles of Risk Model Based on Differentially Expressed Genes in Mesenchymal..., Guo [/bib_ref] [bib_ref] Construction and Validation of a Prognostic Risk Model for Triple-Negative Breast Cancer..., Yan [/bib_ref]. Currently, the roles of m6A, m1A, and m5C regulated genes in HCC progression are not yet fully understood. Therefore, in this study, data from The Cancer Genome Atlas (TCGA) database were used to elucidate the biological functions and network involved in m6A/m1A/m5C regulated genes [bib_ref] The Prognostic Value and Immune Landscapes of a M6a/M5c/M1a-Related LncRNAs Signature in..., Wang [/bib_ref] , and the roles of m6A/m1A/m5C regulated genes in the prognosis of HCC patients were identified. The risk model and nomogram of m6A/ m1A/m5C regulated genes were constructed to determine the prognosis of HCC patients.
# Materials and methods
The Expression Levels of m6A/m1A/m5C Regulated Genes Gene expression data of HCC patients were extracted from TCGA database. A total of 50 normal liver tissue samples and 374 HCC tissue samples were included. The Writer, Reader, and Eraser genes of m6A/m1A/m5C were obtained from the literature [bib_ref] The Prognostic Value and Immune Landscapes of a M6a/M5c/M1a-Related LncRNAs Signature in..., Wang [/bib_ref] , and the obtained m6A, m1A, and m5C regulated genes were merged and de-duplicated. Subsequently, the m6A/ m1A/m5C regulated gene expression data were retrieved in normal and HCC tissues, and the expression levels of m6A/ m1A/m5C regulated genes in HCC tissues were analyzed using the Limma package.
The Relationship Between m6A/m1A/m5C Regulated Genes
The network of 45 m6A/m1A/m5C regulated genes in the STRING database was explored, and Cytoscape (version: 3.8.2) software was used to show the relationship between the proteinprotein interaction (PPI) network of m6A/m1A/m5C regulated genes.
## Gene ontology and kyoto encyclopedia of genes and genomes analyses
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methods are frequently used to assess the biological functions and the signaling mechanisms of polygenes [bib_ref] Evaluation of the Prognostic Value of STEAP1 in Lung Adenocarcinoma and Insights..., Guo [/bib_ref]. In this study, GO and KEGG methods were used to investigate the biological functions and signaling mechanisms associated with the 45 m6A/m1A/m5C regulated genes. In addition, the biological functions and signaling mechanisms involved in the abnormally expressed genes related to the 45 m6A/m1A/m5C regulated genes were determined using GO and KEGG methods.
Identification of m6A/m1A/m5C Regulated Gene Subgroups Using Consensus Clustering
The "ConsensusClusterPlus" package was used to cluster 45 m6A/m1A/m5C regulated genes in 374 HCC tissues extracted from TCGA database into different subtypes. TCGA 374 HCC tissues were divided into cluster1 and cluster2 groups based on the optimal k value. Principal component analysis (PCA) was used to determine the differences between the two groups of patients. The Kaplan-Meier (K-M) survival analysis and logrank test were used to assess the relationship between survival and clinicopathological characteristics of HCC patients in the two cluster groups.
# Cluster-related differentially expressed
Genes Based on the m6A/m1A/m5C Regulated Genes Cluster1 and cluster2 groups of the m6A/m1A/m5C regulated gene subsets were matched with the tissue samples from 374 HCC patients, and the Limma package was used to determine the differentially expressed genes (DEGs) in cluster1 and cluster2 groups. The false discovery rate (FDR) <0.05 and log fold change (FC) = 2 were used to identify DEGs associated with m6A/m1A/ m5C regulated gene subsets.
## Identification of roles m6a/m1a/m5c regulated gene subgroups associated with differentially expressed genes
The relationship between the m6A/m1A/m5C regulated gene subgroups related to DEGs and the prognosis of HCC patients was using univariate Cox regression analysis (p < 0.001). Subsequently, the Limma package was used to investigate the expression levels of DEGs that were modulated by m6A/m1A/ m5C in subgroups of genes associated with prognosis in 50 normal liver tissue samples and 374 HCC tissue samples.
Cluster-Related Genes Based on the m6A/ m1A/m5C Regulated Gene Subgroups Using Consensus Clustering
The expression data of 37 prognosis related DEGs in 374 HCC tissues were extracted. The optimal k value was determined by consensus clustering analysis, and the 374 HCC tissues from TCGA were grouped. PCA was used to determine the differences between subgroups of HCC patients. The K-M survival analysis and log-rank test were used to determine the relationship between prognostic and clinicopathological factors in patient subgroups.
## Construction of a risk model and nomograms associated with m6a/m1a/ m5c regulated genes
The expression data of 45 m6A/m1A/m5C regulated genes were matched and merged with the prognosis data of HCC patients, and the relationship between m6A/m1A/m5C regulated genes and the prognosis of HCC patients was investigated using univariate Cox regression analysis (p < 0.05). The least absolute shrinkage and selection operator (LASSO) regression analysis was used to construct a risk model for m6A/m1A/m5C regulated genes, and the risk score for m6A/m1A/m5C regulated genes was calculated as follows: risk score = S (ExpmRNAn × bmRNAn). Correlation analysis explored the relationship between the risk score and the expression levels of model factors, including YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3. The K-M survival analysis was performed to determine the prognosis of HCC patients in the high-and low-risk groups, as well as the construction of nomogram related to m6A/m1A/m5C regulated genes.
## Identification of m6a/m1a/m5c regulated gene expression in hepatocellular carcinoma tissues
Cancer and normal liver tissues of 12 HCC patients diagnosed through pathology at our hospital were collected between February and April 2022. All 12 HCC patients signed the informed consent, which was reviewed and approved by the ethics committee of our hospital. With the use of a standard qRT-PCR protocol, the expression levels of YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 in 12 HCC tissues and normal liver tissues were determined [bib_ref] Construction of AP003469.4 -miRNAs-mRNAs ceRNA Network to Reveal Potential Biomarkers for Hepatocellular..., Fan [/bib_ref]. The primer numbers for TRMT10C and RRP8 that were purchased from GeneCopoeia (Guangzhou, China) were HQP013870 and HQP097040, respectively. [fig_ref] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes [/fig_ref] shows the primer sequences for the m6A/m1A/m5C regulated genes YBX1, ZC3H13, YTHDF1, YTHDF2, TRMT6, LRPPRC, and IGF2BP3.
## Clinical values of a risk model associated with m6a/m1a/m5c regulated genes
The relationship between the high-and low-risk groups and clinicopathological characteristics of HCC patients were assessed using the log-rank test. To understand the clinical value of the risk model, univariate and multivariate Cox regression analyses were performed to establish the relationship between age, gender, grade, clinical stage, T stage, M stage, N stage, and risk score value and OS of HCC patients.
## Signaling mechanisms of the risk model
Gene Set Enrichment Analysis (GSEA) is commonly used to investigate the signaling mechanisms of risk model [bib_ref] The Value of Erlotinib Related Target Molecules in Kidney Renal Cell Carcinoma..., Zhang [/bib_ref]. HCC patients were divided into the high-and low-risk groups based on the median risk score. With the use of GSEA (version 4.1.0), the effect of the risk model constructed by m6A/m1A/m5C regulated genes on each gene set was determined. GSEA was run for 1,000 cycles (Nominal p < 0.05).
## The relationship between the constructed model and immune
Infiltrating Cells Based on the m6A/m1A/ m5C Regulated Genes
The expression levels of immune cells in the 374 HCC tissues were calculated using the CIBERSORT method. The immune cells of 374 HCC patients were divided into two groups: cluster1 and cluster2 groups. A t-test was used to determine whether there was a difference in the expression levels of HCC immune cells in cluster1 and cluster2 groups. The high-and low-risk models constructed based on the m6A/m1A/m5C regulated genes were combined with the immune cell data of 374 HCC patients, and the expression levels of immune cells in the highand low-risk groups were explored. In addition, correlation analysis investigated the relationship between the risk score and immune cell infiltration in HCC.
The Relationship Between the Constructed Risk Model of m6A/m1A/m5C Regulated Genes and Immune Cell Markers
The expression levels of immune cell markers extracted from 374 HCC tissues in TCGA database were acquired. The risk scores were combined with the immune cell marker gene data of cancer patients, and the expression levels of immune cell markers in the high-and low-risk groups were explored using the t-test. In addition, correlation analysis explored the relationship between the risk scores and immune-infiltrating cell markers in HCC tissues.
# Statistical analysis
The expression levels of m6A/m1A/m5C regulated genes and m6A/m1A/m5C regulated gene subgroup-related genes in HCC tissues were identified using the Limma package. Cox, LASSO, and K-M survival analyses were used to identify m6A/m1A/m5C regulated genes associated with the prognosis of HCC patients. The relationship between models based on m6A/m1A/m5C regulated genes and immune cell infiltration was determined using correlation analysis (p < 0.05).
# Results
## Identification of expression levels of m6a/ m1a/m5c regulated genes in hepatocellular carcinoma tissues
Compared to normal liver tissues, abnormal expression levels of m6A/m1A/m5C regulated genes were observed in HCC tissues [fig_ref] TABLE 2 |: The expression levels of m6A/m1A/m5C regulated genes in HCC tissues [/fig_ref]. The heatmap depicts the expression levels of m6A/m1A/ m5C regulated genes in HCC tissues [fig_ref] FiguresFIGURE 1: 3A-C [/fig_ref]. In HCC tissues, 41 (91.11%) of the m6A/m1A/m5C regulated genes were statistically significant. Based on the fold change, the expression levels of 12 m6A/m1A/m5C regulated genes in HCC tissues are [fig_ref] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes [/fig_ref] AGTTGTTGTCCCTCGTGACC GTCCACTTTGCAGAGCCTTC shown [fig_ref] FiguresFIGURE 1: 3A-C [/fig_ref]. IGF2BP1, IGF2BP3, IGF2BP2, DNMT3B, DNMT3A, DNMT1, NSUN7, NSUN5, ALYREF, METTL3, TRMT6, and TRMT61A were overexpressed in HCC tissues relative to normal tissues.
The Functional Mechanisms and Roles of m6A/m1A/m5C Regulated Genes GO annotation revealed that the m6A/m1A/m5C regulated genes participate in RNA modification and methylation, macromolecule methylation, mRNA methylation and modification, RNA regulation and mRNA stability, ncRNA processing, regulation of mRNA catabolic process, RNA splicing, mRNA catabolic process, mRNA transport, and other processes [fig_ref] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes [/fig_ref]. KEGG analysis involved the microRNAs in cancer, spliceosome, and RNA transport. [fig_ref] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based... [/fig_ref] showed the PPI network between m6A/m1A/m5C regulated genes.
## Evaluation of clinical values of m6a/m1a/ m5c regulated gene subgroups in hepatocellular carcinoma patients using consensus clustering
Based on 45 m6A/m1A/m5C regulated gene expression data, and the optimal k value of 2, 374 HCC patients are divided into cluster1 and cluster2 groups [fig_ref] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based... [/fig_ref]. PCA revealed significant differences between cluster1 and cluster2 groups [fig_ref] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based... [/fig_ref]. The K-M survival analysis demonstrated that the OS of patients in cluster1 was significantly better than that of patients in cluster2 [fig_ref] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based... [/fig_ref]. In addition, there was a statistically significant difference between HCC patients in the two cluster groups in terms of clinical stage, T stage, tumor grade, and fustat status [fig_ref] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based... [/fig_ref].
The Functions and Signaling Pathways of Cluster-Related Differentially Expressed Genes Based on the m6A/m1A/m5C Regulated Genes
Compared to cluster1 group, cluster2 group HCC tissues displayed 371 cluster-related DEGs [fig_ref] TABLE 2 |: The expression levels of m6A/m1A/m5C regulated genes in HCC tissues [/fig_ref] , including 315 overexpressed genes and 56 downregulated genes. shows the top 20 cluster-related DEGs in HCC tissues. The m6A/ m1A/m5C regulated gene subsets related DEGs were found to be involved in signaling release, neurotransmitter transport, drug catabolic process, positive regulation of protein secretion, regulation of secretion, calcium ion regulated exocytosis, hormone transport, regulation of protein secretion, and others using GO analysis (
## Prognostic values and expression levels of cluster-related differentially expressed genes
Univariate Cox regression analysis revealed that 127 DEGs were associated with poor prognosis in HCC patients based on the p < 0.05 [fig_ref] TABLE 3 |: Association of the 37 cluster-related DEGs with the prognosis of HCC patients [/fig_ref] , and 37 DEGs were associated with poor prognosis in HCC patients based on the p < 0.001 [fig_ref] TABLE 3 |: Association of the 37 cluster-related DEGs with the prognosis of HCC patients [/fig_ref]. [fig_ref] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes [/fig_ref] , PLBD1, PRDM9, POU3F2, and FABP6 and [fig_ref] TABLE 3 |: Association of the 37 cluster-related DEGs with the prognosis of HCC patients [/fig_ref] The 37 cluster-related differentially expressed gene subgroups divided the 374 HCC patients into cluster1 and cluster2 groups [fig_ref] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1,... [/fig_ref]. PCA showed significant differences between cluster1 and cluster2 groups [fig_ref] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1,... [/fig_ref]. The K-M survival analysis revealed that the OS of HCC patients in cluster1 group was significantly better than that of the patients in cluster2 group [fig_ref] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1,... [/fig_ref]. There are statistically significant differences between HCC patients in cluster1 and cluster2 groups in terms of clinical stage, T stage, and fustat status [fig_ref] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1,... [/fig_ref]. [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref]. The minimum l value was determined using the LASSO algorithm, and the risk model feature was constructed based on the 9 m6A/m1A/m5C regulated genes [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref]. [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref] shows the relationship between the YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 expression levels in the high-and low-risk groups. Furthermore, significant correlations between the YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 expression levels and the risk score were reported [fig_ref] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1,... [/fig_ref]. [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref] shows the relationship between the risk score and survival time of HCC patients. In the risk model based on expression levels of YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3, the K-M survival analysis revealed a poor prognosis in high-risk HCC patients [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref]. Furthermore, the HCC tissues from our hospital showed high expression levels of YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 . The gene expression trends in TCGA database were consistent with the results of gene expression in our hospital tissues. Therefore, a nomogram incorporating the m6A/m1A/m5C regulated genes YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 was constructed [fig_ref] FIGURE 7 |: Prognostic nomogram based on the 9 m6A/m5C/m1A-regulated genes in HCC patients [/fig_ref].
## Construction
## Clinical values of the risk model
Associated With the m6A/m1A/m5C Regulated Genes
The risk model was related to the tumor grade, clinical stage, T stage, distant metastasis, and fustat status of HCC patients, according to an analysis of clinicopathological characteristics of HCC patients in the high-and low-risk groups . Univariate Cox regression analysis revealed that the clinical stage, T stage, and risk score were risk factors for the dismal prognosis in HCC patients . Multivariate Cox regression analysis revealed that distant metastasis and risk score were risk factors for poor prognosis in HCC patients .
## Signaling mechanisms associated with a high-risk score
A high-risk score is associated with spliceosome regulation, cell cycle, base excision repair, RNA degradation, oocyte meiosis, ubiquitin-mediated proteolysis, pyrimidine metabolism, DNA [fig_ref] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis... [/fig_ref] and [fig_ref] TABLE 5 |: Signaling mechanisms of high-risk score [/fig_ref].
## Immune cell infiltration in hepatocellular carcinoma correlates with the cluster groups and risk model of m6a/m1a/m5c regulated genes
There were significant differences in the expression levels of B cells naive, B cells memory, CD4 memory resting T cells, CD4 memory activated T cells, M0 macrophages, M1 macrophages, resting mast cells, and eosinophils among the 45 m6A/m1A/ m5C regulated gene subgroups . The expression levels of B cells naive, B cells memory, T cells CD8, CD4 memory resting T cells, M0 macrophages, and eosinophils were significantly different between the 37 cluster-related differentially expressed gene subgroups . There were significant differences in the expression levels of the stromal score, CD4 memory resting T cells, follicular helper T cells, activated NK cells, monocytes, M0 macrophages, M1 macrophages, resting mast cells, eosinophils, and neutrophils in high-and low-risk groups . The risk score was significantly correlated with the levels of the stromal score, CD4 memory resting T cells, CD4 memory activated T cells, follicular helper T cells, M0 macrophages, M1 macrophages, and resting mast cells and .
# Discussion
Numerous studies indicate that the expression levels of many genes change during HCC progression [bib_ref] Upregulation of Ubiquitin-Conjugating Enzyme E2T (UBE2T) Predicts Poor Prognosis and Promotes Hepatocellular..., Ren [/bib_ref] [bib_ref] Downregulation of MAGE Family Member H1 Enhances Hepatocellular Carcinoma Progression and Serves..., Wang [/bib_ref] [bib_ref] Elevated SHOX2 Expression is Associated With Tumor Recurrence of Hepatocellular Carcinoma, Yang [/bib_ref]. Wang et al. for example discovered that the expression level of MAGEH1 is significantly downregulated in HCC tissues, which is associated with poor prognosis in HCC patients. MAGEH1 can inhibit proliferation, migration, and invasion and delay HCC progression [bib_ref] Downregulation of MAGE Family Member H1 Enhances Hepatocellular Carcinoma Progression and Serves..., Wang [/bib_ref]. Yang et al. reported that elevated SHOX2 expression is associated with tumor recurrence and TNM stage in HCC patients. Increased SHOX2 expression is observed in HCC cells. SHOX2 expression can be suppressed to inhibit HCC cell proliferation and invasion [bib_ref] Elevated SHOX2 Expression is Associated With Tumor Recurrence of Hepatocellular Carcinoma, Yang [/bib_ref]. Current research demonstrates that RNA modification is associated with the progression of malignant tumors . For example, the level of IGF2BP3 overexpression correlates with cancer progression and survival. IGF2BP3 knockdown inhibits DNA replication in the S phase of the cell cycle, cell proliferation, and angiogenesis by reading the m6A modification of CCND1 and VEGF [bib_ref] RNA N6-Methyladenosine Reader IGF2BP3 Regulates Cell Cycle and Angiogenesis in Colon Cancer, Yang [/bib_ref]. WTAP is highly expressed and is a risk factor for poor prognosis in HCC patients. WTAP can promote cell proliferation and tumor growth in HCC patients. ETS1 is a downstream effector of WTAP. WTAP can induce post-transcriptional repression of ETS1 by regulating m6A modification, which in turn mediates the p21/p27-dependent mechanism involved in the G2/M phase regulation of HCC cells [bib_ref] WTAP Facilitates Progression of Hepatocellular Carcinoma via M6a-HuR-Dependent Epigenetic Silencing of ETS1, Chen [/bib_ref]. The expression level of METTL3 is significantly elevated in HCC tissues and cells. Elevated METTL3 expression is associated with poor OS. When METTL3 expression is inhibited, the ability of HCC cells to invade, migrate, and proliferate is significantly decreased. Mechanistically, METTL3 may regulate the expression level of USP7 via m6A methylation modification, thereby promoting the growth and migration of HCC cells [bib_ref] METTL3 Facilitates the Progression of Hepatocellular Carcinoma by Modulating the M6a Level..., Li [/bib_ref]. This suggests that m6A/ m1A/m5C regulated genes play important roles in HCC progression. In this study, the roles of 45 m6A/m1A/m5C regulated genes were explored in HCC progression, and significant differences in OS, clinical stage, T stage, tumor grade, and fustat status between subgroups of m6A/m1A/m5C regulated genes were observed. The identification of the expression of the risk model genes YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 in HCC tissues was consistent with that in TCGA database. In addition, a high-risk score constructed using YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 was associated with poor prognosis, tumor grade, clinical stage, T stage, distant metastasis, and fustat status and is an independent risk factor for poor prognosis in HCC patients. Preliminary evidence suggests that m6A/m1A/m5C regulated genes play important biological roles in the progression of HCC, which can be used to determine the prognosis and survival of HCC patients. The signaling pathways, including the cell cycle, DNA replication, and WNT are closely associated with cancer progression [bib_ref] ASF1B Serves as a Potential Therapeutic Target by Influencing Cell Cycle and..., Ouyang [/bib_ref] [bib_ref] PAQR3 Inhibits Non-Small Cell Lung Cancer Growth by Regulating the NF-kb/ P53/Bax..., Guo [/bib_ref] [bib_ref] LncRNA SNHG5 Promotes the Proliferation and Cancer Stem Cell-Like Properties of HCC..., Li [/bib_ref] [bib_ref] Targeting CDC7 Potentiates ATR-CHK1 Signaling Inhibition Through Induction of DNA Replication Stress..., Guo [/bib_ref] [bib_ref] CCNB1 Promotes the Development of Hepatocellular Carcinoma by Mediating DNA Replication in..., Rong [/bib_ref] [bib_ref] KIF14 and KIF23 Promote Cell Proliferation and Chemoresistance in HCC Cells, and..., Cheng [/bib_ref]. Previous studies have found that ASF1B is highly expressed in HCC, and elevated ASF1B expression level is associated with poor prognosis in HCC patients. GSEA revealed that ASF1B may regulate the cell cycle, DNA replication, and oocyte meiotic signaling pathway. ASF1B silencing inhibits the growth and cell cycle arrest, induces apoptosis, and reduces the expression levels of PCNA, cyclinB1, cyclinE2, and CDK9 in HCC cells [bib_ref] ASF1B Serves as a Potential Therapeutic Target by Influencing Cell Cycle and..., Ouyang [/bib_ref]. The level of TCF3 expression is significantly increased in HCC tissues. TCF3 expression level correlates with clinical stage, tumor size, TNM stage, grade, OS, disease-specific survival, and progression-free survival (PFS) in HCC patients. TCF3 knockdown inhibits cancer cell proliferation and cell cycle, which is associated with dysregulation of the WNT signaling mechanism (31). m6A/m1A/m5C regulated genes are involved in multiple functions, including RNA modification and methylation, mRNA methylation and modification, regulation of RNA, and mRNA stability. The cell cycle, base excision repair, RNA degradation, oocyte meiosis, DNA replication, homologous recombination, basal transcription factors, pathways in cancer, WNT signaling pathway, and long-term potentiation processes are involved in the construction of a high-risk model for m6A/ m1A/m5C regulated genes. Previous studies indicate that m6A/ m1A/m5C regulated genes are closely associated with HCC progression. However, the mechanism of m6A/m1A/m5C regulated genes in HCC progression remains to be confirmed in future studies. Currently, targeted therapy and immunotherapy are among the treatment options available to HCC patients [bib_ref] Sorafenib Induces Mitochondrial Dysfunction and Exhibits Synergistic Effect With Cysteine Depletion by..., Li [/bib_ref] [bib_ref] PD-1/PD-L1 Immuno-Mediated Therapy in NAFLD: Advantages and Obstacles in the Treatment of..., Lombardi [/bib_ref] [bib_ref] Programmed Cell Death Protein 1 (PD-1)-Inhibition in Hepatocellular Carcinoma (HCC): A Single..., Mahn [/bib_ref]. For example, PD-1 inhibitors are well tolerated by HCC patients. HCC patients have achieved good clinical outcomes. The median OS for PD-1 inhibitor-treated HCC patients is 6.6 months, the median PFS is 5.3 months, and the overall response rate is 30.8%. A patient achieves complete remission [bib_ref] Programmed Cell Death Protein 1 (PD-1)-Inhibition in Hepatocellular Carcinoma (HCC): A Single..., Mahn [/bib_ref]. The relationship between immunotherapy and the immune microenvironment is well established [bib_ref] Immunobiology and Immunotherapy of HCC: Spotlight on Innate and Innate-Like Immune Cells, Ruf [/bib_ref] [bib_ref] M6a Regulator-Associated Modification Patterns and Immune Infiltration of the Tumor Microenvironment in..., Li [/bib_ref]. In this study, the expression levels of CD4 memory resting T cells, follicular helper T cells, activated NK cells, monocytes, M0 macrophages, M1 macrophages, resting mast cells, eosinophils, and neutrophils were found to be significantly different in the risk model constructed by m6A/ m1A/m5C regulated genes YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3. The risk score correlated with levels of the stromal score, CD4 memory resting T cells, CD4 memory activated T cells, follicular helper T cells, M0 macrophages, M1 macrophages, and resting mast cells. In addition, the risks core correlated with the HCC immune cell [fig_ref] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes [/fig_ref] , GZMB, MS4A4A, GATA3, IFNG, and HLA-DQB1. This suggests that the risk model constructed using m6A/m1A/m5C regulated genes is associated with HCC immune cell infiltration.
This study identified the involvement of m6A/m5C/m1A regulated genes and constructed a risk model for HCC using HCC data from TCGA database and our hospital data. In the prognosis in HCC patients. The risk score is associated with HCC stromal score and levels of CD4 memory resting T cells, M0 macrophages, M1 macrophages, resting mast cells, CD4 memory activated T cells, and follicular helper T cells.
# Data availability statement
The data used in database are available from the TCGA website and the data of qRT-PCR by contacting the corresponding authors.
# Ethics statement
The studies involving human participants were reviewed and approved by the ethics committee of Affiliated Hospital of Zunyi Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
# Author contributions
DK, R-SS, and DL designed the study and supervised the manuscript writing process. DL, KL, WZ, and K-WY analyzed the data and wrote the manuscript. DL, D-AM, and G-JJ performed data visualization and edited the language of the manuscript. All authors approve the submission and publication of the manuscript.
[fig] FiguresFIGURE 1: 3A-C) and involved in bile secretion, | m6A/m1A/m5C regulated DEGs in HCC tissues as determined based on fold change. (A) IGF2BP1; (B) IGF2BP3; (C) IGF2BP2; (D) DNMT3B; (E) DNMT3A; (F) NSUN7; (G) DNMT1; (H) NSUN5; (I) METTL3; (J) ALYREF; (K) TRMT61A; (L) TRMT6. DEGs, differentially expressed genes; HCC, hepatocellular carcinoma; ***P < 0.001. [/fig]
[fig] FIGURE 2 |: The clinical values in m6A/m1A/m5C regulated gene subgroups in HCC patients based on consensus clustering. (A-C) The consensus clustering of m6A/m1A/m5C regulated genes. (D) PCA. (E) K-M survival analysis. (F) The relationships between the clinicopathological features and m6A/m1A/m5C regulated gene subgroups. HCC, hepatocellular carcinoma; PCA, principal component analysis; K-M, Kaplan-Meier; *P < 0.05; **P < 0.01. [/fig]
[fig] FIGURE 3 |< 0: The expression levels and roles of cluster-related DEGs. (A-C) The biological functions of cluster-related DEGs. (D) The prognostic value of clusterrelated genes as determined using the Cox analysis. (E, F) The expression of cluster-related genes. BP, biological process; CC, cellular component; MF, molecular function; DEGs, differentially expressed genes; *P < 0.05; **P < 0.01; ***P [/fig]
[fig] FIGURE 4 |: Correlation analysis revealed that immune cell markers IL10, ITGAM, STAT5B, CD68, HLA-DPB1, KIR2DL4, IRF5, CSF1R, CD274, HLA-DRA, CD8B, STAT1, NOS2, ITGAX, CD86, CD8A, BCL6, TGFB1, CD163, CCR8, TBX21, CCL2, CD3E, TNF, CD1C, CD2, HAVCR2, NRP1, STAT5A, CD3D, LAG3, HLA-DPA1, PDCD1, VSIG4, STAT3, GZMB, MS4A4A, The prognostic value of 37 cluster-related DEG subgroups in HCC based on consensus clustering. (A-C) The consensus clustering of 37 clusterrelated DEGs. (D) PCA. (E) K-M survival analysis. (F) The relationships between the clinicopathological features and 37 cluster-related DEG subgroups. HCC, hepatocellular carcinoma; PCA, principal component analysis; K-M, Kaplan-Meier; differentially expressed genes; **P < 0.01; ***P < 0.001. [/fig]
[fig] FIGURE 5 |: The risk model constructed using the LASSO method for predicting the prognosis of patients. (A) Cox method showing the prognostic value of m6A/ m1A/m5C regulated genes. (B, C) The 9 m6A/m5C/m1A-regulated genes identified using least absolute shrinkage and LASSO method. (D-F) The association of high-risk score with the m6A/m1A/m5C regulated genes and prognosis of HCC patients. HCC, hepatocellular carcinoma; LASSO, least absolute shrinkage and selection operator; ***P < 0.001. IFNG, and HLA-DQB1 expression levels were associated with the risk score level in HCC (Figures 10 and S7), and the expression levels of IRF5, ITGAM, CD86, NRP1, CTLA4, STAT5A, HAVCR2, CSF1R, STAT1, CD19, ITGAX, CD68, TGFB1, CD3D, PDCD1, HLA-DPB1, HLA-DRA, TNF, CCR8, IFNG, VSIG4, BCL6, IL10, HLA-DPA1, STAT5B, MS4A4A, STAT6, LAG3, HLA-DQB1, KIR2DL4, STAT3, and IL21 differed significantly between the high-and low-risk groups (Figure 11). [/fig]
[fig] FIGURE 6 |FIGURE 8 |: The expression profile of risk model genes in HCC tissues. (A) YTHDF1; (B) YBX1; (C) ZC3H13; (D) TRMT10C; (E) LRPPRC; (F) YTHDF2; (G) RRP8; (H) IGF2BP3; (I) TRMT6. HCC, hepatocellular carcinoma; The correlation between risk model and the prognosis and clinicopathological characteristics of HCC patients. (A) The relationships between the clinicopathological features and risk model. (B, C) Univariate and multivariate Cox regression analyses for the prognostic factors of HCC patients. HCC, hepatocellular carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001. [/fig]
[fig] FIGURE 7 |: Prognostic nomogram based on the 9 m6A/m5C/m1A-regulated genes in HCC patients. HCC, hepatocellular carcinoma. [/fig]
[fig] FIGURE 9 |FIGURE 10 |: The correlation between the cluster groups and risk model and immune cell infiltration in HCC. (A) The expression levels of immune cells in m6A/m1A/ m5C regulated gene subgroups. (B) The expression levels of immune cells in 37 cluster-related DEG subgroups. (C) The expression levels of immune cells in highand low-risk groups. (D-I) Correlation between the risk model and immune cells. HCC, hepatocellular carcinoma; differentially expressed genes; ns, not statistically significant; future, however, this must be confirmed using additional clinical HCC tissue samples and cell experiments. m6A/m5C/m1A regulated genes are generally involved in the occurrence and development of HCC. m6A/m5C/m1A regulated genes YBX1, ZC3H13, YTHDF1, TRMT10C, YTHDF2, RRP8, TRMT6, LRPPRC, and IGF2BP3 are the influencing factors of poor prognosis in HCC patients. A high-risk score is associated with patient prognosis and is an independent risk factor for poor The correlation between the risk model and HCC immune-infiltrating cell markers. (A) ITGAM; (B) IL10; (C) HLA-DPB1; (D) PDCD1; (E) CD274; (F) IRF5; (G); CSF1R; (H) CD86; (I) TGFB1; (J) CD163; (K) HAVCR2; (L) NRP1; (M) STAT5A; (N) CTLA4; (O) CD3D; (P) LAG3; (Q) HLA-DPA1; (R) VSIG4; (S) STAT3; (T) HLA-DQB1; (U) GZMB; (V) MS4A4A; (W) GATA3; (X) IFNG. HCC, hepatocellular carcinoma. [/fig]
[fig] FUNDINGFIGURE 11 |: Our research was funded by the Zunyi City Joint Fund (Zun Shi Ke He HZ Word (2021) No. 73).SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.918140/ full#supplementary-material The expression levels of immune-infiltrating cell markers in high-and low-risk groups. (A, B) A heatmap showing the expression pattern of immune genes in HCC tissues. (C-J) Violin showing the expression of IL21, CD19, IFNG, ITGAM, CCR8, CD3D, CTLA4, and PDCD1 in HCC tissues. HCC, hepatocellular carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001. [/fig]
[table] TABLE 1 |: The primer sequences of m6A/m1A/m5C regulated genes. [/table]
[table] TABLE 2 |: The expression levels of m6A/m1A/m5C regulated genes in HCC tissues. [/table]
[table] TABLE 3 |: Association of the 37 cluster-related DEGs with the prognosis of HCC patients. [/table]
[table] TABLE 4 |: The effect of m6A/m1A/m5C regulated genes on the prognosis of HCC patients determined using univariate Cox regression analysis. [/table]
[table] TABLE 5 |: Signaling mechanisms of high-risk score. NES, normalized enrichment score; NOM, nominal. Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 918140 [/table]
|
PDCD6 is an independent predictor of progression free survival in epithelial ovarian cancer
Background: Programmed cell death 6 (PDCD6) beside its known proapoptotic functions may be a player in survival pathways in cancer. The purpose of this study is to further explore the roles of PDCD6 in epithelial ovarian cancer.Methods: Lentiviral vector with shRNA for PDCD6 was used to investigate the effects of PDCD6 knockdown on cell growth, cell cycle, apoptosis and motility in ovarian cancer cells. Two hundred twelve epithelial ovarian cancer tissues were analyzed for mRNA expression of PDCD6 using RT-PCR. Associations of its expression with clinical pathological factors, progression free and overall survival were evaluated.Results: PDCD6 is highly expressed in metastatic ovarian cancer cells and positively regulates cell migration and invasion. Significantly, the level of PDCD6 expression in epithelial ovarian cancer correlates with clinical progression. Patients with medium or high levels of PDCD6 mRNA were at higher risk for disease progression, compared to those with low levels (HR, 1.29; P = 0.024 for medium levels; and HR, 1.57; P = 0.045 for high levels) after adjusting for age, disease stage, tumor grade, histologic type and residual tumor size. Kaplan-Meier survival analysis demonstrated similar results. However, no association was found between PDCD6 expression and overall survival.Conclusions: PDCD6 seems to play an important role in ovarian cancer progression and it may be an independent predictor of progression free survival in epithelial ovarian cancer. Further studies are needed to more completely elucidate the molecular mechanisms of PDCD6 involve in ovarian cancer progression.
# Background
Ovarian cancer is a common gynecologic malignancy and a major cause of cancer death among women in the United States. The 5-year survival rate for patients with ovarian cancer is only 35% [bib_ref] Carboplatin plus paclitaxel as first-line chemotherapy in previously untreated advanced ovarian cancer...., Bois [/bib_ref] [bib_ref] Paclitaxel plus carboplatin versus paclitaxel plus alternating carboplatin and cisplatin for initial..., Aravantinos [/bib_ref]. The high mortality of ovarian cancer is related to our inability to detect the disease early and to treat it effectively. Most of ovarian cancer patients ultimately die from tumor recurrence and metastasis, despite the fact that they initially respond to the treatment of cytoreductive surgery followed by paclitaxel and platinum-based chemotherapy.
So, understanding the molecular mechanisms involving the initiation, progression, and metastasis of ovarian cancer is important for the prevention, detection, and treatment of ovarian cancer.
In our previous study, two epithelia ovarian cancer cell lines with low and high metastatic potentials were set up successively, named as HO-8910, and HO-8910 PM respectively [bib_ref] Establishment and Characterization of a Model of Highly Metastasizing Human Ovarian Cancer..., Shenhua [/bib_ref] [bib_ref] Establishment of a highly metastatic human ovarian cancer cell line (HO-8910 PM)..., Shenhua [/bib_ref]. Seventeen different significantly expressed proteins between them were detected by comparative proteomic analysis, of which programmed cell death 6 (PDCD6), also named as apoptosis-linked gene 2 (ALG-2), was identified [bib_ref] Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer..., Su [/bib_ref].
PDCD6, a calcium-binding protein belonging to the penta-EF-hand protein family, participates in T cell receptor-, Fas-, and glucocorticoid-induced programmed cell death [bib_ref] Apoptosis-Linked Gene 2 Binds to the Death Domain of Fas and Dissociates..., Jung [/bib_ref] [bib_ref] The Penta-EF-Hand Protein ALG-2 Interacts with a Region Containing PxY Repeats in..., Shibata [/bib_ref]. However, apoptosis was not blocked in mice deficient for PDCD6, suggesting PDCD6 is functionally redundant [bib_ref] Apoptosis-Linked Gene 2-Deficient Mice Exhibit Normal T-Cell Development and Function, Jang [/bib_ref]. Furthermore, PDCD6 was found up-regulated in lung cancer and hepatoma tissue compared with normal tissue, also suggesting that PDCD6 beside its known proapoptotic functions may be a player in survival pathways [bib_ref] Up-Regulation of ALG-2 in Hepatomas and Lung Cancer Tissue, La Cour [/bib_ref]. Similarly, our previous results showed PDCD6 was over-expressed in HO-8910 PM which has higher metastatic potential, as compared with HO-8910 [bib_ref] Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer..., Su [/bib_ref]. To further explore the function of PDCD6 in ovarian cancer, in this study, we investigated the effects of PDCD6 knockdown by short hairpin RNA (shRNA) on cell growth, cell cycle, apoptosis and motility of ovarian cancer cells. In addition, we analyzed the association between PDCD6 expression, clinical pathological factors and outcome in ovarian cancer patients.
# Materials and methods
Cell culture HO-8910 and HO-8910 PM cells were established by Zhejiang Cancer Hospital [bib_ref] Establishment and Characterization of a Model of Highly Metastasizing Human Ovarian Cancer..., Shenhua [/bib_ref] [bib_ref] Establishment of a highly metastatic human ovarian cancer cell line (HO-8910 PM)..., Shenhua [/bib_ref]. The cells were cultured in RPMI-1640 medium containing 10% newborn bovine serum, supplemented with 100 U/ml penicillin and 125 μg/ml streptomycin, and were incubated at 37°C with 5% CO 2 .
# Western blot analysis
The cells were harvested with 0.02% EDTA and 0.025% trypsin, rinsed three times in phosphate-buffered saline (PBS), and pelleted in 1.5 ml microcentrifugetubes by centrifugation. Whole-cell extracts were prepared with the Nuclear Extract Kit (Active Motif, Carlsbad, California, USA). The protein content of the cell lysate was determined by using the Bradford calorimetric assay method (Bio-Rad, Richmond, California, USA). Fifty μg of cell lysates were directly resolved by 12% SDS-PAGE and were transferred to PVDF membranes (Millipore, MA, USA). Proteins transferred to the membrane were detected by GAPDH and PDCD6 (Abcam Inc, MA, USA) primary antibodies and horseradish peroxidaselinked secondary antibodies (anti-rabbit IgG), and the proteins were then visualized by enhanced chemiluminescence reagents. BioRad Laboratories Quantity One software (BioRad, CA, USA) was used to quantify the blots, GAPDH as a loading control.
Quantitative real-time polymerase chain reaction (qRT-PCR)
RNeasy Mini Kits (QIAGEN Inc., Valencia, CA) were used to extract total RNA from cells and tissue. RNA (5 ng) was reverse transcribed to cDNA using a Super-Script First-Strand Synthesis System for RT-PCR (Invitrogen Corp., Carlsbad, CA). Quantitative real-time PCR was performed to determine the expression level of PDCD6 mRNA in each tumor sample, using GAPDH as an endogenous control for calibration. The primer sequences were designed online (http://www.idtdna. com) and ordered from Invitrogen Corp. (Shanghai, China). The primer sequences were: TGA CCA GTT CCA CGA CAT CCT CAT (PDCD6 forward), TTG GCT CTT TCC ATG TTG TGC TGC (PDCD6 reverse), GAA GGT GAA GGT CGG AGT C (GAPDH forward), and GAA GAT GGT GAT GGG ATT TC (GAPDH reverse). Quantitative real time PCR was carried out using an ABI 7500 real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA). The PCR reaction solution (25 μl) contained 5 ng/μl cDNA, 12.5 μl Power SYBR Green PCR Master Mix (Bioer Technology Inc., Hangzhou, China) and a pair of primers at a final concentration of 5 μM for PDCD6 and GAPDH. Dissociation curve analysis was performed after PCR to confirm the size of PCR products. All tumor samples were analyzed in duplicate along with negative controls. Real-time PCR results were recorded as Ct values (threshold cycle). To adjust for the total amount of cDNA used for analysis in each sample, a ΔCt was calculated based on the difference in Ct values between the target gene PDCD6 and the housekeeping gene GAPDH. ΔCt was further converted to an expression index (EI) based on the formula 2 (-ΔCt) . Inhibition rate of PDCD6 was calculated by formula [1-2 (-ΔΔCt) ]*100%.
## Transfection of lentiviral vectors with shrna for pdcd6
The pGCSIL-GFP-shRNA-PDCD6 lentiviral vectors (pGCSIL, a lentiviral vector) were purchased from Shanghai GeneChem Company and the target shRNA sequences as follows: 5'-cagagggtcgataaagaca-3'. pGCSIL-GFP-lentiviral vector was used as a control. The lentiviral vectors and pHelper were co-transfected into 293 T cells. The culture supernatants were collected, concentrated, and used as a virus stock. HO-8910 PM at 40%-50% confluence was inflected with lentivirus expression shRNA to the human PDCD6 gene or vector control. The shRNA targeting sequences was validated for the most efficient interference of PDCD6 by qRT-PCR and Immunoblotting.
## Cell proliferation assay
To assess cell proliferation effected by PDCD6, HO-8910 PM cells was infected with shRNA-PDCD6 for 72 h. Then the cells were plated at 5 × 10 3 cells/well in 96well microtiter plates and cultured at 37°C with 5% CO 2 . Each 24 h, the cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagent (Sigma, St Louis, MO, USA) according to the manufacturer's instructions. Briefly, add reconstituted MTT in an amount equal to 10% of the culture medium volume. Return to incubator for 2 h. After that, medium was moved, and MTT formazan was resolved in 100 ul acidic isopropanol. Absorbance was measured at a wavelength of 570 nm. Experiments were performed in triplicate, and 3 different experiments were performed for each experimental condition. A cell proliferation curve was constructed by measuring cell growth for 4 consecutive days.
## Cell cycle assay
Cell cycle distribution was monitored by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) using propidium iodine (PI) staining (Becton Dickinson) according to the protocol of the manufacturer. Briefly, the H0-8910 PM cells which were preinfected with shRNA-PDCD6 for 72 h were plated in 6well plates. After cultured for 48 h, cells were collected, washed with PBS, and fixed with 2% paraformaldehyde at room temperature for 30 min. After paraformaldehyde was moved, cell pellets were resuspended by 70% ethanol and stored at -20°C for 2 h. Then spin cells out of fix, pellets were resuspend in PBS with 200 ug/ml RnaseA and incubated at 37°C for 1 h. Added PI to a final concentration of 50 ug/ml for 2 h before analyzing on the flow cytometer.
Software-based cell cycle analysis was performed using ModFit 2.0 (Verity, Topsham, ME).
## Hoechst stain for detecting apoptotic cells
Apoptotic cells were detected by fluorescence imaging using Hoechst 33258 stain (Sigma-Aldrich, St. Louis, MO, USA). Seventy-two hours after transfection, cells were washed twice with PBS, and then incubated with Hoechst reagent (10 mg/ml) for 30-45 min at 37°C in the dark. Morphological changes including a reduction in volume and nuclear chromatin condensation were observed under a fluorescence microscope (Nikon, Tokyo, Japan) and photographed at a magnification of 200×. Apoptotic cells were counted in at least three to four random fields and expressed as percentage of total cells.
## Transwell migration and invasion assay
For transwell migration assays, cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). For invasion assays, cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). In both assays, a number of 2 × 10 5 HO-8910 PM cells treatment in 100 μL serum-free medium were pre-infected with shRNA-PDCD6 for 72 h and added to the upper chamber, while 500 μL medium with 10% FBS were added to the lower chamber. Transwells were incubated for 24 h at 37°C. Cells on the inside of the transwell inserts were removed with a cotton swab, and cells on the underside of the insert filter were fixed and stained. Photographs of four random fields were taken, and the cells were counted to calculate the average number of cells that had transmigrated.
## Patients and follow-up
Patient samples of ovarian cancer were consecutively collected in the Gynecologic Oncology Unit at University of Turin in Italy between October 1991 and February 2000 and all participants gave informed consent. Use of tissue samples was approved with an Ethical Review Committee of University. All ovarian cancer tissues were obtained at the time of cytoreductive surgery. In this study, we identified 212 patients with primary epithelial ovarian cancer who underwent cytoreductive surgery. The average patient age at surgery was 57.6 years (SD, 11.5; range, 26-82). Follow-up information was available for 203 patients who were followed from surgery to June 2001. The overall follow-up time ranged from 0.6 to 114 months, and the median was 31 months. The median disease progression-free survival was 20.6 months. Disease staging was classified according to the criteria of FIGO (the International Federation of Gynecologists and Obstetricians) [bib_ref] Revised FIGO staging for gynaecological cancer, Shepherd [/bib_ref]. Of the 211 patients with information on disease stage, 51 (24.2%) were diagnosed with stage I disease, 12 (5.7%) were stage II, 133 (63.0%) were stage III, and 15 (7.1%) were stage IV. Histological type determined following the World Health Organization (WHO) criteria [bib_ref] Histologic typing of ovarian tumors, Scully [/bib_ref] included serous, endometrioid, mucinous, clear cell, and other epithelial tumors. For data analysis, tumor histotypes were grouped into non-serous (59.9%, n = 127) and serous (40.1%, n = 85). Of the 211 patients with information on tumor grade, most patients (65.4%, n = 138) had grade 3 tumors (poorly differentiated); few had grade 1 or 2 (34.6%, n = 73). After surgery, most of the patients were treated with platinum-based chemotherapy following a standard protocol.
# Statistical analysis
The data from in vitro experiments were expressed as the mean ± SD for 3 independent experiments using three difference preparations. The difference between the means was assessed by Student's t-test. For patient study, PDCD6 mRNA expression was analyzed as a continuous variable and as a categorical variable after grouping by expression index (EI) distribution as low expression (EI < 0.1), middle expression (EI 0.1-1) and high expression (EI > 1). Associations between PCDC6 expression and clinical variables were analyzed using the Chi-square test. Survival analysis was performed for progression-free and overall survival using the Cox proportional hazards regression and Kaplan-Meier curves. All p-values were two-sided, and a probability of 0.05 or smaller was considered statistical significance. SPSS version 11.0 (SPSS Inc., Chicago, IL) was used for data analysis.
# Results
Expression of PDCD6 in ovarian cancer cell lines with different metastatic potential
To confirm our previous proteomic results [bib_ref] Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer..., Su [/bib_ref] , we compared the expression of PDCD6 in H0-8910 and H0-8910 PM cells by western blot. The results showed that PDCD6 expression was higher in H0-8910 PM than that in H0-8910 [fig_ref] Figure 1: A [/fig_ref].
## Knockdown of pdcd6 in ho-8910 pm cells by lentiviral vectors-mediated transfection
In the present study, lentiviral vectors-mediated transfection was used to determine effects of PDCD6 expression silencing on metastatic ovarian cancer cell behavior. Seventy two hours after transfection, HO-8910 PM cells were observed using a fluorescent microscope. The transfection efficiency was evaluated by counting the green fluorescent cells, which was over 95% when the cells were infected with a multiplicity of infection (MOI) of 3. mRNA and protein levels of PDCD6 were detected by RT-PCR and Western blot at 72 h after transfection. The results showed pGCSIL-GFP-shRNA-PDCD6 lentiviral vector was effective in silencing PDCD6 expression and the inhibition rate of PDCD6 was about 80% [fig_ref] Figure 1: A [/fig_ref].
## Influence of ho-8910 pm cell proliferation, apoptosis and cell cycle by pdcd6 expression silencing in vitro
As shown in [fig_ref] Figure 2: A [/fig_ref] , PDCD6 knockdown had no obvious effect on cell proliferation and apoptosis although apoptotic cells were increased slightly and cell growth slowed down a little bit in HO-8910 PM cells infected with shRNA-PDCD6. No significant difference of cell cycle distribution was found.
## Inhibition of ho-8910 pm migration and invasion in vitro by sirna-mediated pdcd6 knockdown
Migration and invasion assay in vitro were carried out to evaluate PDCD6 knockdown on HO-8910 PM cell motility. The result showed that the average number of migrated and invaded HO-8910 PM cells transfected with siRNA significantly decreased in comparison with the Mock group, p < 0.001 and P = 0.050 respectively [fig_ref] Figure 3: A [/fig_ref].
## Correlation of pdcd6 mrna expression with disease characteristics and patient survival
Spearman correlation analysis showed PDCD6 expression was positively correlate with residual tumor size (r = 0.16, p = 0.019) [fig_ref] Table 1: Spearman correlation of PDCD6 mRNA expressions with clinicopathologic features of ovarian cancer [/fig_ref]. Compared the median expressions of PDCD6 by different clinical and pathologic characteristics, no significant difference was found, although PDCD6 expression was slight higher in advanced (stagesIII and IV) than that in early disease [fig_ref] Table 2: Association of PDCD6 mRNA expression with clinical and pathological characteristics of ovarian... [/fig_ref]. Similar results were found when categorical variable was analyzed [fig_ref] Table 2: Association of PDCD6 mRNA expression with clinical and pathological characteristics of ovarian... [/fig_ref] , but significant difference of PDCD6 expression was showed between the patients with residual tumor and without residual tumor after surgery. Patients with residual tumor had significantly higher PDCD6 expression than patients who had no residual tumor (29.3% versus 16.7%, P = 0.050).
PDCD6 mRNA expression was significantly associated with progression free survival [fig_ref] Table 3: Associations of PDCD6 mRNA expression and patient survival a a Associations determined... [/fig_ref]. Patients with medium or high levels of PDCD6 mRNA were at higher risk for disease progression, compared to those with low levels (HR, 1.29; P = 0.024 for mid levels; and HR, 1.57; P = 0.045 for high levels) after adjusting for age, disease stage, tumor grade, histologic type and residual tumor size. Kaplan-Meier survival analysis demonstrates similar results. Patients who had medium or high PDCD6 mRNA had worse progression free survival . No significant association was found between PDCD6 mRNA expression and overall survival although patients with medium or high levels of PDCD6 mRNA were at higher risk for death compared to those with low level.
# Discussion
In our proteomic analysis of ovarian cancer cell lines, we detected 21 significantly different spots (two-fold increase or decrease) through 2-D gel electrophoresis, of which 17 candidate proteins were successfully identified and characterized. These proteins were mainly involved in the regulation of cell growth, proliferation, motility, apoptosis and tumor immunity [bib_ref] Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer..., Su [/bib_ref]. To further study the function of those differentially expression proteins in ovarian cancer, in this study, we selected PDCD6, which had the highest protein score and 100% protein confidence interval [bib_ref] Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer..., Su [/bib_ref]. As expected, PDCD6 overexpression in HO-8910 PM as compared to HO-8910 was confirmed by western blot analysis.
PDCD6, discovered by cDNA library screening for apoptosis-related genes [bib_ref] Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3, Vito [/bib_ref] , is a calcium binding protein which belongs to the penta-EF-Hand family that has the Ca2 + -binding helix-loop-helix structures [bib_ref] A growing family of the Ca2 + -binding proteins with five EF-hand..., Maki [/bib_ref]. PDCD6 functions as a Ca2+ sensor by changing its conformation [bib_ref] Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a..., Maki [/bib_ref]. This conformational change enables PDCD6 to interact with various intracellular proteins containing Pro-rich regions in a Ca2 + -dependent manner, such as Alix (ALG-2-interacting protein X) [bib_ref] The mechanism of Ca2 + -dependent recognition of Alix by ALG-2: insights..., Suzuki [/bib_ref] , annexins VII, XI [bib_ref] The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin..., Satoh [/bib_ref] , Sec31A (SEC31 homolog A) [bib_ref] ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in..., Shibata [/bib_ref] , and TSG101 (tumor susceptibility gene 101) [bib_ref] The mechanism of Ca2 + -dependent recognition of Alix by ALG-2: insights..., Suzuki [/bib_ref]. Original cloning report of PDCD6 cDNA in a screen for genes involved in apoptosis [bib_ref] Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3, Vito [/bib_ref] and early studies indicated PDCD6 participates in T cell receptor-induced programmed cell death in a Ca2 + -dependent manner as a pro-apoptotic factor [bib_ref] Functional cloning of genes involved in Tcell receptor-induced programmed cell death, D'adamio [/bib_ref]. Recent study showed Alix and ALG-2 involved in tumor necrosis factor receptor 1-induced cell death by interacting with procaspase-8 [bib_ref] Alix and ALG-2 Are Involved in Tumor Necrosis Factor Receptor 1-induced Cell..., Mahul-Mellier [/bib_ref]. However, apoptosis was not blocked in PDCD6 deficient mice, suggesting that PDCD6 is functionally redundant [bib_ref] Apoptosis-linked gene 2-deficient mice exhibit normal T-cell development and function, Jang [/bib_ref]. Furthermore, PDCD6 was found to be up-regulated in a variety of human tumors compared to normal tissues of the breast, liver, lung, and colon, especially in metastatic tissues, which suggests that in addition to its known pro-apoptotic function PDCD6 may play a role in cell survival [bib_ref] Up-regulation of ALG-2 in hepatomas and lung cancer tissue, La Cour [/bib_ref] [bib_ref] Genomic markers for malignant progression in pulmonary adenocarcinoma with bronchioloalveolar features, Aviel-Ronen [/bib_ref] [bib_ref] The apoptosis linked gene ALG-2 is dysregulated in tumors of various origin..., La Cour [/bib_ref]. Additionally, in vitro experiments indicated that PDCD6 had an anti-apoptotic action in HeLa cells via facilitating proliferating cells passing through the G2/M cell cycle checkpoints [bib_ref] ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation..., Hoj [/bib_ref]. Different from the previous study, PDCD6 knockdown had no obvious effect on cell proliferation and apoptosis although apoptotic cells were increased slightly and cell growth slowed down a little bit in HO-8910 PM cells infected with shRNA-PDCD6, but did result in decrease in cell motility and invasiveness. These results indicated that over expression of PDCD6 promote both migration and invasion in ovarian cancer cells.
Significantly, our patient study supported the results in vitro. PDCD6 mRNA expression was significantly correlated with residual tumor size. Patients with residual tumor had higher PDCD6 expression than patients with no residual tumor. Furthermore, PDCD6 expression was associated with patient survival; patients with high PDCD6 mRNA had shorter disease progressionfree survival than those with middle or low expression. Our findings suggest that PDCD6 mRNA expression is an independent predictor of ovarian cancer progression free survival.
In the study, we found that high PDCD6 expression was associated with disease progression, but not death. There are a number of possible reasons that may explain this discrepancy. First, PDCD6 may promote disease progression or reduce the effect of adjuvant treatment by stimulating tumor cell migration or invasion, but these effects do not lead to the death of all cases. Some patients may die of other reasons. Second, the study was relatively small and the follow-up time was short. We do not have enough power to find a significant association with overall survival. Third, our study findings are still preliminary. Independent large studies are needed to confirm these results. These findings may be explained by our observations in vitro and vivo which are generally in agreement with the findings of other studies. One study of lung cancer demonstrated that PDCD6 overexpression was indicative of unfavorable prognosis for patients with early stage non-small cell lung cancer or lung adenocarcinoma; the protein may serve as a potential molecular marker for aggressive lung cancer [bib_ref] Genomic markers for malignant progression in pulmonary adenocarcinoma with bronchioloalveolar features, Aviel-Ronen [/bib_ref]. However, no association was found between PDCD6 protein expression measured by immunohistochemical staining and survival of lung, breast or colon cancer patients [bib_ref] The apoptosis linked gene ALG-2 is dysregulated in tumors of various origin..., La Cour [/bib_ref]. And recent study reported that purified recombinant human PDCD6 inhibits vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2 [bib_ref] Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting..., Rho [/bib_ref]. Those inconsistent findings in the literature may be due to the observations made on different cancer cell lines or disease stages. In early stage tumors or tumors other than ovarian cancer, PDCD6 may have a pro-apoptotic effect on the tumors, whereas in our case when the tumor is ovarian cancer or tumors progress to advanced stages (since most of ovarian cancers are grade 3 tumors), the PDCD6 function changes to an opposite direction either due to different regulation or downstream targets, which is anti-apoptotic or pro-migration that leads to shortened progression-free survival.
In the future, we will continue this work from clinic to confirm its prognostic utility as well as from lab experiment to elucidate its molecular mechanism, finding or assembling another clinical study to measure PDCD6 expression in ovarian tumors and to analyze its association with disease progression and overall survival, performing chip assay to identify upstream modulators of PDCD6 as well as its downstream targets or pathways being involved in the PDCD6's effect on tumor progression. With a comprehensive understanding of PDCD6 regulation and function, we may be able to tell what confounding biology is involved in the relationship between PDCD6 expression and ovarian cancer progression.
# Conclusions
In summary, PDCD6 was identified and characterized as differentially expressed proteins between ovarian cancer cells with low and high metastatic property by our previous comparative proteomic study. It was further confirmed to be overexpressed in HO-8910 PM as compared to HO-8910 by western blot. PDCD6 knockdown significantly inhibited migration and invasion of ovarian cancer cells in vitro, supporting the notion that PDCD6 played an important role in ovarian cancer progression. PDCD6 mRNA was detectable in most of ovarian tumor samples, and the expression was correlated with residual tumor size. High PDCD6 expression was found to be associated with unfavorable prognosis of the disease. It may serve as an independent marker for ovarian cancer prognosis. Further studies are needed to more completely elucidate the molecular mechanisms of PDCD6 in ovarian cancer progression.
[fig] Figure 1: A. Western blotting analysis showed protein expression of PDCD6 was higher in HO-8910 PM cells than that in HO-8910 cells. GAPDH was used as a loading control. B. Western blotting analysis showed protein expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was decreased compared with that in control HO-8910 PM-mock. GAPDH was used as a loading control. C. qRT-PCR results showed mRNA expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was inhibited by 80% compared with that in HO-8910 PM-mock. [/fig]
[fig] Figure 2: A. Growth curve of HO-8910 PM cells infected with control or siRNA-PDCD6. At each indicated time point, cell viability was determined and represented as the degree of absorbance 570 nm using the MTT. The mean ± SD absorbance (triplicate wells) for each time point is plotted as a function of the number of days after seeding. B. Cell cycle distribution in shRNA-PDCD6-HO-8910 PM and HO-8910 PM-mock cells. C. Apoptosis analysis in shRNA-PDCD6-HO-8910 PM and HO-8910 PM-mock cells by Hoechst 33258 staining. Normal cells showed normal nuclei blue, while apoptotic cells showed the cell nucleus was chunky, stainded densely and brightly. I and II), 0.28 versus 0.16, p = 0.183, and in poorly differentiated tumors (grade3) than in well-differentiated tumors (grade 1 or 2), 0.29 versus 0.16, p = 0.275. Patients who had residual tumor after surgery had higher PDCD6 expression than patients who had no residual tumor, 0.31 versus 0.18, p = 0.261 [/fig]
[fig] Figure 3: A. Migrated cells in shRNA-PDCD6-HO-8910 PM and HO-8910 PM-mock cells. Magnification in × 100. B. Quantitative analysis for migration and invasion assays of HO-8910 PM infected with shRNA-PDCD6 or control. [/fig]
[table] Table 1: Spearman correlation of PDCD6 mRNA expressions with clinicopathologic features of ovarian cancer [/table]
[table] Table 2: Association of PDCD6 mRNA expression with clinical and pathological characteristics of ovarian cancer Expression Index (EI) > 1; f p-value from Chi-square test; g p-value from Spearman correlation test; h Bold values are statistically significant (p < 0.05) [/table]
[table] Table 3: Associations of PDCD6 mRNA expression and patient survival a a Associations determined by Cox proportional hazards regression and adjusted for age, stage, grade, histotype and residual tumor size. Significance level of p = 0.05 b 95% Wald Confidence Limits c Hazard Ratio (HR) for relapse with respect to low PDCD6 expression d Hazard Ratio (HR) for death with respect to low PDCD6 expressionFigure 4 Kaplan-Meier progression-free survival curves (A) and overall survival curves (B) according to PDCD6 levels: Among the patients, 77 patients had low PDCD6 expression, 86 patients had medium PDCD6 expression, and 49 patients had high PDCD6 expression. [/table]
|
Anodic Electrogenerated Chemiluminescence of Ru(bpy)32+ with CdSe Quantum Dots as Coreactant and Its Application in Quantitative Detection of DNA
In the present paper, we report that CdSe quantum dots (QDs) can act as the coreactant of Ru(bpy) 3 2+ electrogenerated chemiluminescence (ECL) in neutral condition. Strong anodic ECL signal was observed at ~1.10 V at CdSe QDs modified glassy carbon electrode (CdSe/GCE), which might be mainly attributed to the apparent electrocatalytic effect of QDs on the oxidation of Ru(bpy) 3 2+ . Ru(bpy) 3 2+ can be intercalated into the loop of hairpin DNA through the electrostatic interaction to fabricate a probe. When the probe was bound to the CdSe QDs modified on the GCE, the intense ECL signal was obtained. The more Ru(bpy) 3 2+ can be intercalated when DNA loop has larger diameter and the stronger ECL signal can be observed. The loop of hairpin DNA can be opened in the presence of target DNA to release the immobilized Ru(bpy) 32+, which can result in the decrease of ECL signal. The decreased ECL signal varied linearly with the concentration of target DNA, which showed the ECL biosensor can be used in the sensitive detection of DNA. The proposed ECL biosensor showed an excellent performance with high specificity, wide linear range and low detection limit.Electrogenerated chemiluminescence (ECL) is a light emission that arises from the high-energy electron-transfer reaction between electrogenerated species. ECL is attracting more attention due to its low cost, wide linear range, simple instrumentation, and high sensitivity 1 . Since it was first observed in the early 1970s, the Ru(bpy) 3 2+ ECL has been extensively studied and widely used in immunoassays and DNA probe assays 2-9 . Coreactant is often needed in Ru(bpy) 3 2+ ECL and numerous analytes, such as oxalate, amino acids, and tri-n-propylamine (TPA), can serve as coreactants 10-12 . Among them, TPA is mostly used in Ru(bpy) 3 2+ -based ECL biosensor because TPA can produce the highest light emission 4,13,14 . TPA radicals can be generated via direct electrode oxidation to react with Ru(bpy) 3 3+ to form [Ru(bpy) 3 2+ ]* which then decays to produce orange emission centered at 610 nm 15 . However, the oxidation of TPA on the electrode surface is not as fast as desired. Moreover, TPA is highly volatile, poisonous, and low solubility, which makes experimental operation relatively difficult, and this has driven the search for new coreactants of Ru(bpy) 32+. On the other hand, during the introduction of coreactants into the solution, the coreactant-related physicochemical behaviors like diffusion, absorption, as well as its concentration, may complicate the ECL systems. Because ECL reaction involves both electrochemical and chemical reactions, it is possible to find new coreactants using chemically modified electrode. For
immunoassay and DNA analysis, new coreactants with high ECL efficiency, low ECL background and user-friendliness are desired [bib_ref] Coreactants of Tris(2,2'-bipyridyl)ruthenium(II) Electrogenerated Chemiluminescence, Yuan [/bib_ref].
Due to the unique quantum size dependent electrochemical properties of QDs and controllable ECL merits, QDs ECL has become more and more fascinating [bib_ref] Quantum Dots for Live Cells, Michalet [/bib_ref] [bib_ref] Capping of CdSe-ZnS Quantum Dots with DHLA and Subsequent Conjugation with Proteins, Clapp [/bib_ref]. ECL applications of QDs are almost based on the cathodic ECL in the presence of coreactants [bib_ref] Electrochemiluminescence Sensors for Scavengers of Hydroxyl Radical Based on Its Annihilation in..., Jiang [/bib_ref] [bib_ref] CdS Nanocrystal-Based Electrochemiluminescence Biosensor for the Detection of Low-Density Lipoprotein by Increasing..., Jie [/bib_ref] [bib_ref] Enzyme-Quantum Dots Architecture for Highly Sensitive Electrochemiluminescence Biosensing of Oxidase Substrates, Jiang [/bib_ref] [bib_ref] Electrogenerated Chemiluminescence of CdSe Nanocrystals, Myung [/bib_ref] [bib_ref] Ultrasensitive Immunoassay Based on Anodic Near-Infrared Electrochemiluminescence from Dual-Stabilizer-Capped CdTe Nanocrystals, Liang [/bib_ref]. However, other electrochemical characters, such as electrocatalytic behavior of QDs, have not attracted much attention. It has been found during the investigation of anodic ECL of QDs that the cation radical QD +- can be directly produced from the electro-oxidation of QDs [bib_ref] Electrogenerated Chemiluminescence Determination of C-reactive Protein with Carboxyl CdSe/ZnS Core/Shell Quantum Dots, Wang [/bib_ref] [bib_ref] Anodic Electrochemiluminescence of CdTe Quantum Dots and Its Energy Transfer for Detection..., Liu [/bib_ref]. Since the oxidization process of QDs is similar to that of TPA, it is reasonable to speculate that the QDs modified on the GCE might act as a coreactant to generate anodic ECL with Ru(bpy) 3 3+ , which can sufficiently avoid the drawback of the introduction of coreactant in the solution and can find new roles of QDs in ECL investigation.
Nucleic acids have long been considered as a kind of molecule with genetic information. The development of sensitive and selective methods for the detection of trace amount of sequence specific DNA is of great importance in clinical diagnosis, food analysis, and environmental monitoring. To fulfill these requirements, numerous DNA detection systems combined with elegant signal transduction and some amplification strategies based on the hybridization between a DNA probe and its complementary target have been described [bib_ref] Design of a Molecular Beacon DNA Probe with Two Fluorophores, Zhang [/bib_ref] [bib_ref] Bio-Bar-Code-Based DNA Detection with PCR-Like Sensitivity, Nam [/bib_ref] [bib_ref] Electrochemiluminescence Biobarcode Method Based on Cysteamine-Gold Nanoparticle Conjugates, Duan [/bib_ref] [bib_ref] Label-Free Detection of DNA Hybridization Using Surface Enhanced Raman Spectroscopy, Barhoumi [/bib_ref] [bib_ref] Ratiometric Fluorescent Signaling of Small Molecule, Environmentally Sensitive Dye Conjugates for Detecting..., Wang [/bib_ref] [bib_ref] Mercury/Homocysteine Ligation-Induced ON/OFF-Switching of a T-T Mismatch-Based Oligonucleotide Molecular Beacon, Stobiecka [/bib_ref] [bib_ref] Situ Hybridization Chain Reaction Amplification for Universal and Highly Sensitive Electrochemiluminescent Detection..., Chen [/bib_ref]. Recently, ECL technique has been developed to detect DNA. For example, Ru(bpy) 3 2+ /TPA ECL system has been applied in the detection of sequence-specific DNA [bib_ref] Quenching of the Electrochemiluminescence of Tris(2,2'-bipyridine)ruthenium(II) by Ferrocene and Its Potential Application..., Cao [/bib_ref]. However, effective solutions for the above studies are limited, and this has driven the search for developing new ECL-based techniques in DNA detection. It was reported that DNA has the capacity to be intercalated with some small molecules into its grooves with high affinity. As a result, several DNA sensors have been developed by the use of the intercalation of small molecules probes into the DNA structures [bib_ref] Sensitive Detection of Protein by an Aptamer-Based Label-Free Fluorescing Molecular Switch, Li [/bib_ref] [bib_ref] Aptamer-Based ATP Assay Using a Luminescent Light Switching Complex, Wang [/bib_ref] [bib_ref] Signaling Aptamer/Protein Binding by a Molecular Light Switch Complex, Jiang [/bib_ref] [bib_ref] Aptamer-Functionalized Gold Nanoparticles for Turn-On Light Switch Detection of Platelet-Derived Growth Factor, Huang [/bib_ref] [bib_ref] Direct Detection of DNA with an Electrocatalytic Threading Intercalator, Tansil [/bib_ref]. Ru(phen) 3 2+ (phen = 1,10-phenanthroline) and its derivatives have already been successfully intercalated into the grooves of DNA to develop ECL bioassays [bib_ref] Turn-On" Electrochemiluminescent Biosensor for Detecting Hg 2+ at Femtomole Level Based on..., Tang [/bib_ref] [bib_ref] The Electrochemiluminescence of Ruthenium Complex/Tripropylamine Systems at DNA-Modified Gold Electrodes, Liu [/bib_ref] [bib_ref] Analyte-induced Formation of Partial Duplexes for the Preparation of a Label-Free Electrochemiluminescent..., Zhao [/bib_ref].
Herein, we propose a novel strategy for highly sensitive detection of DNA based on Ru(bpy) 3 2+ / QDs ECL system. QDs modified on the electrode can act as coreactant and generate strong anodic ECL with Ru(bpy) 3 2+ in neutral condition. Amino groups functionalized hairpin DNA can be bound to the carboxyl group of CdSe QDs via amide reaction. Ru(bpy) 3 2+ can be intercalated into the loop of hairpin through the electrostatic interaction, resulting in the strong ECL signal due to the reaction between Ru(bpy) 3 2+ and QDs. The loop of the hairpin DNA can be opened in the presence of target DNA, resulting in the release of Ru(bpy) 3 2+ and the decrease of ECL signal. The decreased ECL signal is linearly with the concentration of target DNA, which can be used in the sensitive detection of DNA.
# Results
## Ecl of ru(bpy) 3
2+ at CdSe QDs modified electrode. As reported, the strong anodic Ru(bpy) 3
## 2+
ECL can be observed in the presence of TPA. However, the limitation of TPA solution makes it necessary to explore new coreactants. Previous work revealed that QDs had good catalytic properties besides their good luminescent properties, which had rarely been studied in ECL [bib_ref] Application of Quantum Dots as Analytical Tools in Automated Chemical Analysis: A..., Frigerio [/bib_ref]. Therefore, Ru(bpy) 3 2+ ECL was studied at the CdSe/GCE in the absence of TPA to explore the possibility of QDs as the coreactant.
High resolution transmission electron microscopy image displayed the crystalline feature of CdSe QDs with average size of about 3.7 nm [fig_ref] Figure 1: ECL curves of a bare GCE and a CdSe/GCE in Ru [/fig_ref]. UV-vis absorption and fluorescence spectra of CdSe QDs were recorded and shown in [fig_ref] Figure 2: Cyclic voltammograms of a bare GCE and a CdSe/GCE in PBS with... [/fig_ref]. CdSe QDs showed an absorption peak at 465 nm and a strong fluorescence emission at 565 nm, which was consistent with the previous work [bib_ref] Enhanced Electrochemiluminescence of CdSe Quantum Dots Composited with CNTs and PDDA for..., Jie [/bib_ref].
The fabrication of QDs on a bare GCE was monitored by electrochemical impedance spectroscopy (EIS). Due to the existence of electrostatic repulse force between negatively charged QDs and [Fe(CN) 6 ] 3−/4− , the diameter of Nyquist circle increases with the amount of QDs modified on the electrode, which can indicate the assembly process of QDs. The EIS results of the GCE modified with different amount of CdSe QDs were recorded and shown in [fig_ref] Figure 3: Effect of potential scan rate on the oxidation peak current of Ru [/fig_ref]. It can be concluded that QDs are successfully modified on the GCE.
The ECL behaviors of Ru(bpy) 3 2+ were studied at a bare GCE and a CdSe QDs modified GCE (CdSe/ GCE) in pH 7.4 PBS as shown in [fig_ref] Figure 1: ECL curves of a bare GCE and a CdSe/GCE in Ru [/fig_ref]. In the absence of coreactant, the anodic ECL of Ru(bpy) 3 2+ is extremely weak. One strong anodic ECL peak can be observed at 1.10 V at the CdSe/GCE and the ECL intensity increases nearly 40 times compared with the bare GCE, indicating that CdSe QDs can act as the coreactant to react with Ru(bpy) 3
## 2+
, resulting in the generation of a strong anodic ECL signal. Because carboxylated QDs are easier to immobilize DNA, the new ECL system can be used to fabricate ECL biosensor for the detection of DNA without adding other coreactant [bib_ref] Electrogenerated Chemiluminescence Resonance Energy Transfer between Luminol and CdSe@ZnS Quantum Dots and..., Dong [/bib_ref]. In order to further support the conclusion, several experiments were carried out as shown in. Firstly, the same ECL behavior can be obtained at QDs modified gold electrode, revealing that strong ECL is result from QDs but not from electrode materials. Secondly, weak ECL signal is obtained at the bare GCE in Ru(bpy) 3 2+ solution, while strong ECL signal is obtained in QDs/Ru(bpy) 3 2+ mixing solution. Thirdly, the ECL intensity increased linearly with Ru(bpy) 3 2+ concentration but not QDs. These results can support sufficiently that QDs act as coreactant of Ru(bpy) 3 2+ ECL system. The effects of the amount of QDs modified on the GCE on the ECL signals were studied and shown in [fig_ref] Figure 3: Effect of potential scan rate on the oxidation peak current of Ru [/fig_ref]. The ECL intensity increased with the amount of modified QDs. When the amount of QDs exceeded 20 μ L, the increase of ECL signal leveled off. Because too thick QDs film is not facile for the electron transfer, 20 μ L of QDs is chosen in the fabrication of ECL biosensor. on the bare GCE and the CdSe/GCE were studied in PBS (pH = 7.4) as shown in [fig_ref] Figure 2: Cyclic voltammograms of a bare GCE and a CdSe/GCE in PBS with... [/fig_ref]. At the bare GCE, a reversible redox peak observed at 1.10 V should be attributed to the oxidation-reduction of Ru(bpy) 3
## 2+
. When the CdSe/GCE was studied in PBS without Ru(bpy) 3
## 2+
, the QDs starts to oxidize at 0.70 V. This conclusion is also supported by the reported results [bib_ref] Electrogenerated Chemiluminescence Determination of C-reactive Protein with Carboxyl CdSe/ZnS Core/Shell Quantum Dots, Wang [/bib_ref] [bib_ref] Anodic Electrochemiluminescence of CdTe Quantum Dots and Its Energy Transfer for Detection..., Liu [/bib_ref]. When the CdSe/GCE was studied in PBS containing Ru(bpy) 3 2+ , one apparent oxidation peak starts at 0.50 V and has the peak potential at 1.05 V, which could be assigned to the oxidation of Ru(bpy) [bib_ref] Electrogenerated Chemiluminescence. 62. Enhance ECL in Bimetallic Assemblies with Ligands That Bridge..., Richter [/bib_ref] 2+ because the peak current increased with the increase of Ru(bpy) concentration. The oxidation current of Ru(bpy) 3 2+ enhanced more than two orders of magnitude while the reduction peak nearly disappeared, suggesting that electrochemical reaction of Ru(bpy) 3 2+ could be catalyzed at the CdSe/GCE. Because the oxidation of QDs occurred at the same potential range, it is reasonable to deduce that the oxidation product of Ru(bpy) 3 2+ (Ru(bpy) 3 3+ ) can react with the oxidation product of QDs (QDs +- ), resulting in the decrease of reduction peak of Ru(bpy) 3
## 3+
. Meanwhile, the onset potential of the oxidation peak of Ru(bpy) 3 2+ at the CdSe/GCE located at ~0.50 V which is much less positive than that at the bare GCE (~0.85 V). The apparent increase in the oxidation current as well as the lower onset oxidation potential suggested that the QDs modified on the GCE exhibited good electrocatalytic effect on the oxidation of Ru(bpy) 3
## 2+
.
The effect of potential scan rate on the oxidation peak of Ru(bpy) 3 2+ at the CdSe/GCE was studied as shown in [fig_ref] Figure 3: Effect of potential scan rate on the oxidation peak current of Ru [/fig_ref]. The oxidation current increased with the increase of potential scan rate. The peak current changed linearly with the potential scan rate, revealing that the oxidation process of Ru(bpy) 3 2+ on the CdSe/GCE is adsorption controlling process.
## Spectral investigation of the interaction between qds and ru(bpy) 3
## 2+
. UV-vis absorption, Fluorescence (FL), and ECL spectrum were recorded to explore the interaction between Ru(bpy) 3 2+ and QDs. In, the mixture of QDs/Ru(bpy) [bib_ref] Electrogenerated Chemiluminescence. 62. Enhance ECL in Bimetallic Assemblies with Ligands That Bridge..., Richter [/bib_ref] 2+ exhibited a merging of absorption from separated QDs and Ru(bpy) 3 2+ in the range of 400-500 nm. Likewise, Ru(bpy) 3 2+ and QDs in the mixture also keep their FL properties as shown in. These results suggested that no chemical reaction occurred between QDs and Ru(bpy) 3
## 2+
. Thus, the light emission should be result from the reaction between the electrogenerated Ru(bpy) 3 3+ and QDs. There has no overlap between the absorption peak and the emission peak, revealing that energy transfer cannot occur between Ru(bpy) 3 2+ and QDs. In, the ECL spectrum included the maximum emission peak at 600 nm and a weak shoulder peak at 500 ~ 550 nm. The former agrees with the FL spectrum of Ru(bpy) 3 2+ while the latter agrees with the FL spectrum of QDs [fig_ref] Figure 2: Cyclic voltammograms of a bare GCE and a CdSe/GCE in PBS with... [/fig_ref]. The former ECL peak is more intense than the latter one, revealing that Ru(bpy) 3 2+ is the main emitter of the present ECL system while QDs can also emit weak light. Therefore, the results from the spectral investigation suggested that Ru(bpy) in the mixture is the main emitter while QDs in the mixture can not only generate weak ECL but also act as coreactant to generate strong ECL with Ru(bpy) 3 2+ .
ECL mechanism of Ru(bpy) 3 2+ at the CdSe/GCE. It has been drawn from the ECL spectrum that the main luminophor of anodic ECL is Ru(bpy) 3 2+ while QDs can also generate weak emission. According to the ECL mechanism of Ru(bpy) 3 2+ /TPA system as well as the electrochemical results, it is reasonable to propose that the electro-oxidation of Ru(bpy) 3 2+ to Ru(bpy) 3 3+ can be catalyzed by QDs. QDs can also be electro-oxidized at the same potential to generate strong reductive species QDs +- . Ru(bpy) 3 3+ can react with QDs +- to generate excited state of Ru(bpy) 3 2+ , which can emit light at ~600 nm. The mechanism of ECL process can be as follows: It was reported that the dissolved oxygen participated in the anodic QDs ECL in the species of O 2 −- , which could inject electrons into the hole injected QDs and generate anodic ECL [bib_ref] Anodic Electrochemiluminescence of CdTe Quantum Dots and Its Energy Transfer for Detection..., Liu [/bib_ref]. Therefore, the extremely weak ECL peak inshould be generated as follows:
[formula] ( ) − → ( ) ( ) + ++ → + ( ) + − - - ⁎ QDs O QDs O 5 2 2 → + (∼ ) ( ) ⁎ hv QDs [/formula]
QDs 550 nm 6
Fabrication of ECL biosensor. Previous studies have revealed that Ru(phen) 3 2+ and its derivatives can be intercalated into the grooves of double-stranded DNA through electrostatic interaction, which can be used in ECL bioassay [bib_ref] Turn-On" Electrochemiluminescent Biosensor for Detecting Hg 2+ at Femtomole Level Based on..., Tang [/bib_ref] [bib_ref] The Electrochemiluminescence of Ruthenium Complex/Tripropylamine Systems at DNA-Modified Gold Electrodes, Liu [/bib_ref] [bib_ref] Analyte-induced Formation of Partial Duplexes for the Preparation of a Label-Free Electrochemiluminescent..., Zhao [/bib_ref]. However, the amount of Ru(phen) 3 2+ that can be intercalated into the grooves of DNA is low, which limits the detection sensitivity. Therefore, in the present study, a hairpin DNA was used to integrate Ru(bpy) 3 2+ through electrostatic interaction. The interaction between Ru(bpy) 3 2+ and DNA was monitored by UV-vis and FL spectra as shown in [fig_ref] Figure 5: Schematic representation of the modification of the GCE and the detection of... [/fig_ref]. FL spectra didn't change while the UV-vis absorption peak at 250 nm changed when Ru(bpy) 3 2+ was intercalated into DNA, revealing that Ru(bpy) 3 2+ could be intercalated into DNA through the electrostatic interaction but not the chemical reaction. [fig_ref] Figure 5: Schematic representation of the modification of the GCE and the detection of... [/fig_ref] depicted the protocol of the proposed ECL biosensor based on the assembly strategy of DNA and QDs. First, QDs were modified on a bare GCE with the help of PDDA. Ru(bpy) 3 2+ was intercalated into the loop of hairpin DNA through electrostatic interaction to fabricate a probe. The probe could be immobilized on the QDs modified electrode through the interaction between -COOH groups of QDs and -NH 2 groups of DNA. The strong ECL signal can be obtained by the reaction between Ru(bpy) 3 2+ and QDs. In the presence of the target DNA, the loop of hairpin DNA can be opened and the intercalated Ru(bpy) 3 2+ can be released from the modified electrode, which resulted in the decreased ECL signals. As an effective tool for the characterization of the interface properties, electrochemical impedance spectroscopy (EIS) was used to monitor the biosensor fabrication process as shown in [fig_ref] Figure 6: Nyquist diagrams of electrochemical impedance spectra recorded from 0 [/fig_ref]. The impedance spectra of all modified process consisted of a semicircle at high AC modulation frequency and line at low AC modulation frequency, demonstrating that the electrode process was controlled by electron transfer at high frequency and by diffusion at low frequency. The charge transfer resistance (R ct ) which equals the diameter of semicircle reflects the restricted diffusion of the redox probe through the electrode surface. When QDs were modified on a bare GCE, R ct increased greatly due to the electrostatic repulsive force between negatively charged QDs and [Fe(CN) 6 ] 3−/4− . The R ct decreased when the Ru(bpy) 3 2+ intercalated probe DNA was connected on the QDs modified electrode due to the electrostatic attraction between the positively charged probe and [Fe(CN) 6 ] 3−/4− . When the target DNA was introduced, the loop of hairpin DNA was opened and Ru(bpy) 3 2+ was released from the probe. On the other hand, the insulating effect of DNA could perturb the interfacial charge transfer. As a result, the R ct was greatly increased again. In order to confirm the above speculation, the Zeta potential results of QDs and DNA probe were recorded and shown in [fig_ref] Figure 6: Nyquist diagrams of electrochemical impedance spectra recorded from 0 [/fig_ref]. It can be found that QDs and probe DNA are negatively charged while Ru(bpy) 3 2+ intercalated probe DNA is positively charged, which can support our conclusion. These results suggested a successful stepwise fabrication of the proposed ECL biosensor. The ECL responses of the modified electrodes in different stages were examined as shown in [fig_ref] Figure 7: ECL responses of a bare GCE, a CdSe/GCE, a probe1/CdSe/GCE, a probe2/CdSe/GCE,... [/fig_ref]. Extremely weak ECL was observed at the CdSe/GCE, which is due to the anodic ECL of QDs between the dissolved oxygen. When the probe DNA (probe 1) was modified on the QDs, the intense ECL signal was obtained due to the ECL reaction between QDs and Ru(bpy) 3
## 2+
. When the hairpin DNA with smaller loop was used as the probe (probe 2), the increased ECL signal is weaker than the hairpin DNA with larger loop, suggesting that Ru(bpy) 3 2+ could be intercalated into the loop of hairpin DNA. The ECL signal of the biosensor at 1.10 V remained at an almost constant value during 20 consecutive cyclic potential scanning as shown in the inset of [fig_ref] Figure 6: Nyquist diagrams of electrochemical impedance spectra recorded from 0 [/fig_ref]. The stable ECL signals can facilitate the ECL sensor design. When the target DNA was hybridized with the hairpin DNA, the loop of hairpin was opened and the intercalated Ru(bpy) [bib_ref] Electrogenerated Chemiluminescence. 62. Enhance ECL in Bimetallic Assemblies with Ligands That Bridge..., Richter [/bib_ref] 2+ was released, resulting in the reduced ECL signal. The inhibiting effect of the target DNA on ECL signal of the biosensor can be used in the determination of DNA.
Analytical performance of the biosensor. The quantitative behavior of the fabricated ECL biosensor for DNA detection was assessed by measuring the dependence of the Δ ECL upon the concentration of the target DNA under the optimized experimental conditions as shown in [fig_ref] Figure 8: The ECL signals of the biosensor incubated with different concentrations of target... [/fig_ref]. With the increase of the concentration of the target DNA, the ECL of Ru(bpy) 3 2+ decreased as the result of opening the [fig_ref] Figure 8: The ECL signals of the biosensor incubated with different concentrations of target... [/fig_ref]. The detection limit for target DNA was estimated to be 1.9 × 10 −16 mol L −1 (3σ ). The comparison of the various ECL biosensors was listed in . It can be found that the present biosensor is superior to most of other reported ECL biosensors.
Specificity of the biosensor. In order to investigate the specific response of the biosensor to DNA, control experiments were performed by incubating the biosensor in several aqueous solutions containing complementary (target) DNA, single-base mismatched DNA, three-base mismatched DNA, and noncomplementary (random) DNA, respectively. [fig_ref] Figure 9: Comparison of the ECL intensity changes for the sensors hybridized with target... [/fig_ref] showed the comparison of the ECL intensity changes of the biosensors for the same concentration of different DNA. It can be found that a great ECL signal change (Δ ECL) was observed after the ECL sensor was incubated in a complementary DNA, which was due to the loose of Ru(bpy) 3 2+ when the loop was opened. The ECL signal change for noncomplementary DNA was very small, attributed to the fact that the loop of hairpin DNA cannot be opened. The responses to the one-base mismatched DNA and the three-base mismatched DNA was only 15% and 5% of that for complementary target DNA. The comparison indicated that this method had high sequence specificity, and this detection approach had potential application in single nucleotide polymorphism analysis. The possible application of the biosensor in real samples detection was evaluated by recovery experiments determined in human serum samples. Two human serum samples were spiked with known concentrations of target DNA and were determined using the calibration curves of [fig_ref] Figure 8: The ECL signals of the biosensor incubated with different concentrations of target... [/fig_ref]. The obtained recovery values range from 95 to 102%, demonstrating that the satisfactory results can be obtained in real samples . Discussion Taken together, CdSe quantum dots can catalyze the electrochemical oxidation of Ru(bpy) 3 2+ and strong anodic ECL signal can be observed at the CdSe QDs modified glassy carbon electrode in neutral condition, suggesting that CdSe QDs can act as novel coreactants for Ru(bpy) 3 2+ ECL system. This work has several key meritorious novelties. Firstly, CdSe QDs can act as coreactant of Ru(bpy) 3 2+ instead of TPA, which reveals new characters of QDs in ECL application. Secondly, the immobilization of QDs coreactant on the electrode can avoid the use of coreactant in solution and simplify ECL system. Thirdly, good biocompatibility of QDs is facile for the fabrication of ECL biosensor. Greatly improved sensitivity is realized via intercalation of more Ru(bpy) 3 2+ into the loop of hairpin DNA through the electrostatic interaction. The strong ECL between Ru(bpy) and QDs results in a sensitive ECL biosensor for DNA without signal amplification. The inherent selectivity of the probe endows the biosensor with high base discrimination ability. The proposed approach provides a promising detection platform for genetic analysis and clinic biomedical applications. The electrochemical measurements were recorded with CHI 660D electrochemical workstation (CH Instruments Co., China). The ECL emission measurements were conducted on a model MPI-M electrochemiluminescence analyzer (Xi' An Remax Electronic Science & Technology Co. Ltd., China) at room temperature, and the voltage of the photomultiplier tube (PMT) was set at -800 V in the process of detection. All experiments were carried out with a conventional three-electrode system, including a modified GCE as the working electrode, a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode, respectively. A commercial 5 ml cylindroid glass cell was used as ECL cell and was placed directly in front of the PMT. High resolution transmission electron microscopy (HRTEM) was obtained by a JEOL-2100 transmission electron microscopy (JEOL, Japan). The UV-vis absorption spectra were obtained on a Shimadzu UV-3600 spectrophotometer (Shimadzu, Japan). The fluorescence measurements were carried out on a RF-5301PC FL spectrophotometer (Shimadzu, Japan). The ECL spectrum was obtained by collecting the ECL data at 1.10 V during cyclic potential sweep with 10 pieces of filter at 425, 450, 475, 500, 525, 550, 575, 600, 625, and 650 nm, respectively.
# Methods
Preparation of CdSe QDs modified electrodes. CdSe QDs were synthesized following the literature procedures [bib_ref] Dopamine Detection Based on Its Quenching Effect on the Anodic Electrochemiluminescence of..., Liu [/bib_ref]. The QDs were precipitated out with ethanol, centrifuged, dried under vacuum and kept in a refrigerator at 4 °C for further use. A glassy carbon electrode (3 mm in diameter) was mechanically polished with alumina pastes of 0.3 μ m, and then cleaned thoroughly in an ultrasonic cleaner with alcohol and water sequentially. After it was dried with blowing N 2 , 20 μ L of QDs solution was spread on the working electrode and dried at the room temperature to fabricate QDs modified GCE (denoted as CdSe/GCE).
Preparation of ECL biosensor. The Ru(bpy) 3 2+ intercalated DNA probe (probe/Ru(bpy) 3
## 2+
) was synthesized according to the literature 48 . 5 μ M probe DNA and 1.5 × 10 −4 mol L −1 Ru(bpy) [bib_ref] Electrogenerated Chemiluminescence. 62. Enhance ECL in Bimetallic Assemblies with Ligands That Bridge..., Richter [/bib_ref] 2+ were mixed at the volume ratio of 1:1 homogeneously, and kept at 4 °C for 15 h to intercalate Ru(bpy) 3 2+ into the loop segment of the probe DNA. 10 μ L of 1% PDDA was dropped on the surface of the working electrode and dried in the air. Then 20 μ L of CdSe QDs was spread on it and dried at room temperature. The modified electrode was immersed in pH 7.4 0.1 mol L −1 PBS containing 5 mM EDC and 10 mM NHS for 30 min to active the carboxylic group of the QDs. 10 μ L of the probe/Ru(bpy) 3 2+ was deposited on the activated electrode and incubated overnight at 4 °C. The electrode was washed by tris-HCl buffer twice to remove the unbonded probe. Afterward, the electrode was immersed in 1% BSA solution for 1 h to block the nonspecific binding sites on the surface. The resulted electrode was incubated with target DNA with different concentrations for 2 h at 38 °C. The obtained electrode was washed twice with tris-HCl-EDTA (TE) buffer and used to measure the ECL response.
[fig] Figure 1: ECL curves of a bare GCE and a CdSe/GCE in Ru(10 −4 mol L −1 ; PBS, 0.1 mol L −1 ; pH, 7.4; scan rate, 100 mV s −1 . If not mentioned additionally, all high voltages applied to the PMT were maintained at -800 V. [/fig]
[fig] Figure 2: Cyclic voltammograms of a bare GCE and a CdSe/GCE in PBS with and without Ru(bpy) 10 −4 mol L −1 ; PBS, 0.1 mol L −1 ; pH, 7.4; scan rate, 100 mV s −1 . The inset is the enlarged CV of a bare GCE in PBS with Ru(bpy) 3 2+ . [/fig]
[fig] Figure 3: Effect of potential scan rate on the oxidation peak current of Ru(bpy) 3 2+ at the CdSe/GCE. Ru(bpy) 3 2+ , 1.5 × 10 −4 mol L −1 ; PBS, 0.1 mol L −1 ; pH, 7.4. The inset is the linear relationship between peak current of Ru(bpy) 3 2+ and potential scan rate. [/fig]
[fig] Figure 4: (A) UV-vis absorption spectra of QDs, Ru( [/fig]
[fig] Figure 5: Schematic representation of the modification of the GCE and the detection of target DNA. [/fig]
[fig] Figure 6: Nyquist diagrams of electrochemical impedance spectra recorded from 0.01 Hz to 10 5 Hz for [Fe(CN) 6 ] 3−/4− (10 mM, 1:1) in 10 mM pH 7.4 PBS containing 0.10 mol L −1 KCl at a bare GCE, a CdSe/ GCE, a probe/CdSe/GCE, and a target/probe/CdSe/GCE. Scientific RepoRts | 5:15392 | DOi: 10.1038/srep15392 hairpin segment and releasing Ru(bpy) 3 2+ . The Δ ECL varied linearly with the logarithm of target DNA concentration over the range of 5.0 × 10 −16 -5.0 × 10 −12 mol L −1 with the correlation coefficient of 0.997 as shown in the inset of [/fig]
[fig] Figure 7: ECL responses of a bare GCE, a CdSe/GCE, a probe1/CdSe/GCE, a probe2/CdSe/GCE, and a target/probe1/CdSe/GCE in 0.1 mol L −1 PBS (pH 7.4). The inset is ECL emission from the probe1/CdSe/ GCE under continuous cyclic voltammetry for 20 cycles. [/fig]
[fig] Figure 8: The ECL signals of the biosensor incubated with different concentrations of target DNA (0, 0.5 fM, 1 fM, 10 fM, 100 fM, 0.5 pM, 1 pM, 5 pM). The inset is the corresponding logarithmic calibration curve, Δ ECL stands for the inhibited ECL signal of the modified electrode after the incubation in different concentration of target DNA. Scientific RepoRts | 5:15392 | DOi: 10.1038/srep15392 [/fig]
[fig] Figure 9: Comparison of the ECL intensity changes for the sensors hybridized with target DNA, onebase mismatched target DNA, three-base mismatched target DNA, and random sequence DNA in the same concentration (5 pM). Scientific RepoRts | 5:15392 | DOi: 10.1038/srep15392 [/fig]
|
A Model of Hormonal Regulation of Stamen Abortion during Pre-Meiosis of Litsea cubeba
Figure S1. The high conservation of similar functional genes in sequence (nucleic acid sequence or protein sequence) among different species, and functional annotation of unigene. . The relationship of transcriptome samples and expression distribution from the stage of LC_M1/F1 to LC_M3/F3 were assessed by the Pearson correlation coefficient and boxplot.
[fig] Figure S3: The enrichment analysis on KEGG pathway of DEGs in L. cubeba. (A) The top of 20 GO terms enrichment in LC_M1_vs_LC_F1; (B)The top of 20 pathway enrichment in LC_M1_vs_LC_F1; (C) The top of 20 GO terms enrichment in LC_M2_vs_LC_F2; (D)The top of 20 pathway enrichment in LC_M2_vs_LC_F2. [/fig]
[fig] Figure S4: The expression trend of 459 DEGs were analyzed by Short Time-series Expression Miner software (STEM) in female (LC_F1, LC_F2, LC_F3) and male flowers (LC_M1, LC_M2,LC_M3) respectively. [/fig]
[table] Table S1 Table S3 Table S5: Primers used in this study. List of GO terms related to stamen development genes. Selected reaction monitoring conditions for protonated or deprotonated plant hormones ([M+H] + or[M-H] -).Table S6. List of 459 DEGs related to hormone. IAA: indole acetic acid, GA:gibberellin, SA:salicylic acid, JA :jasmonic acid, ETH:ethylene, ABA: abscisic acid, BR :brassinosteroid, CTK: cytokinine. [/table]
[table] Table S7: Up-and down-regulated DEGs of profile 0 and 7 in female and male flowers. [/table]
|
Mitochondrial DNA Aberrations and Pathophysiological Implications in Hematopoietic Diseases, Chronic Inflammatory Diseases, and Cancers
Mitochondria are important intracellular organelles that produce energy for cellular development, differentiation, and growth. Mitochondrial DNA (mtDNA) presents a 10-to 20-fold higher susceptibility to genetic mutations owing to the lack of introns and histone proteins. The mtDNA repair system is relatively inefficient, rendering it vulnerable to reactive oxygen species (ROS) produced during ATP synthesis within the mitochondria, which can then target the mtDNA. Under conditions of chronic inflammation and excess stress, increased ROS production can overwhelm the antioxidant system, resulting in mtDNA damage. This paper reviews recent literature describing the pathophysiological implications of oxidative stress, mitochondrial dysfunction, and mitochondrial genome aberrations in aging hematopoietic stem cells, bone marrow failure syndromes, hematological malignancies, solid organ cancers, chronic inflammatory diseases, and other diseases caused by exposure to environmental hazards.
# Introduction
Mitochondria are maternally inherited and are usually described as the powerhouse of the cell. In addition to their role in the generation of cellular energy in the form of ATP, mitochondria are involved in additional functions, such as signaling, apoptosis, cellular differentiation, growth, as well as cell cycle control. The number of mitochondria in a cell can range from one to several thousand, and this range is determined by the tissue type and organism. Intriguingly, mitochondria possess their own DNA, called mitochondrial DNA (mtDNA), and the extent of alterations that occur to the mtDNA during development affects the presence and emergence of mtDNA mutations in the cell [bib_ref] Mitochondrial DNA mutations in disease, aging, and neurodegeneration, Reeve [/bib_ref] [bib_ref] Mitochondrial DNA mutations in patients with myelodysplastic syndromes, Shin [/bib_ref].
Human mtDNA is composed of 16,569 nucleotide bases and encodes 13 polypeptides of the electron transport chain, 22 transfer RNAs, and 2 ribosomal RNAs. mtDNA encodes approximately 3% of all mitochondrial proteins and is present in multiple copies-usually 10 [bib_ref] Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes, Shin [/bib_ref] contains a control region termed the D-loop, which contains considerable genetic variations. In fact, the D-loop forms the basis of forensic medicine in human identification and has been a useful tool in molecular anthropological studies on human origins [bib_ref] Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes, Shin [/bib_ref] [bib_ref] Mitochondrial DNA in aging and disease, Wallace [/bib_ref].
The origin of mitochondria is unknown; however, one of the most plausible explanations is the endosymbiosis hypothesis. This hypothesis posits that free-living bacteria had colonized proto-eukaryotic cells over the course of evolution, thereby establishing a symbiotic relationship. Primitive eukaryotic cells with intracellular mitochondria capable of metabolizing oxygen possessed selective advantage in an oxygen-rich environment. The electron transport chain within the mitochondria produces far more energy per molecule of glucose consumed, than anaerobic respiration. The oxidative phosphorylation process carried out in the mitochondria produces 38 molecules of ATP, compared to 2 ATPs by anaerobic glycolysis, and facilitates the conversion of toxic oxygen into water, providing those cells with mitochondria a protective biological advantage [bib_ref] Mitochondria: hub of injury responses in the developing brain, Hagberg [/bib_ref].
However, the disadvantage of oxidative phosphorylation is the formation of reactive oxygen species (ROS), such as singlet oxygen and hydroxyl radicals, which damage cellular components such as lipids, proteins, and DNA. A normally functioning electron transport chain has a 2% ratio of ROS produced for every electron transported. However, under disease or aging conditions, large quantities of ROS are generated, and this can contribute to cellular deterioration, as well as senescence [bib_ref] Mitochondria, oxygen free radicals, and apoptosis, Raha [/bib_ref].
Compared to the nuclear genome, mtDNA has a modified genetic code and lack introns and protection from histones, resulting in limited repair capacity. The proximity of mtDNA to sites of ROS generation suggests that mtDNA may be more susceptible to mutations compared to nuclear DNA [bib_ref] Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes, Shin [/bib_ref]. Accelerated mtDNA mutation rates can result in features indicative of premature aging, consistent with the view that loss of mitochondrial functions is a major causal factor in aging [bib_ref] Mitochondrial DNA in aging and degenerative disease, Berdanier [/bib_ref]. mtDNA damage and the resultant mitochondria dysfunction has been implicated in a wide range of human diseases, such as constitutional mitochondrial diseases, chronic degenerative/inflammatory diseases, and cancer.
This review summarizes and presents recent publications illustrating the heterogeneity that exists within mtDNA sequences in the Korean population, as well as mtDNA aberrations and their pathological implications in hematological malignancies, solid cancers, chronic inflammatory diseases, and diseases due to environmental hazards such as benzene and polyaromatic hydrocarbons.
## Heterogeneity of mtdna sequences in the korean population
Our laboratory examined sequence variations in the mtDNA control region, transfer RNA leucine1 (tRNA leu1), and cytochrome b (CYTB) genes to investigate the characteristics of mtDNA polymorphisms and haplogroups in the Korean population. Small deletion mutations exist only in the hypervariable (HV) region. A phylogenetic tree revealed a wide range of diversity in mtDNA sequences with 17.7% to 99.9% identification [fig_ref] Figure 1: Phylogenetic tree of 70 unrelated Korean individuals based on direct sequencing results... [/fig_ref]. The population exhibited a high level of length heteroplasmy (mixture of wild type and mutant sequences) in the 16184-16193 and 303-315 homopolymeric C (poly-C) regions from the mtDNA control region. Some of the most common polymorphisms found in all subjects were 73A > G, 263A > G, 3107delC, and 15326A > G from HV2, HV1, tRNA leu1, and CYTB genes, respectively. The most common haplogroup in the population was D4, found in approximately 16% of the population, followed by A, B, B4a, D5, G1a, and M10 (each 6%). Haplogroup D4 was further categorized into subgroups D4a, D4b, D4e, D4g, D4h, and D4j. However, these data from the Korean population do not correlate with haplogroup distributions described in previous studies and are more similar to the general pattern and frequency of haplogroups of the Japanese population than that of the Han Chinese population. The distinction between polymorphisms, including both common and novel mutations, is also poorly defined. For these reasons, we have strived to build a database for mtDNA sequences representing different age groups in the Korean population [bib_ref] Profiling of length heteroplasmies in the human mitochondrial DNA control regions from..., Shin [/bib_ref].
During the analysis of mtDNA HV1 and HV2 sequences from Korean donors, we experienced extreme difficulties when we attempted to sequence beyond the poly-C regions, and therefore we postulated that a possible reason was a high degree of length Korean donors Database Korean donors Nucleotide substitutions heteroplasmy . The length heteroplasmies in the HV regions of mtDNA from blood cells were examined in 70 healthy Korean donors. Interestingly, all subjects displayed length heteroplasmies in both the HV1 and HV2 regions. Closer examination of the HV2 length heteroplasmies indicated that 84% of these donors exhibited a minimal 303-315 poly-C tract frame shift of 1 bp. Sixteen percent of the donors possessed poly-C tract frame shifts of 2 bp or more. The mtDNA copy number in the donor group with major length variants (two or more frame shifts) was about twice as low as in the group with only a 1-bp frame shift. A wide range of mtDNA polymorphisms as well as new sequence variants in each age group were found; however, there was no significant correlation between the number of mtDNA mutations and an increase in donor age. Therefore, these results do not correlate with the hypothesis that the number of mtDNA mutations is in direct proportion to age, or related to the phenomenon of aging. The mutation rate of mtDNA is at least 10-fold higher than that of nuclear DNA. This higher mutation rate is attributed to the lack of protective histones, inefficient DNA repair capacity, proximity of ROS generated by the electron transport chain, and unique structural characteristics that favor mutational events [bib_ref] Profiling of length heteroplasmies in the human mitochondrial DNA control regions from..., Shin [/bib_ref].
## Mtdna aberrations in hematological malignancies
## Role of mitochondria in hematopoiesis
The heme biosynthetic pathway originates in the mitochondria, and after a few intermediate steps in the cytoplasm, is returned to its original location in the mitochondria. It has been speculated that sideroblastic anemia is caused by an enzyme defect in the heme biosynthetic pathway, which leads to a shortage of heme precursors and thereby impairs the utilization of iron that is imported into the mitochondria. In erythroblasts, virtually all the iron that enters the cell via the transferrin receptor enters into the mitochondria, where it is incorporated into protoporphyrin IX to produce heme. Heme is then exported from the mitochondria to combine with globin chains synthesized on cytoplasmic ribosomes [bib_ref] From sideroblastic anemia to the role of mitochondrial DNA mutations in myelodysplastic..., Gattermann [/bib_ref]. In the case of defective protoporphyrin synthesis, the imported iron lacks its reaction partner and would therefore accumulate in the mitochondria [fig_ref] Figure 3: Schematic diagram of the heme biosynthetic pathway [/fig_ref].
## Aging of the hematopoietic system and mitochondrial dysfunction
Hematopoietic stem cells (HSCs) have a very high turnover rate; nonetheless, they are not protected from age-related damages.
Aging of the hematopoietic system is exhibited through increased incidence of myeloid proliferative diseases, such as MDS and cancer, and through deterioration of the adaptive human immune system. Since HSCs are responsible for sustaining the blood system throughout life, age-related changes could be due to functional deterioration in HSCs. Although HSCs are able to sustain blood production for multiple life cycles, as demonstrated by serial transplantation in mice, phenotypically and functionally they undergo dramatic changes during the aging process [bib_ref] Mechanisms of hematopoietic stem cell aging, Ergen [/bib_ref]. The most profound effect is on the adaptive immune system, where there is a marked decline in lymphoid function in the elderly. Additionally, aging causes overproduction of myeloid cells, which leads to a pro-inflammatory environment [bib_ref] Mechanisms of hematopoietic stem cell aging, Ergen [/bib_ref] [bib_ref] Mitochondrial DNA mutations in disease and aging, Park [/bib_ref]. Advanced aging is accompanied by a number of clinically significant conditions arising from the hematopoietic system, including diminution and decreased competence of the adaptive immune system, elevated incidences of certain autoimmune diseases, increased hematological malignancies, and elevated incidences of age-associated anemia. As with most tissues, the aged hematopoietic system also exhibits reduced capacities for regeneration and recovery back to normal homeostasis after injury or stress [bib_ref] Mechanisms of hematopoietic stem cell aging, Ergen [/bib_ref]. The mechanisms underlying aging of the hematopoietic system vary and include intrinsic and extrinsic factors associated with the aging environment that act together to adversely influence the production and functions of hematopoietic effector cells [fig_ref] Figure 4: Schematic representation of aging hematopoietic stem cells [/fig_ref]. However, increasing evidence suggests that age-dependent cellular and molecular alterations within the most primi- . Demonstration of the difficulties involved in sequencing the homopolymeric C (poly-C) tracts in mitochondrial DNA (mtDNA) control region. Many mtDNA length heteroplasmies are localized in the hypervariable (HV) 2 poly-C tract, which is located between nucleotide positions (np) 303 and 315 (303CCCCCCCTCCCCC315 which is abbreviated as 7CT5C) (A and C). Another poly-C tract variant is located between np 16184 and 16193 (16184CCCCCTCCCC 16193, 5CT4C) in the HV1 region (B and D). This intractability against sequencing beyond these poly-C tracts in the HV regions is most likely attributable to the existence of more than one length of mtDNA. tive hematopoietic stem cell compartments may significantly contribute to hematopoietic deterioration during aging [bib_ref] Marked mitochondrial DNA sequence heterogeneity in single CD34+ cell clones from normal..., Shin [/bib_ref].
The classical pathway for the mitochondrial theory of aging is that by-products of mitochondrial phosphorylation (ROS: superoxide anion, hydrogen peroxide, and hydroxyl radicals) results in damages to mitochondrial macromolecules including the mtDNA. When these factors impair the apparatus for mitochondrial energy generation beyond a functional threshold, proteins are released from the mitochondria that activate the caspase pathway and lead to apoptosis. The aging process can introduce excessive mutations in the mtDNA via error-prone polymerase γ, which in turn can sufficiently impair mitochondrial functions, without causing further increase of ROS [bib_ref] Mitochondrial free radical theory of aging: Who moved my premise?, Liu [/bib_ref] [bib_ref] A midlife crisis for the mitochondrial free radical theory of aging, Stuart [/bib_ref] [bib_ref] Updating the mitochondrial free radical theory of aging: an integrated view, key..., Barja [/bib_ref]. It has been suggested that while somatic mtDNA mutations accumulate with age in post-mitotic tissues, the mutations are diluted and lost in continuously proliferating tissues such as the bone marrow. However, on average, 25% of individual CD34 + clones from the adult bone marrow showed mtDNA heterogeneity or sequence differences from the aggregate mtDNA sequences of total bone marrow cells from the same individual. In contrast, only 1.6% of single CD34 + clones from cord blood showed mtDNA sequence variation from the aggregate pattern. Thus, it appears that the age-dependent accumulation of mtDNA mutations appears relatively frequently in mitotically active human tissues, and thus poses important implications in the aging pro-cess in hematopoietic tissues [fig_ref] Figure 5: Schematic representation of mtDNA mutations in hematopoietic stem/progenitor cells and their clonal... [/fig_ref] [bib_ref] Mitochondrial free radical theory of aging: Who moved my premise?, Liu [/bib_ref] [bib_ref] A midlife crisis for the mitochondrial free radical theory of aging, Stuart [/bib_ref].
## Mitochondrial aberrations in bone marrow failure syndromes
Bone marrow failure syndromes encompass a group of disorders that are either inherited or acquired. These diseases are HSC disorders that can involve all cell lines: erythroid for red blood cells, myeloid for white blood cells, or megakaryocytic for platelets. The MDS are a heterogeneous group of hematological diseases characterized by bone marrow failure and are associated with increased risk of malignant transformation. Cytogenetic abnormalities are highly prevalent and in fact correlate with the prognosis and progression of leukemia [bib_ref] Mitochondrial DNA mutations in patients with myelodysplastic syndromes, Shin [/bib_ref]. The more aggressive categories of MDS, especially from secondary exposure to alkylating drugs, topoisomerase inhibitors, and radiation, share similar risk factors with acute leukemia and show stereotypical chromosomal abnormalities. Functionally relevant point mutations were found in the mitochondrial RNA and polypeptide-encoding genes of 50% of the patients with MDS. Their increasing mutation load correlates with MDS and the development of AML. Several point mutations for Leber's hereditary optic neuropathy occur in the bone marrow and may exert a synergistic effect on bone marrow stem cells via the apoptotic pathway. We systematically analyzed the entire mitochondrial genome by gene amplification and direct sequencing of samples from 10 patients with MDS. Overall, no increase was observed in the number of mtDNA genes harboring polymorphisms or mutations in the patients examined, compared to 8 healthy controls; however, there were a few more mtDNA changes as a result of amino acid alternations in MDS. Thirty novel mutations-all nucleotide substitutions-were found distributed throughout the mitochondrial genome among the 10 patients, with 5 mutations resulting in amino acid alternations. None of the mutations in the control group produced amino acid alterations. We were unable to confirm previously described mutations in sideroblastic anemia or 'hot spots' in the cytochrome c oxidase (CO) I and II genes. Our data indicated that mitochondrial genomic instability does not play a major role in MDS and also does not correlate with previous reports of significant or widespread mitochondrial mutations in this disease [bib_ref] Mitochondrial DNA mutations in patients with myelodysplastic syndromes, Shin [/bib_ref]. Modest changes in mutation numbers and mitochondrial microsatellites may be evidence of increased mutagenesis in mtDNA, or a reflection of limited clonality among HSCs in the bone marrow failure syndrome [bib_ref] Mitochondrial DNA mutations in patients with myelodysplastic syndromes, Shin [/bib_ref] [bib_ref] Mitochondrial DNA aberrations of bone marrow cells from patients with aplastic anemia, Kim [/bib_ref].
Aplastic anemia is considered a heterogeneous disease based on the numerous different putative etiologies, such as idiopathic, secondary causes (radiation, drug and chemicals, viruses and immune diseases), paroxysmal nocturnal hemoglobinuria, and pregnancy [bib_ref] Mitochondrial DNA aberrations of bone marrow cells from patients with aplastic anemia, Kim [/bib_ref]. Complete mtDNA nucleotide sequences were analyzed in bone marrow specimens taken from 9 Korean patients who were afflicted with aplastic anemia, as well as 8 healthy individuals. Several polymorphisms and novel mutations were found throughout the entire mtDNA genome in both the patient group and the control group. In patients with aplastic anemia, 12 mutations produced amino acid alterations; however, none of the mutations found in the control group produced amino acid alterations. More heteroplasmic mutations and nonsynonymous mtDNA changes were observed in patients with aplastic anemia. The number of mtDNA aberrations in bone marrow cells from patients with aplastic anemia (25.6 ± 14.3, mean ± SD) was significantly higher compared to that in controls (12.8 ± 7.3) (P = 0.019). These data suggested an association between mtDNA aberrations and aplastic anemia [bib_ref] Mitochondrial DNA aberrations of bone marrow cells from patients with aplastic anemia, Kim [/bib_ref].
## Mtdna aberrations in leukemia and leukemia stem cells
mtDNA can be easily damaged owing to the lack of histone pro-teins, inefficient repair ability, and close vicinity to the respiratory chain and ROS production [bib_ref] Somatic mitochondrial DNA mutations in adult-onset leukaemia, He [/bib_ref]. mtDNA damage is more extensive and persists longer than nuclear DNA damage [bib_ref] Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA..., Yakes [/bib_ref]. Pathogenic mutations can affect certain mtDNA copies in a cell, resulting in heteroplasmy (combination of mutated and wild type mtDNA) or homoplasmy (uniformity of sequences within an individual cell), which affects the entire mtDNA repertoire within the cell. The somatic mutation process in leukemia is complex, leading to diverse levels of genetic alterations due to either an intrinsic aspect of leukemia pathophysiology or chemotherapy effects [bib_ref] Mitochondrial DNA sequence variation in single cells from leukemia patients, Yao [/bib_ref]. Thus, it appears that somatic mutations may accumulate at varying rates in differentiated cells and progenitor cells from the same individual [bib_ref] Mitochondrial DNA sequence variation in single cells from leukemia patients, Yao [/bib_ref]. At diagnosis, some leukemic patients present mtDNA alterations that were comparable to controls. The mtDNA sequence is relatively homogenous in CD34 + cells from cord blood, and much more heterogeneous in CD34 cells from adults aged 25-57 yr. The observed complex pattern in leukemia patients suggests that mutations emerge at a relatively high rate in leukemic cell mtDNA, with homoplasmy and clonal expansion (expression of the fixed mtDNA mutation in a substantial proportion of progeny) occurring over months rather than decades [bib_ref] Mitochondrial DNA sequence variation in single cells from leukemia patients, Yao [/bib_ref].
## Mtdna aberrations in acute myeloid leukemia
Numerous novel mtDNA mutations were found only in primary AML cells (unpublished data). This observation provided insights into the analysis of mitochondrial proteins and development of novel chemotherapeutic agents. Aberrant mitochondrial protein expression was found in primary AML cells, and this could prove to be helpful in the identification of new molecular biomarkers and the development of novel anti-cancer agents. The mitochondrial protein alteration in primary AML cells imply that the mutation process in mtDNA is increased due to the high level of oxidative stress that occurs during ATP synthesis Recent studies suggested that mitochondrial genomic instability (mtGI), which consists of substitutions, deletions, length heteroplasmic mutations, and mtDNA copy number alteration, is observed in primary AML cells. As a result of mtGI, enzyme activities of the mitochondrial respiratory chain complexes are reduced. Our data indicated that many mutations and polymorphisms (a total of 606 mtDNA sequence variants) identified were from the bone marrows, buccal mucosa, and blood samples of 48 patients with AML.
## Mtdna aberrations in acute lymphoid leukemia
By comparing entire sequences of the mitochondrial genomes of normal buccal epithelium and leukemic cells, a recent study found that 4 out of 6 patients (67%) with adult-onset ALL harbored leukemic cell-specific mtDNA mutations. Bone marrow samples taken from ALL patients at different stages of the disease indicated that the A15296G mutation, as observed in the presentation and relapse of bone marrow samples, is a clonal marker for ALL, making it a suitable biomarker for the detection and monitoring of the disease during treatment. Not many studies have been conducted on mtDNA aberrations in ALL; therefore, additional investigation is necessary to reveal the spectra of mtDNA mutations and their corresponding pathophysiological and clinical implications.
Polymorphisms around the H-strand replication origin (nucleotides 150 to 199) and conserved sequence block II (nucleotides 299 to 317) in mitochondrial genes were associated with leukemia biology and treatment response. T-cell ALL patients were more likely to exhibit high levels of length heteroplasmies at the nucleotide position 303 poly-C tract. The T199C polymorphism was associated with increased risk for ALL in the Malaysian population. Patients with the T152C polymorphism showed better treatment responses than those patients without the polymorphism. No differences were observed in mtDNA contents between diagnostic ALL samples and controls; however, there was a significant reduction in mtDNA content after treatment, especially in patients with polymorphisms located in the origin of H-strand replication. Somatic mutations were found in 13% (9 of 76) of patients, suggesting an association to leukemogenesis. This study suggested that polymorphisms affecting transcriptional control affect mtDNA replication. In childhood ALL, decreased mtDNA copy number after treatment may confer susceptibility to chemotherapy.
## Mtdna aberrations in chronic lymphocytic leukemia
In both untreated and previously treated CLL patients, several nucleotide alterations were identified in the D-loop region. The role of mtDNA mutations in oncogenesis and chemosensitivity is unclear; however, increases in mtDNA mutations were observed during ROS generation. mtDNA mutations in primary leukemia cells are caused by DNA-damaging chemotherapeutic agents, resulting in heteroplasmy. After chemotherapy, new heteroplasmic mutations (G-C/G), identified by sequencing analysis, altered the CO II gene at nucleotide position 7762 [bib_ref] Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and..., Carew [/bib_ref].
Mitochondrial defects have been implicated in the development and progression of cancer for several decades. The groundbreaking work by Warburg illustrated that cancer formation is precipitated by damage to the respiratory machinery, which re-sults in an increase in glycolytic ATP production as compensation; some researchers claim that the observed effects are associated with mtDNA mutations [bib_ref] Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and..., Carew [/bib_ref].
The mitochondrial respiratory chain produces intracellular superoxide radicals, and ROS generation is known for causing mtDNA mutations. Due to this biological consequence, patients who had undergone chemotherapy exhibited increased superoxide generation in CLL cells and increased mtDNA mutations. In conclusion, instabilities among B-CLL patients demonstrate neutrality to DNA functions and likely do not promote tumorigenesis [bib_ref] High mitochondrial DNA stability in B-cell chronic lymphocytic leukemia, Cerezo [/bib_ref].
## Mitochondrial aberrations in leukemia stem cells
Cancer stem cells have recently been discovered in colon cancer and brain tumors, and they have been shown to demonstrate resistance against anti-cancer drugs and radiotherapy. Thus, it is imperative that novel drugs and treatment strategies that can effectively target cancer stem cells are identified and developed. Leukemic stem cells (LSCs) may play a pivotal role in the pathogenesis of hematological malignancies such as AML. Due to the slow division process and long interphase of LSCs as compared to normal stem cells and hematopoietic cells, many anti-cancer therapeutics are not effective in LSC treatment. Our laboratory has been examining the growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSCs, by comparing healthy donors and AML patients by using single cell sorting and various culture systems (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA, USA).
A total of 384 normal hematopoietic stem cells (CD34 + CD38 + /CD38 -) were obtained from the peripheral blood and cord blood donated by four donors, using a single cell sorter. Individual single cells were cultured in 96-well plates with each well containing 100 µL of serum media, 100 ng/mL of stem cell factor, 100 ng/mL of Flt-3, 100 ng/mL of thrombopoietin, and 50 ng/mL of G-CSF for five days. There were 768 single CD34 + CD38 -LSCs and 384 single CD34 + CD38 + cells obtained from 3 AML patients. The growth and proliferation capacities of normal HSCs and LSCs were determined in terms of plating efficiency (number of wells in which more than two cells had proliferated/ total number of cells in 96-well plate culture × 100 (42/288), 3.6% (7/192), and 8.0% (23/288). These results directly corroborated the properties of quiescence and slow division exhibited by LSCs. In addition, the plating efficiency of normal HSCs was shown to vary between healthy donors (unpublished data).
Single AML stem cells exhibited significantly lower plating efficiency, implying the properties of quiescence and slow division exhibited by the cells. AML stem cells possessed significant alterations of length heteroplasmy in the mtDNA control region, which may lead to a decline in mitochondrial biogenesis (reduction of mtDNA copy number) and dysfunction of mitochondrial ATP synthesis. These findings supported the introduction of aggressive compensatory mechanisms by the mitochondria of AML stem cells, and also implied potential strategies for the development of novel LSC-targeting therapeutics.
## Mtdna aberrations in non-hodgkin's lymphoma
The mtDNA copy number was increased in patients with CLL, Burkitt lymphoma, Epstein-Barr virus-transformed lymphoblastoid cell lines, and T-cells activated via the T-cell receptor. It was recently reported that the fact that peripheral white blood cells possessed higher mtDNA copy numbers as compared to healthy individuals is associated with future risk of non-Hodgkin's lymphoma (NHL). There exists a dose-response relationship between tertiles of mtDNA copy number and the risk of acquiring NHL (odds ratio
## Mtdna sequence alterations and new therapeutic targets in multiple myeloma
Cancer cells are more prone to harbor altered mitochondrial electron transport chains compared to normal cells, leading to a state of metabolic oxidative stress and in turn more efficient for ROS production [bib_ref] Mitocans, a class of emerging anti-cancer drugs, Sj [/bib_ref].Therefore, it follows that therapies which can specifically target elevated ROS production in cancer cells should be able to induce apoptosis. Examples of such agents are anti-cancer drugs which target mitochondria [bib_ref] Mitocans, a class of emerging anti-cancer drugs, Sj [/bib_ref] [bib_ref] Mitocans as anti-cancer agents targeting mitochondria: lessons from studies with vitamin E..., Neuzil [/bib_ref].
To study mtDNA aberrations in multiple myeloma (MM) cells, we investigated the rates of mtDNA mutations in MM cells from bone marrow aspirate specimens at the initial diagnosis. mtDNA mutations were detected exclusively in the CD133 + myeloma cells, but not in the CD33 + bone marrow cells from the same patients.
Using total bone marrow cells from 6 MM patients,CD133 + MM cells and CD33 + cells were used as normal internal controls and the isolation of both was performed using a single cell sorting system (FACS Aria cell sorter, BD Biosciences). Highly selective cell sorting procedures were used to target cell populations with unique expressions of CD133 and CD33 markers. A total of 3 out of 6 patients (50%) displayed MM cell-specific mtDNA mutations in the HV segment of the control region as length heteroplasmic and substitution mutations, which were not found in the corresponding CD33 + cells from the same patients. MM cell-specific length heteroplasmic mutations were detected in np 16184-16193 poly-C track (two patients) in the HV1 region and np 12385-12391 poly-C in the ND1 gene. These findings suggest the potential of developing new molecular markers for minimal residual disease monitoring, and could be used as new therapeutic agents to target mitochondria in MM and provide new insights into MM mitochondria-related molecular pathophysiology.
## Usefulness of mtdna minisatellite markers after allogeneic stem cell transplantation
Chimerism analysis is an alternative technique for monitoring minimal residual disease. However, timely therapeutic intervention is critical to prevent possible relapse or rejection. It is therefore imperative to closely monitor chimerism after allogeneic stem cell transplantation (SCT). Short tandem repeat (STR) markers are commonly used because of the high polymorphic nature between individuals. The STR assay generates quantitative results by using fluorescence-labeled primers and a capillary electrophoresis system. However, when the target DNA content is low or degraded, STR assays are imprecise and have low sensitivity. Careful approach and consideration is required because of this potential increase in imprecision, particularly with a small proportion of donor cells. To circumvent these problems, 6 mtDNA minisatellite (mtMS) markers were developed based on our publications describing the mtDNA genetic diversity among the Korean population. These markers were constructed to monitor the extent of donor cell engraftment in SCT recipients, and also to evaluate their usefulness and sensitivity compared with the nuclear DNA markers [bib_ref] Mitochondrial DNA minisatellites as new markers for the quantitative determination of hematopoietic..., Shin [/bib_ref]. The selected mtMS markers were assessed by using clinical samples and in vitro mixing to test the usefulness, sensitivity, and precision. Chimerism studies were carried out between 14 and 114 days after SCT by using 7nuclear DNA markers (STR-D12S391, VNTR-D1S80, STR-D18S51, STR-F13A1, STR-HUM FABP2, STR-HUM RENA-4, and STR-Amelogenin) and 7mtMS markers, including direct sequencing of the mtDNA HV region. The 303 poly-C and 16184 poly-C mtMS proved to be useful as nuclear DNA D12S391 and D1S80 markers. Meanwhile, the mtMS markers showed a higher sensitivity than the nuclear DNA-STR markers, possibly due to preferential amplification of mtDNA. After allogeneic hematopoietic stem cell transplantation (allo-HSCT), mtMS markers were used in multiple capacities for monitoring mixed chimerism and prognosis. By analyzing the 303 poly-C mtDNA marker and nuclear D18S51 STR marker using variable mixed blood cell concentrations, mtMS markers presented higher accuracies' sat monitoring mixed chimerism than nuclear STR markers, especially in unrelated transplantations and under inappropriate sampling conditions. Longitudinal follow-up after allo-HSCT revealed that chimerism precisely reflected the status of engraftment or relapse during the clinicopathological course. Moreover, changes in mtMS marker levels in recipients before allo-HSCT were associated with detrimental clinical outcomes. The mtDNA copy number per cell is relatively high, and sequencing the mtDNA is easier than the diploid nuclear DNA because of the haploid nature of mtDNA, which leads to preferential amplification.
Moreover, mtDNA typing is used primarily in cases where the nuclear DNA is too degraded or cannot be recovered in sufficient quantities for sequencing to proceed. These facts provide plausible reasons as to why the mtMS markers showed higher sensitivities and are considered more useful than the nuclear DNA markers [bib_ref] Forensic applications of mitochondrial DNA, Butler [/bib_ref].
## Mtdna aberrations in solid cancers
## Mtdna aberrations in colorectal cancer
Most studies on mtDNA mutations in colorectal cancer lack clinical relevance and include only case-control and case-database comparisons. Our previous work investigated tissue-specific mtDNA mutations in colorectal cancer, and evaluated their clinical relevance from a collection of 54 matched colorectal cancer and adjacent normal tissue samples [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref]. Over a half of the patients (59%) harbored cancer tissue-specific mtDNA mutations. The patterns of mtDNA mutations encompass substitutions (13%), mtDNA minisatellite instability (mtMSI) (20%), and both mutations combined (26%). mtMSI in colorectal cancer occurred mainly in the 303 poly-C (35%) and 16184 poly-C (19%) minisatellites. mtDNA copy number and hydrogen peroxide level were significantly increased in colorectal cancer tissues [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref]. The numbers of mtDNA large deletions significantly declined in colorectal cancer tissues compared with those from matched normal mucosa (P = 0.03). The activities of the mitochondrial respiratory chain enzyme complexes I, II, and III in colorectal cancer tissues were reduced. In addition, the mtDNA haplogroup B4 may be closely associated with colorectal cancer risk. The patient group harboring cancer tissue-specific mtDNA mutations showed larger tumor sizes (P = 0.005) and more advanced TNM stages (P = 0.002). Thus, mtDNA mutations in colorectal cancer may evoke risk factors that induce negative outcomes and in turn promote tumorigenesis [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref].
The numbers of mtDNA large deletions decreased significantly in colorectal cancer tissues compared with those from matched normal mucosa. These findings implied an active selection against mtDNA with the 4,977 bp deletion, or represent a population of tumor cells that does not undergo the typical aging response and subsequent tumor formation [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref].
mtDNA haplogroups are frequently found in various cancers, metabolic diseases, aging, and some neurodegenerative diseases. Patients with the mtDNA haplogroup M exhibited increased risks for developing breast cancer. Additionally, the mtDNA haplogroup D4a was associated with increased risks of thyroid cancer [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref]. On the other hand, no significant correlations between any mtDNA haplogroups and colorectal cancer were found. However, this study remarked that the mtDNA haplogroup B4 may be closely associated with risks of colorectal cancer among the Korean population. Nevertheless, the number of patients included in our study was limited, and thus further investigation which includes a larger cohort is warranted to elucidate the correlations between mtDNA haplogroups and colorectal cancer risk.
Furthermore, our previous study investigated clinicopathological values according to the existence of mutations, as well as specific mutation types [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref]. Patients who harbor both the substitution and mtMSI showed larger tumor sizes and advanced TNM stages, compared to patients with only one type of mutation (substitution or mtMSI) or no mutations at all. In conclusion, owing to the elevated levels of ROS in cancer tissues, most colorectal cancer patients harbored cancer tissue-specific mtDNA mutations, specifically, a type of substitution and minisatellite alteration. mtDNA mutations may be associated with advanced stages, and is a potential risk factor indicative of poor treatment outcome and prognosis in colorectal cancer [bib_ref] High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated..., Lim [/bib_ref]. corresponding peripheral blood samples (83.45 ± 60.36 and 39.0 ± 24.38, respectively) (mean ± SD) differed significantly (P < 0.001) from our previous study on bladder cancer [bib_ref] Analysis of fluorescence in situ hybridization, mtDNA quantification, and mtDNA sequence for..., Yoo [/bib_ref]. The mtDNA copy numbers in the urine samples from patients with high-grade and low-grade bladder tumors (81.83 ± 67.78 and 86.49 ± 46.69, respectively) did not differ significantly (P = 0.589). The mtDNA copy numbers in urine samples were much higher than those in the corresponding peripheral blood samples. mtDNA mutations were present in 80% of the D-loop regions in bladder cancer patients. This report further supported the significance of genetic alterations in urothelial bladder carcinoma, and the clinical utility of mtDNA quantitative polymerase chain reaction, mtDNA sequencing, and capillary electrophoresis for diagnostic purposes in patients with bladder cancer [bib_ref] Analysis of fluorescence in situ hybridization, mtDNA quantification, and mtDNA sequence for..., Yoo [/bib_ref].
## Mtdna aberrations in bladder cancer
## Mtdna aberrations in chronic inflammatory diseases
## Mtdna aberrations in barrett's esophagus
Carcinogenesis is a long-term, multistep process driven by genetic and epigenetic changes in susceptible cells, which gain a selective growth advantage and undergo clonal expression. Barrett's esophagus (BE) is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA). With respect to morphology, the carcinogenetic process of Barrett's mucosa progresses through increasing grades of epithelial dysplasia. At the present time, intraepithelial neoplasia, also called dysplasia, is the only marker that can be used to define patient populations who are at high risks of cancer. During the malignant transformation from BE to dysplasia and EA, prevalence of these molecular changes is increased. Although for the majority of identified genes, the predictive or prognostic value remains unclear. Nearly all of the new markers have not yet been validated in prospective controlled or randomized studies [bib_ref] Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence..., Lee [/bib_ref].
Even though many molecular events take part in the neoplastic transformation of Barrett's mucosa, only a few (i.e., changes in DNA ploidy, increased proliferation, and alteration of the p53 gene) have been identified as potential promoters of carcinogenesis. However, the molecular mechanism underlying the progression from BE to EA remains to be elucidated, while most studies on mtDNA mutations in BE have been only performed with dysplasia, and there the data is insufficient. We investigated new molecular events (BE tissue-specific mtDNA alterations/instabilities) in the mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissues (NT) and Barrett's esophageal tissues (BT) from 34 patients afflicted with Barrett's metaplasia but present no dysplasia. Eighteen patients (53%) exhibited mtDNA mutations that were not found in the adjacent NT. The mtDNA copy number was approximately 3 times higher in BT than in the adjacent NT. In fact, the activities of the mitochondrial respiratory chain enzyme complexes were impaired in the Barrett's metaplasia tissues that did not present dysplasia. ROS levels in BT were significantly higher than the corresponding NT samples. Therefore, it is suggested that high levels of ROS in BT can cause mtDNA mutations, which play an important role in disease progression and tumorigenesis in BE [bib_ref] Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence..., Lee [/bib_ref]. Oxidative damage has long been correlated to mucosal damage of the gastrointestinal tracts and their ensuing carcinogenesis. Furthermore, despite treatment with anti-secretory medications for reflux esophagitis, many patients failed to achieve complete mucosal healing, and instead suffered from sustained symptoms or development of BE. This implied that additional damaging factors or impaired mucosal resistance is involved in the pathogenesis.
Chronic inflammation that is mainly induced by the gastroesophageal reflux disease is the major cause of BE. An increase in the level of ROS production frequently occurs in chronic inflammatory cells and tissues. Thus, the current study detected BT-specific mtDNA alterations under the assumption that mtDNA aberrations are caused by abundant production of ROS in chronically inflamed BT. mtDNA mutations were detected exclusively in BT samples but not in the adjacent NT samples. These mtDNA mutations were observed as base substitutions and length heteroplasmies in the control regions. Oxidative stress elicited by chronic inflammation increases the number of mtDNA mutations in BT, and may correlate with a precancerous status. The levels of ROS were significantly higher in the BT supernatants compared to those from the adjacent NT. This high level of ROS damages the mitochondria, leading to mtDNA mutations. mtDNA control regions known as 'hot spots' of gene mutations contain the mtDNA production-regulating fraction and the HV regions, because mutations at these locations can cause various degenerative diseases and tumors [bib_ref] Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence..., Lee [/bib_ref].
Importantly, mutations in the control regions may alter the rates of DNA replication by modifying the binding affinities of significant transactivating factors. These mtDNA alterations in BT may further impair the respiratory chain defect and result in an increase in mtDNA copy number to compensate for the ATP deficiency. During this perturbation, mitochondria may produce a large quantity of ROS, which causes the vicious cycle observed in other chronic inflammatory diseases [bib_ref] Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue, Park [/bib_ref]. BT-specific mtDNA mutations frequently occur in both the mtDNA control and minisatellite regions due to excessive production of ROS. High levels of ROS in BT may contribute to the development of mtDNA mutations, which would play a crucial role in the pathophysiology of BE and further progression to EA. Although many risk factors are considered to be attributable to BE and EA, antioxidant treatments appear to be the preferring therapeutic option that is primarily used for prevention or treatment of BE, and can act in a preventive capacity in disease progression [bib_ref] Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence..., Lee [/bib_ref].
## Mtdna aberrations in nasal polyps
We investigated, in 23 patients, the possibility that mtDNA mutations promoted inflammatory or chronically damaged nasal polyp tissues. There were 13 patients (57%) who displayed nasal polyp tissue-specific mtDNA mutations in the HV segments of the control regions as well as the CYTB gene, which were not found in the corresponding blood cells and adjacent normal tissues. In np 303-315 homopolymeric poly-C track (39%), np 514-523 CA repeats (17%), and np 16184-16193 poly-C track (30%) nasal polyp tissue-specific length heteroplasmic mutations were also detected. The average mtDNA copy number was approximately 3 times higher in nasal polyp tissues compared to the corresponding peripheral blood cells and adjacent non-polyp tissues. The level of ROS was significantly higher in the nasal polyp tissues compared to the corresponding control samples. Increases in the level of ROS in nasal polyp tissues may be attributable to the mtDNA mutations and contribute to the vicious cycle of the pathophysiology of nasal polyps [bib_ref] Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue, Park [/bib_ref]. A nasal polyp is a relatively common affliction, although the underlying mechanism is currently unknown. A number of causes have been suggested, including chronic inflammation, resistance to aspirin, pollution, damaged epithelium, damaged DNA, and food allergies. Various studies have reported dissimilarities in gene expression in unaffected tissue and nasal polyp tissues [bib_ref] Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue, Park [/bib_ref].
ROS is commonly released in chronic inflammatory tissues. These ROS and free radicals target the mtDNA. With inflammation and excess stress, ROS production can overwhelm the antioxidant system, resulting in mtDNA damages. Nasal polyps most often accompany chronic inflammation, such as the accumulation of focal exudates in tissues, and the proliferation of submucosal tissues in nasal and paranasal sinuses. In conclusion, heteroplasmic mtDNA mutations in nasal polyp tissues frequently occur in both the mtDNA control and coding regions as base substitutions in coding and non-coding regions, and as base insertions/deletions within the control regions in the poly-C tract. The high levels of ROS produced by nasal polyp tissues may induce mtDNA impairment, which can lead to mtDNA mu-tations. Importantly, mutations in the control regions may alter the rate of DNA replication by modifying the binding affinity of significant transactivating factors. These mtDNA alterations in nasal polyp tissues may further damage the respiratory chain and increase mtDNA copy numbers in order to compensate for the ATP deficiency. During this perturbation, mitochondria may produce a large quantity of ROS, which can cause the vicious cycle observed in other chronic inflammatory diseases [bib_ref] Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue, Park [/bib_ref].
## Mtdna aberrations in helicobacter pylori-peptic ulcer
As a result of Helicobacter pylori infections, ROS are commonly released in the inflamed gastric mucosa. It is postulated that mtDNA mutations arise in inflamed or chronically damaged gastroduodenal epithelial cells. Results had shown that mtDNA mutations associated with H. pylori infections occurred in both the mtDNA control and coding regions in peptic ulcer tissues. Approximately half of the patients who were examined presented heteroplasmic mtDNA mutations. The high levels of ROS generated by H. pylori infections caused mtDNA damages leading to mtDNA mutations in peptic ulcer tissues. To compensate for the deficiency in ATPs, the mtDNA copy number is increased. During this perturbation, mitochondria produce a large quantity of ROS, which causes the vicious cycle observed in peptic ulcer diseases [bib_ref] Association between Helicobacter pylori-related peptic ulcer tissue and somatic mitochondrial DNA mutations, Lee [/bib_ref].
## Mtdna aberrations in chronic atrial fibrillation
Many studies had reported that various types of somatic mtDNA mutation were found in cardiac tissues. We showed that mtDNA mutations, including small deletions and tissue-specific length heteroplasmic mutations, occurred in both the control and coding regions of mtDNA from patients with chronic atrial fibrillation (cAF). Oxidative injury and deletion of mtDNA in the cardiac muscles are elevated in patients with cAF, which may contribute to the impairment of the bioenergetic function of mitochondria, and the induction of the vicious oxidative cycle attributable to the pathogenesis of atrial myopathy in cAF patients. In conclusion, 9 novel mutations were found in the control and coding regions of mtDNA. Interestingly, 2 patients (50%) had tissue specific to length heteroplasmic mutations from 16184 poly-C and CA repeats, starting at nucleotide 514, which were not found in blood cells. A 9-bp deletion around the mtDNA CO II gene was also identified in the tissues and blood cells from the patients examined. These findings strongly suggested that somatic mtDNA mutations are associated with cAF. Rapid atrial depolarization in cAF may result in higher oxygen consumption and oxidative workload, thus accelerating aging by accumulating so-
## Mitochondria and environmental medicine
Benzene damages the bone marrow and causes a reduction in red blood cells, leading to anemia. It can also cause excessive bleeding and compromise the immune system, thus increasing chances of infection. Benzene causes leukemia and is associated with other blood cancers and pre-cancers of the blood [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref]. Benzene directly causes an increase in the generation of intracellular ROS, which subsequently induces changes in mitochondrial mass, mtDNA content, and the proteome. In addition, benzene also causes oxidative stress, leading to an increase in mitochondrial mass and mitochondrial membrane potential.
Benzene exposure in humans and animals has been shown to result in structural and the amount of chromosomal aberrations that occur in lymphocytes and bone marrow cells, indicating that benzene is a genotoxin. The mitochondrial genome is highly vulnerable to DNA damage caused by ROS and mutagens, and exhibits higher mutation rates in comparison to the nuclear genome. Furthermore, the lengths of time it requires to correct DNA damages are much longer in the mitochondrial genome. A number of factors contribute to the vulnerability of mtDNA, including the absence of histones, which provide packaging and protection of nuclear DNA, and the error-prone replication and repair systems of mitochondrial genes. Therefore, our previous work had targeted the mitochondrial genome and proteome to identify biomarkers associated with benzene exposure [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref]. Results of the present study showed that after shortterm benzene exposure, the mitochondrial mass and mtDNA copy number increased. These findings are consistent with a previous report which showed an increase in mtDNA content in the tissues of aged rats and elderly human individuals. It was considered that during oxidative stress, the number of mtDNA increased in order to compensate for declining respiratory functions; however, the molecular mechanism underlying the increase in mtDNA associated with decreased respiratory functions under aging and oxidative stress conditions remains unclear.
The mitochondrial membrane potential reflects the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation-the driving force behind ATP production-and is a key indicator of cell viability. The present study demonstrated that the mitochondrial membrane potential could be a novel biomarker for ben-zene toxicity in hematopoietic tissues and cell lines. In comparison to nuclear DNA, mtDNA intrinsically exhibits a 10-to 20fold higher susceptibility to genetic alterations. Therefore, mtDNA is regarded as a useful molecular target to monitor genotoxicity caused by environmental hazards [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref].
We identified biomarkers which can indicate the exposure of benzene in blood cells and hematopoietic tissues [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref]. The mitochondrial content and membrane potential increased dramatically after three weeks of direct benzene exposure. The hydrogen peroxide level, mtDNA copy number, and numerous protein markers increased after a few weeks of benzene treatment, compared to the group not treated with benzene. However, the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, decreased after exposure to benzene. Thus, increased mitochondrial mass, mtDNA copy number, and the hnRNP A2/B1 protein can be used as biomarkers for benzene-related toxicity and hematotoxicity [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref].
Moreover, the mitochondrial proteome was studied to determine whether biomarkers associated with exposure to benzene could be identified. The results indicated that the hnRNP A2/B1 protein may be a novel biomarker associated with benzene exposure. hnRNPs are abundant and exhibited "multi-tasking" features by playing a central role in RNA metabolism; the levels of hnRNPs significantly decreased after exposure to benzene. These proteins are involved in packaging nascent RNA, alternative RNA splicing, mRNA export from the nucleus, and cytoplasm trafficking, stability, and translation. In addition, hnRNPs may play a poorly defined role in telomere maintenance [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref].
Our previous work showed that after benzene exposure, there was a decline in hnRNP A2/B1 levels and cell cycle arrest, which led to decreased numbers of primary blood cells and cell lines. hnRNP B1, an RNA binding protein, is overexpressed in early-stage lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. Plasma hnRNP B1 mRNA has been reported to be a useful noninvasive marker for the detection of lung cancer [bib_ref] Mitochondrial DNA copy number and hnRNP A2/B1 protein: biomarkers for direct exposure..., Eom [/bib_ref].
In summary, in response to elevated intracellular ROS levels, the direct exposure to benzene by primary blood cells and cell lines in vitro was associated with increased mitochondrial mass and higher mtDNA copy number. These results were consistent with the observation that mitochondrial mass and mtDNA contents are increased for the cell to respond to endogenous or exogenous oxidative stress. In addition, results of the present study identified several useful biomarkers associated with benzene exposure, including a novel hnRNP A2/B1 protein.
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous envi-ronmental toxicants found in air, water, plants, and soils [bib_ref] Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic..., Raychoudhury [/bib_ref].
Owing to their widespread dispersion in the environment and the adverse health effects associated with PAH exposure (e.g., carcinogenesis and endocrine disruption), there has been increasing concern in the human health field regarding PAH exposure. Although the adverse effects associated with each individual PAHs are not entirely analogous to one another, the United States Environmental Protection Agency has designated 32 PAH compounds as priority pollutants (www.epa.gov). The toxicities of PAHs are determined by their structures. Among the 32 PAH compounds, benzopyrene is notable as the first chemical carcinogen to be discovered [bib_ref] Fifty years of benzo(a)pyrene, Phillips [/bib_ref] [bib_ref] Biomarkers of exposure to polycyclic aromatic hydrocarbons from environmental air pollution, Castaño-Vinyals [/bib_ref].
## Conclusions and future directions
Mitochondria are important micro-organelles that produce the energy fueling cellular development, differentiation, and growth.
Owing to the lack of introns in mtDNA, the rate of genetic mutation is 10-to 20-fold higher in mtDNA compared to nuclear DNA. The mtDNA repair system is inefficient, rendering the mitochondrial genome more susceptible to ROS produced during the ATP synthesis process within the mitochondria. ROS are commonly released during chronic inflammation, and mtDNA is one of the targets for ROS and free radicals. ROS-induced inflammation and excess stress can overwhelm the antioxidant system and lead to mtDNA damage. This oxidative stress plays an important role in mitochondrial dysfunction and mitochondrial genome aberrations, precipitating in various human diseases including hematological malignancies, solid cancers, chronic inflammatory diseases, and diseases caused by environmental hazards.
[fig] Figure 1: Phylogenetic tree of 70 unrelated Korean individuals based on direct sequencing results of mitochondrial DNA (mtDNA) control region. The study population exhibited marked mtDNA sequence diversity. The percent identification distribution was 17.7 to 99.9 among healthy Korean donors. [/fig]
[fig] Figure 3: Schematic diagram of the heme biosynthetic pathway. Heme synthesis begins in the mitochondria, and after several intermediate steps in the cytoplasm, returns to the mitochondria. [/fig]
[fig] Figure 4: Schematic representation of aging hematopoietic stem cells (HSCs) and disease phenotypes. Instabilities of nuclear and mitochondrial genomes, and their altered transcriptions, including epigenetic changes, are associated with HSC aging, resulting in the development of age-related diseases.Abbreviations: mtDNA, mitochondrial DNA; ROS, reactive oxygen species; HSC, hematopoietic stem cell. [/fig]
[fig] Figure 5: Schematic representation of mtDNA mutations in hematopoietic stem/progenitor cells and their clonal expansion. mtDNA mutations arise in hematopoietic stem cells as a result of intracellular reactive oxygen species (ROS). These mutations are clonally expanded into progenitor cells and daughter cells. www.annlabmed.org http://dx.doi.org/10.3343/alm.2015.35.1.1 [/fig]
|
Efficacy of a Standardised Patient Simulation Programme for Chronicity and End-of-Life Care Training in Undergraduate Nursing Students
Citation: Escribano, S.; Cabañero-Martínez, M.J.; Fernández-Alcántara, M.; García-Sanjuán, S.; Montoya-Juárez, R.; Juliá-Sanchis, R. Efficacy of a Standardised Patient Simulation Programme for Chronicity and End-of-Life Care Training in Undergraduate Nursing Students. Int.
# Introduction
The European framework for higher education supports the promotion of lifelong learning, knowledge acquisition, and the assessment of professional competencies [1], understanding them as the degree to which students use their knowledge, attitudes, and judgment associated with their profession to resolve arising situations. One of the most used and successful methods to facilitate learning and the development of competencies in health sciences students is simulation-based education (SBE). SBE allows students to identify aptitudes that they must improve and offers them an opportunity to repeatedly practice and develop their skills in a safe environment. This reduced the occurrence of adverse events secondary to high levels of stress or anxiety in nursing students.
Over the last decade, SBE has been implemented to help develop communication skills in clinical settings. Communication skills are part of the patient-health professional interpersonal relationship and directly affect the therapeutic process; they are a core element in the provision of support and care in palliative care as well as for other complex patients. In the context of communication skills training, perceived self-efficacy and student attitudes towards these skills are two variables of great importance. Self-efficacy refers to a student's perceived ability to communicateand is an important predictor of their motivation, learning, and performance. Positive attitudes towards communication are associated with a higher level of satisfaction and an improved quality of delivered care by students.
Therefore, students might receive specific training in emotionally complex situationsprior to their incorporation into professional teams. Inadequate training in palliative and chronic care could lead to greater emotional distancing, difficulty in decision making, and avoidance behaviours both in these situations as well as with patients and their families.
In this sense, the use of standardised patients (SPs) has considerably increased in communication skills training programmes for health sciences students, especially in the areas of oncology and palliative care. SPs provide students with realistic interactions and responses to controlled and evaluable situations through protocolised simulations and the use of validated assessment methods. In addition, studies have shown that the use of trained actors confers greater realism and fidelity to the scenarios presented in communication skills training programs, especially in the context of emotionally complex situations. Some authors have studied how participation in end-of-life care simulation programs involving SPs endows trainees with more positive attitudes towards the care of dying patients and significantly improves their self-efficacy.
These results have also been reflected in studies in other subject areas. For example, a study with a quasi-experimental design in the field of mental health showed that simulations improve students' attitudes and enhanced their confidence and self-efficacy. Other quasi-experimental design studies in the field of emergency medicine found significant and favourable relationships in the dimensions of satisfaction, confidence, and motivation following the performance of high-fidelity simulations by students. Therefore, the benefits of high-fidelity simulations in terms of student learning calls for further research in this field.
The current academic literature reflects a growing interest in communication skills training through simulations with SPs. However, the variability in the methodologies used (simulation with actors, trained professionals, students, or a combination of all the above), the different designs (quasi-experimental, exploratory, or mixed studies), and the variability in the sample sizes (with a range between n = 12 and n = 630)highlight the lack of protocolised structures and properly validated tools,. Thus, it is important to evaluate the effectiveness of these programme types by applying quantitative methodologies and by using tools to obtain objective, valid, and reliable information. This will allow for the adequate assessment of student attitudes towards the communication skills set out in their training curriculum.
Therefore, there is growing interest in exploring specific simulation scenarios with SPs in end-of-life care education in nursing students, especially focusing on SP simulation programmes. Thus, the object of this current research study was to evaluate the effectiveness of a simulation programme using SPs in complex scenarios linked to chronic and end-of-life situations in terms of the variables of attitude, self-efficacy, and communication skills among nursing students.
# Materials and methods
## Design and participants
A quasi-experimental study was carried out in a single group with pre-and postintervention measurements. To clearly report the elements of the study we followed the extended guidelines of the Consolidated Standards of Reporting Trials (CONSORT) for simulation-based reports. All eligible participants (n = 204) who completed the simulation were fourth-year students in the nursing undergraduate course during the academic period of 2019/2020 at the University of Alicante. The groups were formed according to the enrolment of fourth-year nursing students in one of the 12 laboratory practice groups (A-L) of each theoretical subject in the first quarter.
Each group (comprising 17-18 students) freely divided themselves up into a total of 6 subgroups, each with 2-3 members. Therefore, a total of 72 subgroups were created in order to implement the SP intervention. These subgroups remained unchanged throughout the training except in 2 cases, because of abandonment of the programme by one participant in each subgroup, leaving 2 members in each subgroup in these cases. The natural opportunity presented by the enrolment of nursing students into this course was used to obtain a convenient sample.
## Intervention programme: a high-fidelity simulation
A simulation programme with SPs was implemented between September and December 2019. The home care simulation classrooms in the Faculty of Health Sciences of the University of Alicante were used. As described in , the objective of the programme was to train the students in non-technical skills (communication) for complex care situations in the context of chronicity and end-of-life care. The teaching team that implemented the programme designed 12 cases with contents related to grief (n = 3), decision making and coping in difficult situations (n = 3), states of acute confusion in the context of chronicity (n = 2), bad news (n = 1), the pact of silence (n = 1), pain management (n = 1), and contexts in the last days of life (n = 1). . Description of the high-fidelity simulation intervention using standardised patients implemented in this study.
## Aims
To train effective communication skills in healthcare students.
## Target population
Fourth-year nursing students.
## Theoretical basis
Patient-centred care.
## Context
Chronicity in different stages of life and end-of-life care.
## Duration of each session 2.5 h
The programme comprised 8 sessions, each lasting 2.5 h . The first two sessions were dedicated to preparing the students prior to the simulation scenario training. The objectives of these initial sessions were for the students to understand the educational intervention and cases that would be presented and to generate a known and safe environment for the groups by promoting group dynamics. Two cases were randomly assigned to each subgroup (i.e., 6 per training group), with the aim of developing an adequate theoretical appreciation of the intervention types. Two weeks before the start of the training, the students shared their understanding of the cases they had been assigned with the rest of their group. The remaining 6 sessions were specific training sessions and followed a standardised structure which had been agreed upon by the research team. Each subgroup actively participated in 4 different simulation scenarios, while they participated in the rest of the scenarios (n = 20) both passively as observers and actively during the debriefing. An absence rate of up to 20% was permitted. Three professional actors who were experts in improvisation participated as the SPs. Prior to completing the scenario, all the actors were given a description of each case and both the objectives of the interaction with the students and the appropriate interventions for each of the simulations were explained to them. In addition, all the programme teachers involved received specific training about the interventions (lasting 4 h) which addressed content related to group management, the structure of the simulation sessions, protocols to follow, and the management of problem situations during the simulation and debriefing sessions. The session structure for each scenario was: pre-debriefing, presentation of the case by the students, simulation, and a structured group debriefing during the simulation session, as suggested in the literature.
## Variables and instruments
A data collection form was built which included the following questionnaires: A questionnaire about sociodemographic characteristics related to communication skills, which included the variables of gender, age, nationality, and training in communication skills through two questions: "Have you received training in social/communication skills during your nursing degree training?" and "Have you received training outside your nursing degree in social/communication skills?".
The attitudes towards communication were evaluated using the Spanish version (Attitudes Towards Health Communicationof the Attitudes Towards Medical Communication Scale. This one-dimensional instrument comprises 11 assessable items measured on a 5-option Likert-type response scale from "strongly disagree" (1) to "strongly agree". The overall score ranges from 11 to 55 points, with higher scores reflecting more positive attitudes towards communication. The Spanish version of the scale has an adequate internal consistency score of 0.75.
Self-efficacy in communication skills was evaluated by employing the Spanish version (manuscript in preparation) of the Self-Efficacy of Communication Skills (SE-12) scale. This one-dimensional scale contains 12 items assessed on an 11-point Likert-type response scale ranging from "very insecure" (1) to "very secure". The overall SE-12 score can range from 12 to 120 points, with higher scores indicating greater confidence in one's own communication skills. This scale has a high internal consistency (Cronbach alpha = 0.95) and adequate test-retest reliability (intraclass correlation coefficient = 0.71) in its original version, and has been validated in Spanish students with an internal consistency of 0.94 (manuscript in preparation).
Communication skills were evaluated through the Health Professionals Communication Skills Scale (EHC-PS), an 18-item instrument comprised of 4 communication skills dimensions: empathy, informative communication, respect, and social ability/assertiveness. The EHC-PS is assessed on a Likert-type response scale with 6 response options ranging from "almost never" = 1, to "many times" = 6. The internal consistencies of the EHC-PS dimensions were 0.77 for empathy, 0.78 for informational communication, 0.74 for respect, and 0.65 for social ability/assertiveness. When validated in nursing students, this scale showed excellent internal consistency (0.88) and moderate convergent validity (r = 0.35, p < 0.001) with the attitudes towards communication.
## Procedure
The data were collected both during the first session (September 2019) and after the implementation of the entire programme (December 2019) through an electronic Internetbased form (Google Forms) which was disseminated to the students through the institution's internal platform known as "UACloud". Along with the questionnaires described above, the form also included detailed information about the study, an express request for informed consent from the participants, and details about the voluntary nature of their participation in the research and about the treatment of the collected data.
To achieve a high post-intervention response rate, a standardised methodology was followed in which we sent three email reminders, one week apart each, encouraging participation and containing a link to the questionnaire. A total of 161 students completed the post-intervention questionnaire (loss rate = 21.17%).
This project was approved by the Bioethics Committee at the University of Alicante (file number UA-2018-10-24) and all participants signed their consent form to participation. All the data were treated confidentially, and participation did not impact the students' grades. The study followed the criteria established by the Declaration of Helsinki and the Standards of Good Clinical Practice of the European Union.
# Data analysis
Descriptive analyses of the sociodemographic variables and the outcome variables were carried out. For the attitude, self-efficacy, and perceived communication skills variables, we first checked the assumption of normality for each one by examining the Kolmogorov-Smirnov test. Next, we checked whether there were any significant differences in the scores at the baseline by applying Student's t-tests for the gender variable and Pearson tests for the age variable. Effect sizes were calculated for variables with statistically significant differences, and their threshold values were interpreted as small (0.20), moderate (0.50), and large (0.80) effects.
An analysis of variance with repeated measures (ANOVA MLG) was performed to compare the scores obtained between the pre-and post-intervention timepoints, including the sociodemographic variable of gender as a covariate when this factor had presented significant differences at baseline. Eta squared was calculated as a measure of the effect size for analysis of variance (ANOVA) models and its threshold values were interpreted as small (0.04), moderate (0.25), and large (0.64) effects. All the statistical analyses were carried out using SPSS software (version 25.0; IBM Corp., Armonk, NY, USA).
# Results
Of the total of 161 students who completed the questionnaire at both study timepoints, 83.2% were female (n = 134). The mean participant age was 22.93 years (SD = 5.44, range = 20-48) and 97.5% (n = 157) had Spanish nationality. Most of the students had received initial training in communication skills during their undergraduate nursing training (95.7%, n = 154), and only four (2.48%) students reported not having received any prior training in communications in any context. None of the participants had previous experience with simulation programs.
## Communication skills variables
Before the simulation, our results showed moderate-high scores for the attitudes (M = 52.54; SD = 2.45), self-efficacy (M = 81.73; SD = 14.08), and communication skills (M = 87.41; SD = 8.08) perceived by the students No statistically significant differences were found according to participant age or any of the variables measured at baseline. All our other results are presented in . . Descriptive statistics of the main variables at baseline and differences depending on sex and age (n = 161).
## Effect of the simulation programme
When comparing the scores obtained at the beginning and end of the intervention programme (n = 161), we observed that both self-efficacy and the overall score for perceived communication skills had improved at the end of the simulation programme (F = 165.41; p < 0.001 and F = 50.54; p < 0.001, respectively), with a moderate effect size. The scores for all the dimensions of the communication skills scale improved after the intervention, except for the informational communication dimension (F = 1.20; p = 0.28). No improvement was observed for the attitudes variable (F = 0.67; p = 0.42, . . Pre-and post-intervention comparisons of the variables assessed in the sample.
## Pre-m (sd)
Post
# Discussion
The objective of this study was to evaluate whether a simulation programme using SPs to expose nursing students to situations involving patient chronicity and end-of-life contexts effectively improved their scores for the variables of attitude, self-efficacy, or communication skills. Our results indicate that the simulation programme proposed had improved the communication skills and self-efficacy related to communication, although there had been no improvement in the attitudes towards the communication variable.
The number of participants in our sample and the cohort composition were similar to other analogous studies. As is usual in studies on populations of nursing students, the percentage of female participants in this work was very high. Of note, most of the students participating in this study had received prior specific training in communication skills either during their instruction in nursing or externally. This factor may have impacted their baseline communication levels in our study, resulting in moderate-elevated scores for attitude, self-efficacy, and perceived communication skills. This is unusual, given that some previous studies have shown that nursing students frequently lack communication skills when they finish their undergraduate training, especially those related to complex situations of chronicity or end-of-life contexts.
Here, we found some differences at baseline with respect to gender, with women obtaining higher attitudes towards communication scores compared to men at the beginning of the study. This effect has already been reported in previous studies, such as the work by Cleland, Foster, and Moffaton medical students in Scotland, or that by Alotaibi and Alsaeedion nursing students in Saudi Arabia. Löffler-Stastka et al.pointed out that women tended to have better outcomes towards communication with patients in regard to psychosocial factors, while men tended to feel more insecure in this respect. These authors concluded that these differences had been present since the time of the participants' undergraduate training.
Regarding their perceived communication skills, the women in our study had better scores in the informational communication dimension than the men. However, there are discrepancies in the academic literature in this regard. For example, Graf et al.detected differences in the communication skills of medical students based on gender, with women standing out for non-verbal language and empathy. Similar results were found in a study by Strekalova et al.on nursing students from the USA, which found that women had greater skill in showing empathy than men. However, no differences were found in the communication skills of men and women in the study carried out by Shafakhah et al.on nursing students from Iran. It should also be noted that many studies conducted on nursing students did not consider gender differences given the relatively low proportion of male students usually found in these populations. Therefore, future work must continue examining the influence of gender roles on communication skills in nursing.
Regarding the changes produced in this work after the implementation of the programme, our results showed that self-efficacy in relation to communication increased after completion of the simulation programme. This finding is consistent with previous studies that used similar methodologies. For example, Karlsen et al.showed how nurses from Norwegian intensive care units described feeling more confident in their communication skills after participating in a simulation programme. Similarly, Kortes Miller et al.evaluated the effect of a simulation using a smart mannequin with informal caregivers, and concluded that self-efficacy related to communication skills with patients at the end of their lives had improved.
It is important to remember that according to the theory of Bandura, self-efficacy influences the acquisition, development, and retention of new skills. People with high levels of self-efficacy tend to attain higher levels of achievements and are more persistent in reaching these goals when faced with difficulties. Therefore, achieving high levels of self-efficacy in communication could help to improve the communication skills of nursing students in the future. In this line, a study conducted with nursing students in the United States showed a significant improvement in self-reported confidence and in the usage of communication skills after a simulation-based program with SPs.
In another vein, and in line with Bar-Sarmiento et al., the simulation programme we described in the present study also significantly improved the communication skills perceived by the nursing students in our cohort. In a study conducted on medical students in France, the participants also showed improved communication skills compared to those who followed the standard training programme. Some studies have suggested that communication skills should not only be assessed from the student's own point of view, but that structured external assessments such as the Objective Structured Clinical Examination (OSCE)should also be employed. For decades, the OSCE has been used for multiple purposes during the training of nursing students, including training in communication skills. However, recent research suggests that students' perceptions of their communication skills often tend to coincide with those of external evaluators, and so both perspectives must be considered.
Finally, no improvement was observed for the attitudes towards communication variable. These results differ with respect to what is reflected in other studies in which simulation programs improved attitudes towards communication, especially in the context of end-of-life communication. Nevertheless, we must consider that the initial scores on this scale were already quite high relative to the maximum possible score, and so there was not much room for improvement. Other studies carried out on health sciences professionals using the same scale have reported similar scores. Notwithstanding, the correlation between attitudes towards communication and the self-acquisition of skills was weak, meaning that this factor should be considered but should not be, a priori, a determining factor in these assessments.
Interestingly, during the COVID-19 pandemic, higher education institutions were forced to implement simulations as part of their learning methods. These included online virtual simulations of patient consultations or simulated clinical experiences, generally with satisfactory results in terms of student learning. However, few studies used SP simulations during the pandemic, and these studies did not provide data on the effect that this intervention had on students. The limitations caused by the COVID-19 pandemic meant that the implementation of simulations with SPs to help train nursing students was necessary so that students could be in contact with real patients.
In terms of the specific limitations of this work, it should be noted that, for teaching organisation reasons, the effect in the intervention group could not be compared to a control group. Thus, more experimental designs should be implemented in the future through the use of clinical trials. In addition, a notable percentage of participants (21.17%) did not complete the final questionnaire in this study. Previous work involving SPs have assumed that a maximum drop-out rate of 20% is adequate. Thus, although the losses we experienced in this current work were not exceptionally high, it would nonetheless be advisable to try to improve these response rates in future research in this field. Despite this, the final sample size was quite large, exceeding that of many previous studies with standardised patients.
# Conclusions
In conclusion, the simulation programme with SPs we implemented to address complex scenarios linked to chronic and end-of-life patient situations improved the communication self-efficacy and communication skills (empathy, respect, and assertiveness) of the nursing students included this work. The effect sizes were moderate, suggesting that the improvement was significant. In the baseline assessment, females had better scores for attitudes toward communication and in the process of informative communication compared to male students. In future work it will be important to analyse the influence of gender and attitudes towards communication as variables in the learning of communication skills in nursing students, as well as to introduce specific exercises to address and improve their attitudes toward communication and informative communication with patients. Institutional Review Board Statement: The study was conducted according to the guidelines set out in the Declaration of Helsinki and was approved by the Bioethics Committee at the University of Alicante (file number UA-2018-10-24).
Informed Consent Statement: Informed consent was obtained from all participants involved in the study.
# Data availability statement:
The data supporting the conclusions of this article will be available from the corresponding author upon request. |
Reconstruction of Dorsal Wrist Defects
QUESTIONS 1. What are the 3 most common skin malignancies of the hand and the wrist? 2. What are the challenges of dorsal wrist reconstruction? 3. What reconstructive options are used to repair dorsal wrist defects? 4. What are the potential complications of dorsal wrist reconstruction?
# Discussion
The differential diagnosis for the most common malignant skin lesions of the upper extremity includes, in order of incidence, squamous cell carcinoma, basal cell carcinoma, and melanoma. These cancers can typically be differentiated by their characteristic appearance and presentation; however, practitioners should be alerted to any atypical skin lesion. Although these are the most common malignancies seen on the dorsal arm, it is important to keep the differential broad, as the treatment can vary depending on the diagnosis. An excisional or incisional biopsy should be performed along with any indicated imaging studies for invasive tumors prior to any definitive reconstructive therapy.
The visible location and function of the dorsal wrist pose multiple challenges in reconstruction. The surrounding anatomy is responsible for both hand and forearm functions. Reconstructive efforts must preserve and protect underlying structures including bone, joints, muscles, tendons, nerves, and vasculature. The tenets integral to dorsal wrist reconstruction are that of durable and pliable coverage to withstand the constant use and movement of the wrist, repairing functional anatomy units, as well as correcting aesthetic challenges of matching skin texture, pliability, thickness, and coloring.
The reconstructive ladder should be considered when planning dorsal wrist reconstruction. Primary closure is the most desirable option, but for larger defects this is often not a feasible option. Healing by secondary intention is a viable option for smaller defects without any vital structure exposure. Skin grafting is a practical option when the defect does not involve bone or tendon lacking periosteum or peritenon, respectively. The surgeon should note that skin grafts may be susceptible to breakdown if the repair is in the dorsal wrist crease, an area of frequent motion and a location of watches and bracelets. Local and regional flaps are commonly the option of choice for dorsal wrist reconstruction. They can be used to cover vital structures, are more durable than grafts, have good color match, and can be sensate. Examples of these flaps include local cutaneous or perforator, posterior interosseous, radial forearm, ulnar forearm, and pronator quadratus flaps. Larger defects of the forearm may merit more extensive reconstruction using distant options such as the groin flap or free flaps.
It is important to keep potential complications in mind when considering each reconstructive route. Split-thickness skin grafts tend to contract and stiffen in comparison with full-thickness grafts, making them less suitable for dorsal wrist reconstruction. In addition, skin grafts can be less durable over time than other options. Distant flaps have subsequent donor sites that incur increased morbidity. As with all reconstructions, partial or complete flap loss may occur if tissue perfusion is poor. The plastic surgeon should ensure an optimized patient and wound bed prior to dorsal wrist reconstruction. In the case of malignancy, surgical margins should be negative and any traumatic wound should be thoroughly debrided to healthy tissue. Finally, scar direction and placement are important to consider as longitudinal scars that cross the dorsal wrist crease are prone to webbing. |
A Rare Case of Type I RenalTubular Acidosis with Membranous Nephropathy Presenting as Hypokalemic Paralysis
Type 1 renal tubular acidosis (RTA), or distal RTA (dRTA), is a disorder of renal tubular acidification, which is generally asymptomatic but may rarely present as hypokalemic paralysis. Here, we report the case of a young male who presented with sudden onset weakness of all 4 limbs and a 2-month history of swelling of the legs. An investigation revealed hypokalemia, metabolic acidosis, and nephrotic syndrome. Additional analyses revealed normal anion gap metabolic acidosis with a positive urine anion gap and dRTA. Renal biopsy showed evidence of membranous nephropathy (MN). The patient's weakness improved with potassium supplements. Normalization of the serum potassium level and disappearance of proteinuria were established with an ACE inhibitor and potassium supplementation. This case is an unusual combination of dRTA with MN coupled with the rare presenting symptoms of hypokalemic paralysis and medullary nephrocalcinosis.
# Introduction
Type 1 renal tubular acidosis (RTA), or distal RTA (dRTA), is a disorder of renal tubular acidification, characterized by hyperchloremic metabolic acidosis with a normal serum anion gap and a persistently high urinary pH. Hypokalemia has been reported in 28-53% of patients with type 1 RTA [bib_ref] Hypokalemic paralysis associated with distal renal tubular acidosis, Hattori [/bib_ref]. dRTA is generally asymptomatic but rarely may present as hypokalemic paralysis. Other than the hereditary and idiopathic varieties, most cases of dRTA are secondary to systemic disorders such as Sjögren's syndrome, hyperglobulinemia, chronic active hepatitis, or lupus. In nondiabetic adults, membranous nephropathy (MN) is the most common cause of nephrotic syndrome, accounting for up to one-third of biopsy diagnoses. MN in adults is most often idiopathic (approximately 75% of cases). About onethird of cases are secondary to infection, rheumatologic disorders, neoplasm, and exposure to drugs and toxins. Here, we report a hitherto unusual combination of dRTA with MN presenting as hypokalemic paralysis and medullary nephrocalcinosis.
## Case report
A 20-year-old male presented with a 2-month history of on and off swelling of both legs and sudden onset weakness of all 4 limbs without any history of loss of consciousness, seizure, headache, sensory loss, or bowel and bladder involvement. There was no history of such a weakness. Additionally, there was no history of dryness of the mouth and eyes (suggestive of Sjögren's syndrome), no joint pain, oral ulcers, hair loss, or photosensitivity (suggestive of lupus). A neurological examination revealed normal cranial nerves and higher mental functions. Power in the upper and lower limbs was 2/5 and 1/5, respectively. His deep tendon reflexes were diminished, with no plantar response; however, sensory function remained intact.
The patient's laboratory profile was as follows: hemoglobin: 10.8 g/dl, total leukocyte count: 14,300/mm 3 , platelet count: 2.8 × 10 5 /mm 3 , urinary protein: 3+, urinary sugar: 0, urine microscopy: white blood cell count: 4-6/high-power field, red blood cell count: 2-4/high-power field, urinary pH: -6.5, 24-hour urinary protein: 3.3 g/day, serum albumin: 2.1 g/dl, serum cholesterol: 526 mg/dl, serum triglycerides: 346 mg/dl, C3: 79.6 mg/dl (normal range: 80-160), C4: 62.8 mg/dl (normal range: 20-40), HBsAg: negative, anti-HCV: negative, HIV I and II: negative, anti-Ro and anti-La antibodies: negative, blood urea: 29 mg/dl, serum creatinine: 1.2 mg/dl, random blood sugar: 88 mg/dl, corrected serum calcium: 9.4 mg/dl, serum sodium: 143 mEq/l, serum potassium: 2.0 mEq/l, serum chloride: 120 mEq/l, serum PO4: 4.3 mEq/l, arterial blood gas: pH 7.2, pCO2: 31 mm Hg, pO2: 104 mm Hg, HCO3: 12.5 mEq/l, anion gap: 10.5 mEq/l (normal range: 10-12).
The urine anion gap (UAG) [Na + + K + -Cl -(70 + 25 -65 = 30)] was positive. Urinary pH was <5.5 and the fractional excretion of bicarbonate (FeHCO3) was found to be 2, after intravenous infusion of sodium bicarbonate. Thus, the diagnosis of dRTA was established. Ultrasonography showed normal-sized kidneys with nephrocalcinosis of the medulla (suggestive of type 1 RTA), mild right-sided pleural effusion, and moderate ascites. A renal biopsy showed a thickened basement membrane with subepithelial spikes [fig_ref] Figure 2: Kidney biopsy specimen shows a thickened basement membrane with subepithelial spikes, diagnostic... [/fig_ref]. The interstitium showed mild mononuclear cell infiltrate and fibrosis with tubular involvement [fig_ref] Figure 3: Diffuse tubulointerstitial mononuclear infiltrate [/fig_ref]. Immunofluorescence was positive for IgG and C3 [fig_ref] Figure 4: Immunofluorescence shows IgG positivity and fine, granular deposition of IgG along the... [/fig_ref]. Thus, the diagnosis of MN was made.
## Treatment
For hypokalemic paralysis, the patient was initially treated with 60 mEq potassium chloride (KCl) orally, followed by 30 mEq KCl orally every 30 min until serum potassium had normalized. After potassium supplementation, the patient's weakness improved within 2 days and he was able to walk again. Repeat investigations showed serum potassium: 4.8 mEq/l (normal), serum chloride: 112 mEq/l, arterial blood gas: pH 7.25, HCO3: 15.7 mEq/l, anion gap: 11.3 mEq/l (normal 10-12). Due to proteinuria and hypokalemia, 5 mg enalapril b.d. was started. After 24 h, proteinuria was less than 4 g/day and a renal function test was found to be normal; immunosuppressive therapy was not initiated, as per KDIGO guidelines. The patient responded well to treatment with 5 mg enalapril b.d. and 10 ml potassium citrate t.d.s. Normal potassium levels and no proteinuria have been observed to date (24 months of follow-up). ANA (antinuclear antibody), dsDNA, anti-Ro, and anti-La antibody tests were done to establish the etiology, but were found to be negative. Schirmer's test was also negative (more than 10 mm of moisture on the filter paper in 5 min was noted).
# Discussion
Hypokalemic paralysis is primarily the result of either hypokalemic periodic paralysis (HPP) caused by an enhanced shift of potassium into cells or secondary HPP resulting from excessive gastrointestinal or urinary potassium loss. Secondary HPP requires higher doses of KCl [bib_ref] A simple and rapid approach to hypokalemic paralysis, Lin [/bib_ref]. Our patient required a large dose of KCl for the correction of hypokalemic paralysis. In the presence of hypokalemia, urinary potassium <20 mmol/day suggests extra renal loss, whereas urinary potassium >20 mmol/day suggests renal loss, as was the case in our patient. The UAG represents an indirect index of urinary NH4+ excretion in patients with hyperchloremic metabolic acidosis [bib_ref] Renal tubular acidosis: the clinical entity, Soriano [/bib_ref]. The UAG is defined by the equation: UAG = Na + + K + -Cl - [bib_ref] Renal tubular acidosis, Penny [/bib_ref]. In our patient, the UAG was positive (30 mmol/l), suggestive of renal loss of potassium.
Though RTA is a tubulointerstitial kidney disease, it may also be seen in primary glomerulopathies. Primary glomerulopathies may cause damage to tubules and the interstitium with functional derangement. In glomerular diseases associated with proteinuria, tubular cells may be activated by elevated levels of normal and novel urinary proteins. Activated tubular cells may synthesize proinflammatory mediators, especially monocyte chemoattractant molecules (e.g. MCP-1, RANTES, complement component, and fractalkine) and fibrosis-promoting molecules (e.g. endothelin, TGF-β, and angiotensin II). Damage to the tubular basement membrane facilitates the passage of tubular-derived products into the interstitium and peritubular capillaries spaces. In the distal nephron, protein casts may obstruct urinary flow and aggravate tubulointerstitial damage [6]. Glomerulus-derived cytokines activating tubular epithelial cells reduce peritubular blood flow, leading to tubulointerstitial ischemia and hyperfunction of remnant tubules. Peritubular ischemia contributes more to tubular damage than proteinuria in immune-mediated glomerulonephritis . Occasionally, the same inflammatory mechanism of glomerulonephritis may also affect the tubules. For example, dRTA occurring in immunological disorders such as Sjögren's syndrome and systemic lupus erythematosus is thought to result from an interstitial nephritis which various investigators have ascribed to mononuclear cell infiltration, deposition of antigen-antibody complexes along tubular basement membranes, and the formation of antibodies directed against the tubular basement membrane. A literature search revealed cases in which features of dRTA preceded other manifestations of systemic lupus erythematosus by 4-6 years [8]. In our patient, lupus and Sjögren's syndrome were ruled out. A case of dRTA with MN has been reported . HPP due to proximal RTA with membranoproliferative glomerulonephritis has also been reported . But dRTA with MN presenting as hypokalemic paralysis has not been reported. Long-term correction of the hypokalemia in our patient was important not only to overcome the neuromuscular impairment, but also to prevent further deterioration of kidney function. It has been suggested that hypokalemia stimulates interstitial deposition of complement components (C3 and C5ba), which is conducive to the aggravation of renal interstitial damage .
About 30-35% of patients with idiopathic MN eventually undergo spontaneous remission of nephrotic syndrome; therefore, it is reasonable to delay specific immunosuppressive therapy for at least 6 months, instead utilizing supportive therapy including RAS blockade unless the patient has unexplained rapid deterioration in kidney function or there are complications related to uncontrolled nephrotic syndrome. Our patient was treated with RAS blockade and responded well. KDIGO recommend that initial immunosuppressive therapy be started only in patients with nephrotic syndrome and when at least one of the following conditions is met: urinary protein excretion persistently exceeds 4 g/d and remains at over 50% of the baseline value, and/or it does not show progressive decline despite antihypertensive and antiproteinuric therapy during an observation period of at least 6 months (1B; level 1: recommend; B: moderate quality of evidence).
dRTA is a clinical syndrome consisting of hypokalemia, hyperchloremic metabolic acidosis, inability to lower urinary pH below 5.5 during acidemia nephrolithiasis, and nephrocalcinosis. Hypokalemia is frequently seen in dRTA [12] and may sometimes be severe enough to produce muscle weakness, as in our case. Both hypercalciuria and especially hypocitraturia contribute to the development of nephrocalcinosis and nephrolithiasis . It is very evident that our patient had dRTA (type 1 RTA) with medullary nephrocalcinosis. Correction of acidosis with alkali therapy (usually potassium citrate) generally improves both calcium and potassium balance, and prevents stones and nephrocalcinosis. In contrast to the high alkali requirement in proximal RTA, only daily acid load (1-2 mEq/kg/day) has to be replenished with alkali therapy in dRTA.
# Conclusion
Primary glomerulopathies may cause tubulointerstitial damage, which may subsequently lead to the development of RTA. RTA may be the first manifestation of glomerulopathy, preceding other clinical features. We have reported the case of an unusual association of dRTA with MN, with the rare presentation of hypokalemic paralysis, which was successfully treated.
[fig] Figure 2: Kidney biopsy specimen shows a thickened basement membrane with subepithelial spikes, diagnostic of MN. Methenamine silver stain. [/fig]
[fig] Figure 3: Diffuse tubulointerstitial mononuclear infiltrate (arrows show mononuclear cells infiltrating the base of the tubule) and mild fibrosis. [/fig]
[fig] Figure 4: Immunofluorescence shows IgG positivity and fine, granular deposition of IgG along the outer surface of the capillary walls. [/fig]
|
Optimal temperature overshoot profile found by limiting global sea level rise as a lower-cost climate target
This PDF file includes:Note S1. This auxiliary material contains three supplementary figures and one supplementary table, which are referred to in the main manuscriptFig. S1. Historical evolution and future projections as simulated with our three-layer ocean model.Fig. S2. Simulated ocean-temperature change with an instantaneous quadrupling of atmospheric CO 2 (relative to preindustrial conditions) and then held fixed over 300 years.Fig. S3. Simulated global thermosteric SLR with an instantaneous quadrupling of atmospheric CO 2 (relative to preindustrial conditions) and then held fixed over 300 years.Table S1. Default parameters for the three-layer ocean model.
## This pdf file includes:
Note S1. This auxiliary material contains three supplementary figures and one supplementary table, which are referred to in the main manuscript . Historical evolution and future projections as simulated with our three-layer ocean model.. Simulated ocean-temperature change with an instantaneous quadrupling of atmospheric CO 2 (relative to preindustrial conditions) and then held fixed over 300 years.. Simulated global thermosteric SLR with an instantaneous quadrupling of atmospheric CO 2 (relative to preindustrial conditions) and then held fixed over 300 years. . Default parameters for the three-layer ocean model. (d) give median values and likely ranges for projections of global-mean surface warming in 2100 from IPCC AR5 .2, and global sea-level rise and its rate in 2100 from IPCC AR5 .5.
## S note
which are referred to in the main manuscript.These plots and tables have been created alongside with the plots in the paper to provide further information and insight. We derive parameters from a 300-year simulation with an abrupt 4×CO 2 simulation of MPI-ESM-LR. . Default parameters for the three-layer ocean model.
## Supplementary figures and tables
|
Prevalence, Incidence and Ecological Determinants of Diabetic Retinopathy in Iran: Systematic Review and Meta-analysis
Purpose: To estimate the pooled prevalence and incidence of diabetic retinopathy (DR) in Iran and to investigate their correlations with the Human Development Index (HDI), healthcare access (i.e., density of specialists and sub-specialists), and methodological issues. Methods: Electronic databases such as PubMed, Embase, Scopus, Web of Science, Google Scholar, and local databases were searched for cohort and cross-sectional studies published prior to January 2018. Prevalence and incidence rates of DR were extracted from January 2000 to December 2017 and random effects models were used to estimate pooled effect sizes. The Joanna Briggs Institute critical appraisal tool was applied for quality assessment of eligible studies. Results: A total of 55,445 participants across 33 studies were included. The pooled prevalence (95% CI) of DR in diabetic clinics (22 studies), eye clinics (4 studies), and general population (7 studies) was 31.8% (24.5 to 39.2), 57.8% (50.2 to 65.3), and 29.6% (22.6 to 36.5), respectively. It was 7.4% (3.9 to 10.8) for proliferative DR and 7.1% (4.9 to 9.4) for clinically significant macular edema. The heterogeneity of individual estimates of prevalence was highly significant. HDI ( < 0.001), density of specialists ( = 0.004), subspecialists ( < 0.001), and sampling site ( = 0.041) were associated with heterogeneity after the adjustment for type of DR, duration of diabetes, study year, and proportion of diabetics with controlled HbA1C. Conclusion: Human development and healthcare access were correlated with the prevalence of DR. Data were scarce on the prevalence of DR in less developed provinces. Participant recruitment in eye clinics might overestimate the prevalence of DR.
# Introduction
Diabetic retinopathy (DR) is the leading cause of vision loss in adults aged 20-74 years, and remains one of the foremost causes of blindness and visual impairment worldwide.Despite significant development in the prevention and control of DR, the proportion of DR increased by 7.7% among all declining causes of blindness between 1990 and 2015.The prevalence of DR strongly correlates with both the duration of diabetes and the level of glycemic control.Therefore, timely management of DR stemming from screening programs, appropriate referral for treatment, and improving healthcare accessibility are important in preserving vision in diabetics.Although the treatment of DR can decrease the risk of visual loss by 60%, it imposes a heavy cost to the healthcare system.Despite some improvements in diagnostic assessment and treatment options,the lack of qualified healthcare services along with a Westernized lifestyle have caused the burden of DR to be high and on the rise in developing countries.DR is a pressing public health matter, probably due to suboptimal access to diabetes care services such as eye care professionals and eye care services, especially in low-to middle-income countries.Low human development might be another correlate of the increasing burden of DR. To the best of our knowledge, no study has investigated the correlation between human development and DR. The only study on this subject assessed the association between the Human Development Index (HDI) and the number of studies published on DR.HDI is abstracted from income, education, and life expectancy markers and ranks areas into different levels of human development.Despite the high prevalence of diabetes, there are few reliable national studies on the incidence, prevalence, and correlates of DR in developing countries.Of note, Iran is a country in transition, having a high variety of healthcare access options and human development as well as a huge variation in the prevalence and incidence of DR across its geographic regions.Therefore, besides assessing the prevalence and incidence of DR, their adjusted correlations with HDI and healthcare access were investigated in the current observational study.
# Methods
## Protocol and registration
The Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines were followed.The study protocol was approved by the Shiraz University of Medical Sciences (ethical approval code: IR.SUMS.MED.REC.1397.256).
## Eligibility criteria
Observational studies (prospective or retrospective cohort and cross-sectional) were included if they provided sufficient information about the incidence and prevalence of DR and clinically significant macular edema (CSME). No restriction was applied on the year of publication or type of diabetes, and all studies published in Persian or English language were included. These two languages covered all studies published about the Iranian population and we did not find studies in other languages.
congresses that were held during the study period around the world and specifically in Iran, and we selected and hand searched the abstract books that were obtainable as much as possible. We also searched university websites for thesis and reports that were related to the subject during the study period. References of all included studies were also searched for potentially eligible studies. In cases where the full text of an article was unavailable, the corresponding author was contacted. Documents were catalogued using Endnote X4.
## Study selection
In case of repeated publications from one study, the newest publication was included. Titles, abstracts, and the full texts of retrieved articles were reviewed independently by two experts in the field and eligible articles were selected. Studies on children, pregnant women, or non-Iranian populations were excluded as well as experimental studies, secondary studies, letters, case-reports, case-series, commentaries, and editorials. Clinical trials were also excluded. Studies with a non-representative sample or irrelevant comorbidities were also excluded. Also, in the case of disagreement regarding exclusion or inclusion of a study, a consensus was reached through discussion between the authors.
## Data extraction and risk of bias assessment
We performed a comprehensive literature review and designed a conceptual framework. An excel (MS Office) data sheet based on clinical principles was prepared for data extraction using our framework. The following variables were included in the data extraction form: first author, publication year, study year, study location (i.e., province/district), urbanization ratio (number of participants from the urban area/number of participants from the rural area; based on the data presented in each individual study), sampling site (diabetes clinic, eye clinic, or general population), sampling design (random, multistage, convenient, unknown), study design (cross-sectional, cohort), sample size (overall and for subgroups), age range (or mean age), duration of study, gender (or female/male ratio), diagnostic methods of DR,proportion of diabetics (in population-based studies), type of diabetes (or type I/type II ratio), type of DR (i.e., non-proliferative diabetic retinopathy and its stage (mild, moderate, or severe), proliferative diabetic retinopathy (PDR), CSME, mean HbA1C (overall, among DR patients, and among non-DR patients), number of DR patients (or estimated prevalence and its standard error), number of incident cases of DR (or cumulative incidence rate; only for cohort studies), number of person-years of follow-up (in cohort studies), duration of DM (overall, among DR patients, and among non-DR patients), mean age at the onset of DM, mean duration of DR, and proportion of patients with newly diagnosed DM. The aforementioned data were extracted (if available) and 25% of the extracted data were randomly cross-checked by another author. Also, in the case of disagreement regarding data extraction, a consensus was reached through discussion between the authors. Since several studies had not reported exact values for some variables, more than 30 disagreements on the most appropriate estimates for these missing values were discussed in the team.
The Joanna Briggs Institute ( JBI)'s critical appraisal toolwas applied for quality assessment of eligible studies. Quality assessments and critical appraisals were performed by two different authors independently. In the case of disagreement regarding the quality score, a consensus was reached through discussion between the authors (4 studies out of the included 33 studies).
## Data on human development and healthcare access
The density of specialists and subspecialists (numbers of specialists and subspecialists in the healthcare system of each province to the total population in that province) was retrieved from a recent reliable report by Haghdoost et alas indices of healthcare access. Density ratios were categorized into quantiles. Haghdoost et al gathered the number of physicians based on the questionnaires filled out by medical universities all around Iran. Their study was a part of a project to define the national treatment map of Iran in 2025 (Naghsh-e Rahe Darman-e Iran). In order to control the precision of completing the questionnaire, their data was crosschecked with the Medical Registry Information System and different medical insurance companies.HDI indices (less developed, moderately developed, and developed) were retrieved for each province according to the study performed by Safaeipour et aland used as an independent variable in meta-regression modeling.
## Data preparation and statistical analysis
Point estimates of prevalence were extracted or calculated as the number of patients with DR divided by the number of patients with DM. Additionally, prevalence estimates of DR in the population, regardless of diabetic status, were estimated as the number of participants with DR divided by the number of total participants in populationbased studies. A 95% confidence interval (CI) for all individual point estimates of prevalence was estimated where it was not mentioned.Cumulative incidence proportions (per 100 person-years) were calculated as the number of new DR cases divided by the number of at-risk person-years (i.e., number of study subjects multiplied by number of follow-up years).
To ensure the independence of point estimates in primary studies as well as to prevent repeated counting of participants in primary studies, only one of the overall or subgroup point estimates of each primary study was included in the meta-analysis and meta-regression models. The heterogeneity of individual estimates of prevalence was assessed according to the I-square statistic above 50%.In cases of high heterogeneity, correlates were conceptualized. Then, the significance and magnitude of their correlation were investigated using the random-effects meta-regression technique.
Subgroup analyses were performed according to the most important correlates of heterogeneity if applicable (i.e., sampling site, geographical location, HDI and healthcare access). Due to persistent heterogeneity (even after subgroup analysis), individual estimates of prevalence and incidence were pooled using the DerSimonian and Laird randomeffects modeling method. Publication bias was investigated using Begg's and Egger's tests. Data analysis and calculations were performed using Stata software, version 11.2 (Stata Corporation, College Station, TX, USA). A two-sided < 0.05 was considered as statistically significant. We also provided appropriate tables and graphs for showing our results (i.e., included studies, shortage in studies, methods applied for the diagnosis of DR by primary studies, study flowchart, and study forest plot by HDI categories). The study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO), under the registration number CRD42018104626.
# Results
The initial search resulted in 2,153 records. Of these, 426 records were included in the fulltext review process, and finally, 33 studies were included in the meta-analysis.
## Overview of included studies
Thirty cross-sectional and three cohort studies were included in the analysis, representing an overall number of 55,445 diabetic patients including 17,155 patients with DR []. Among cross-sectional studies, seven had a populationbased sampling design representing 24,623 participants including 5,657 diabetic patients and 2,049 patients with DR. Included cohort studies represented 1,174 diabetic patients (equivalent to 5,400 person-years) and 613 incident cases of DR.
## Assessment of heterogeneity in individual estimates for prevalence of diabetic retinopathy
In cross-sectional studies conducted in diabetes clinics, individual estimates for the prevalence of DR were significantly heterogeneous for overall diabetics (I-square, 99.5; < 0.001; the heterogeneity was similar between genders [I-square, 99.4; < 0.001]). Also, individual estimates for the prevalence of DR were highly heterogeneous in studies conducted in eye clinics (I-square, 98.0; < 0.001) and population-based studies (I-square, 95.7; < 0.001). Additionally, in three cohort studies, the estimates of individual cumulative incidence of DR were statistically heterogeneous with a highly significant I-square of 97.9 ( < 0.001).
## Determinants of dr prevalence (correlates of heterogeneity of individual estimates)
According to the random-effects meta-regression model, HDI (as an ordinal variable of tertile of HDI; adjusted OR: 0.12, 95% CI: 0.05 to 0.34, < 0.001), density of specialists (as an ordinal variable including quintile of density ratio; Risk of bias was not associated with heterogeneity ( = 0.683). Type of diabetes was not a significant determinant of heterogeneity for individual estimates of DR prevalence ( = 0.10).
Due to the lack of enough data to achieve individual estimates of cumulative incidence, metaregression modeling for cumulative incidence was not applicable. Due to significant heterogeneity among studies, the analysis was performed through subgroup analysis based on the most important correlates of heterogeneity.
## Prevalence of dr among patients referred to diabetic clinics
The overall pooled prevalence rate of DR among diabetic patients was
## Prevalence of dr among patients referred to eye clinics
The overall pooled prevalence of DR among diabetic patients was
## Prevalence of dr among patients in population-based studies
The overall pooled prevalence of DR was
## Prevalence of dr in the general population
The overall pooled prevalence of DR (according to the population-based studies) was 3.6% (95% CI: 2.4, 5.0) and 3.5% (95% CI: 1.5, 6.4) among male subjects and 3.6% (95% CI: 2.1, 5.6) among female subjects.
## Cumulative incidence rate of dr in diabetics
The overall pooled cumulative incidence rate (per 100 person-years) of DR was 11.7% (95% CI: 8.0, 15.9) and 9.6% (95% CI: 7.8, 11.8) among male subjects and 8.7% (95% CI: 7.5, 10.0) among female subjects []. The pooled cumulative incidence rates (per 100 person-years) for NPDR and PDR were 11.7% (95% CI: 9.1, 14.9) and 0.2% (95 % CI: 0.0, 1.1), respectively.
## More details on the association of hdi and the prevalence of dr
The estimation of linear correlation coefficient between HDI and the prevalence of DR among diabetics was -0.18 with a of 0.029 (a relatively low but significant linear correlation) [].
## Reporting and methodological shortcomings
One study used the survey data analysis technique to consider multistage sampling design.Two studies reported age-adjusted estimates.Only 33.3% (n = 11) of the studies reported their DR results by gender [].
## Assessment of dr
According to the current results, 54.54% (n = 18) of studies performed indirect ophthalmoscopy with pupillary dilatation for the evaluation of DR. Others probably used the same method; however, this matter was not clearly stated [].
## Publication bias
There was no significant publication bias (Begg's test = 0.824; Egger's test = 0.075) in the current study.
# Discussion
In the current study, the pooled prevalence and incidence of DR including PDR and NPDR among Iranian diabetic subjects referred to diabetes clinics and eye clinics and the pooled prevalence of DR among diabetics retrieved form populationbased studies were determined based on the English language studies only. Persian articles were excluded due to poor quality score in our assessment. We found no publication bias among the included studies. The human development, access to subspecialists and specialists, and site of study sampling had a correlation with the prevalence of DR among diabetics.
Studies from other parts of the world reflect significant differences in the prevalence of DR depending on the factors such as ethnicity, demography, and access to healthcare systems.According to the current results, an increase in HDI was independently correlated with lower DR prevalence. In order to avoid the bias of ecological inference fallacy, this finding could only be interpreted from an ecological point of view. Therefore, we cannot directly relate the DR prevalence in individuals to the HDI in each province. However, HDI may potentially be a good index of quality of diabetes care in developing countries such as Iran at a national level. This finding is consistent with previous evidence from low-income regions of developed countries.The prevalence of DR and PDR/NPDR ratio were higher in eye clinics in comparison to the DM clinics. It might be due to the higher detection rate of DR, especially PDR in eye clinics.In other words, more advanced patients are usually referred to the eye clinics. The density of subspecialists was negatively correlated with the prevalence of DR. This could be due to health literacy, greater access to healthcare services in regions where subspecialists work, leading to more effective diabetes control. This finding is in accordance with previous reports.Of note, many confounding factors such as social factors could be associated with the density of subspecialists and prevalence of DR. It might also be due to the interest of subspecialists to be in areas with higher socioeconomic status in which inhabitants may also have a better access to healthy food, causing more controlled DM and less prevalence of DR.
The pooled population-based prevalence of DR among diabetics was close to the maximum estimates of DR prevalence reported in developing countries such as Pakistan (29% vs 25-29%), while it was lower than the prevalence reported in developed countries.Population aging and Westernized lifestyle could have been among the factors increasing the prevalence of diabetes and DR in Iran and other developing countries.In addition, the higher prevalence of DR among male subjects could be attributed to lifestyle habits such as cigarette smoking, which is consistent with studies conducted in India and Nepal.The pooled prevalence of DR among diabetics referred to eye clinics was significantly higher than that in diabetes clinics. Routine screening by an ophthalmologist is not a common practice in developing countries.Thus, this higher prevalence may be due to higher rates of referral for patients presenting with visual symptoms. There is a need for improving screening programs in primary healthcare services and communication between primary healthcare providers and ophthalmologists to ensure diabetics receive timely ophthalmic examinations.
Based on the results of the current study, the estimation of DR prevalence among diabetics in eye clinics may not be a good indicator of DR prevalence among diabetics; however, a comparison of DR prevalence in diabetic and eye clinics could help determine the sensitivity and specificity of referrals to eye clinics by clinicians and over-/underutilization of eye care in developing countries.
Although, overall and gender-specific pooled prevalence rates of DR among diabetics referred to diabetes clinics were relatively higher than those in population-based studies, these differences were not meaningful. In addition, the female/male ratio of estimated DR prevalence in DM clinics studies was almost equal to the population-based studies (88% vs 80%). Therefore, considering the difficulties in conducting robust population-based studies, it is reasonable to estimate the prevalence of DR in diabetes clinics, especially in developing counties such as Iran.
The pooled estimate of DR incidence among diabetics in Iran was less than recent reports from developed countries such as Canada, the UK, and Spain.Moreover, the estimated incidence was relatively higher than India, South Korea, and Denmark.According to the current study, the available data on the incidence of DR in Iran is inadequate; further studies are needed to determine the incidence of DR in Iran.
Among the eligible studies included in the current meta-analysis, only one study reported the prevalence of DR among Type I diabetics.Accordingly, the pooled estimates provided in the current study are mostly representative of the prevalence of DR among Type II diabetics. More studies on the incidence and prevalence of DR among Type I diabetics are needed.
The results of this study showed that there is a lack of research on the prevalence of DR in less developed provinces, such as Sistan and Balochistan, Bushehr, Hormozgan, Khorasan, Khozestan, Kermanshah, Kordistan, Kohkiloieye, Chaharmahal and Bakhtiyari, and Lorestan. Missing data from these provinces could have affected the results of the current study. In addition to the paucity of data in some provinces, there is a significant heterogeneity among studies, which means that making an accurate single prevalence estimate of DR is not possible. The pooled prevalence of DR was 29%. However, the prevalence rate varied from 9% to 64.1%. Thus, it might not be completely representative of the actual DR prevalence.
In addition to the aforementioned factors, the method used for detecting DR is a major factor that can influence prevalence estimates. It is crucial to know which specific diagnostic method (i.e., dilated fundus examination, direct or indirect ophthalmoscopy, digital imaging, etc.) was used in each study.The ophthalmic examination methods were not adequately explained in at least 12 included studies. In the course of DR, patients might develop maculopathy with no significant change in vision and may not seek medical care. Optical coherence tomography (OCT) is an important test in the evaluation and management of diabetic macular edema and can detect subclinical macular edema. Missing OCT evaluation could lead to many diabetics remaining undiagnosed in the early stage of DR.The current study has some limitations. The pooling of data from different sources introduced potential sources of heterogeneity that could impact accuracy. Various studies could have different inclusion criteria, sample selection, or study protocols. For example, the sample from a diabetes clinic differs from an eye clinic or population-based studies. The pooled estimate of DR prevalence by gender in the current study was sometimes not compatible with the total prevalence in the subgroups, which was due to the lack of reporting DR by gender in some studies. Studies in which the diagnosis of DM was based on patient selfreport, without lab test confirmation, could have led to an overestimation of DR prevalence due to the exclusion of undiagnosed diabetes from the study sample. Although it is desirable to have the HDI and density of specialists and subspecialists at the time of each study to calculate the correlations and perform the analyses, only two up-to-date and available studies were used for estimating the value of both mentioned variables. Also, the absence of studies from the eastern, western, and southern regions of Iran could have also affected the validity and generalizability of the current findings. Although our findings on the prevalence and incidence of DR may not be generalizable to all countries, it could be useful for developing countries, especially regions with similar socioeconomic, demographic, cultural, and geographic conditions.
## Summary
Despite the scarcity of research in less developed regions, a reasonable estimate for the prevalence of DR among Iranian diabetic subjects is around 30% (29% among female and 37% among male subjects). HDI, density of specialists and subspecialists, and sampling site were independently correlated with the prevalence of DR in Iran. The most reliable evidence on DR prevalence is likely to be retrieved from diabetes clinics in developing countries. Furthermore, providing a list of essential items for reporting the descriptive epidemiology of DR and performing studies in less developed regions could generate stronger evidence for health policy programs. |
A Microsatellite Guided Insight into the Genetic Status of Adi, an Isolated Hunting-Gathering Tribe of Northeast India
Tibeto-Burman populations of India provide an insight into the peopling of India and aid in understanding their genetic relationship with populations of East, South and Southeast Asia. The study investigates the genetic status of one such Tibeto-Burman group, Adi of Arunachal Pradesh based on 15 autosomal microsatellite markers. Further the study examines, based on 9 common microsatellite loci, the genetic relationship of Adi with 16 other Tibeto-Burman speakers of India and 28 neighboring populations of East and Southeast Asia. Overall, the results support the recent formation of the Adi subtribes from a putative ancestral group and reveal that geographic contiguity is a major influencing factor of the genetic affinity among the Tibeto-Burman populations of India.
# Introduction
Northeast India has always been a hotspot for population geneticists due to its unique and strategic geographic location and the presence of linguistically, culturally and demographically diverse populations practicing varied occupations (from huntinggathering to settled agriculture). Due to their relative geophysical isolation (flanked by the Eastern Himalayas in the northern and the Bay of Bengal in the southern region), leading to limited external gene flow, these diverse populations retain a unique population structure which in turn is expected to reflect in their gene pools.
This region exhibits linguistic diversity (represented by Tibeto-Burman, Austro-Asiatic and Indo-European language families) which can be attributed to diverse socio-cultural influences, extensive population interactions and putative long history of migrations experienced by the region in the past [bib_ref] People and Languages, Rapson [/bib_ref]. The Tibeto-Burman speaking populations predominate the region, about 2% of the total Indian population [bib_ref] Structure of human populations in India, Malhotra [/bib_ref] , representing a significant component of the biological diversity of the peopling of India. They exhibit vast diversity with respect to culture, language, subsistence economy and population structure variables like size, growth, distribution, marriage patterns and degree of endogamy [bib_ref] Northeast India: A profile, Majumdar [/bib_ref]. These populations are of significance in understanding the peopling of India and in comprehending the relationship prevailing among the regional populations, as well as the relationship of these populations with the neighboring East/ Southeast Asian groups to whom they are morphologically, ethnohistorically and linguistically affiliated [bib_ref] People and Languages, Rapson [/bib_ref] [bib_ref] Northeast India: A profile, Majumdar [/bib_ref]. In view of their importance, many researchers have earlier attempted, using classical and molecular genetic markers, to address various population genetic issues pertaining to these regional groups. The studies were however sporadic and restricted to only few regional populations [bib_ref] Haemoglobin E in six populations of Assam, Das [/bib_ref] [bib_ref] The genetic composition of the people in Eastern India, Roychoudhury [/bib_ref] [bib_ref] Investigations on the variability of haptoglobin, transferrin and Gc polymorphisms in Assam, Walter [/bib_ref] [bib_ref] Haemoglobin E distribution in ten endogamous population groups of Assam, Deka [/bib_ref] [bib_ref] Genetic relationships of the populations in eastern India, Roychoudhury [/bib_ref] [bib_ref] Genetic heterogeneity in northeastern India: Reflection of tribe-caste continuum in the genetic..., Kumar [/bib_ref] [bib_ref] Patterns of genetic diversity at the nine forensically approved STR loci in..., Dutta [/bib_ref] [bib_ref] Genetic relationships among some tribal groups inhabiting the north eastern, eastern and..., Chakrabarti [/bib_ref] [bib_ref] The emergence and dispersal of Mongoloids, Roychoudhury [/bib_ref] [bib_ref] Mitochondrial DNA analysis reveals diverse histories of tribal populations from India, Cordaux [/bib_ref] [bib_ref] The northeast Indian passageway: a barrier or corridor for human migration?, Cordaux [/bib_ref] [bib_ref] Genetic structure and affinity among eight ethnic populations of eastern India: based..., Kashyap [/bib_ref]. In this regard, the Tibeto-Burman speaking populations inhabiting the easternmost tip of northeast India, Arunachal Pradesh, (sharing the international border between India and Bhutan, Tibet, Myanmar) were hardly dealt with and hence there exist a dearth of population genetic studies in this region [bib_ref] Genetic kinship among an isolated Adi tribe of Arunachal Pradesh: isonymy in..., Maji [/bib_ref] [bib_ref] Genetic heterogeneity among three Adi tribes of Arunachal Pradesh, India, Krithika [/bib_ref] [bib_ref] Intertribal and temporal allele-frequency variation at the ABO locus among Tibeto-Burman-speaking Adi..., Krithika [/bib_ref] [bib_ref] Antiquity, geographic contiguity and genetic affinity among Tibeto-Burman populations of India: a..., Krithika [/bib_ref]. However, Arunachal Pradesh is of importance from a population genetic perspective, as this region has experienced cultural contacts and population interactions due to multitude waves of migration, during different periods, from the adjoining regions.
Arunachal Pradesh (situated between latitude 26u309N and 29u309N and longitude 91u309E and 97u309E) is the abode of 26 major Tibeto-Burman speaking tribes and 110 sub-tribes and minor tribes, majority claiming their descent from the Tibetan region during different time periods (evident from the available ethno-historical accounts and folklore tradition). One of the largest tribe of the region is Adi, a collective tribe distributed in the temperate and sub-tropical regions within the districts of West Siang, East Siang, Upper Siang, Upper Subansiri and Dibang Valley in central Arunachal Pradesh. They share similar physical features of that of East Asian populations and speak Adi dialects which belong to North-Assam branch of Tibeto-Burman sub-linguistic family. The ethno-history suggests their origin from southern regions of Tibet (China) and traces their migration and settlement history of their ancestors (the 'Tani' group) at different time periods during about 5 th -7 th century AD. There are about 12 sub-tribes of Adi, categorized under two
# Results
Adi tribe, a Tibeto-Burman speaking population of northeast India, is of importance in understanding the genetic affinity among the Tibeto-Burman speaking tribes of India and neighboring populations of East/Southeast Asia. The results of the genetic affinity and diversity among Adi sub-groups, their differentiation as well as sub structuring have been presented in this study. Also, the genetic relationship of Adi with neighboring Tibeto-Burman populations of north and northeast India as well as with the linguistically divergent populations of East/Southeast Asia have been discussed.
## Extent of microsatellite diversity
The allele frequency distribution of the 15 STR loci among the studied six Adi sub-groups has been previously reported [bib_ref] Genetic diversity at 15 microsatellite loci among the Adi Pasi population of..., Krithika [/bib_ref] [bib_ref] Genotype profile for fifteen tetranucleotide repeat loci in two Tibeto-Burman speaking tribal..., Krithika [/bib_ref] [bib_ref] Allele frequency distribution at 15 autosomal STR loci in Panggi, Komkar and..., Krithika [/bib_ref] Genetic differentiation and sub-structuring among Adi tribe
The locus-wise results of the exact test of population differentiation are shown in . Pair wise comparison of the sub-tribes of Adi reveal most significant difference (at 12 loci: D5S818, FGA, D21S11, D7S820, D3S1358, THO1, D13S317, D16S539, vWA, D18S818, D2S1338, D19S433) between Adi Pasi-Upper and Adi Panggi. Adi Pasi-Upper also shows significant differences at 9 loci with Adi Pasi-Lower (FGA, D7S820, D3S1358, THO1, D13S317, vWA, TPOX, D2S1338, D19S433) and at 8 loci with Adi Komkar (D21S11, D7S820, D3S1358, D13S317, D16S539, TPOX, D18S818, D19S433). Least significant difference is shown by Adi Padam with Adi Pasi-Lower (at locus D2S1338) and Adi Komkar (at locus D7S820). Overall, Adi Pasi-Upper shows significant difference, at loci D7S820 and D13S317, with all the other study populations. Among the analyzed 15 STR loci, D8S1179 shows no significant difference among the Adi sub-tribes and also CSF1PO and D5S818 show significant difference only in one pair of populations (between Adi Pasi-Lower-Adi Panggi and Adi Pasi-Upper -Adi Panggi respectively).
AMOVA results, presented in [fig_ref] Table 3: Genetic differentiation of Adi populations based on AMOVA [/fig_ref] , reveal that irrespective of any grouping, 2.38% of variation is attributable to differences among populations, while 11.6% of variation result from differences among the individuals within populations. The corresponding F ST value of 0.02379 indicates a low degree of genetic differentiation, among the studied groups, which might probably be attributed to the recent formation of the different factions from a common ancestral group. Two important factors that might possibly have played a key role in the genetic differentiation of Adi are: fission due to inter-tribal conflicts and relative geographic isolation of the formed contemporary Adi sub-tribes. So AMOVA analyses were performed to understand the relative influence of both these factors towards the genetic differentiation of Adi [fig_ref] Table 3: Genetic differentiation of Adi populations based on AMOVA [/fig_ref]. The grouping of populations based on their geophysical location (F SC : 0.02885) as well as their ethno-history (F SC : 0.02743) did not reveal any significant differences among the groups. In both cases, the variation among the populations and within the groups was around 2.8% and among the individual within the populations was around 11.6% as in case of the single group analysis. The variation within individuals was found to be around 86% at different levels of analyses.
To understand the extent of sub structuring among the Adi subtribes we have performed structure analysis with different values of K. Simulation summary for K = 2 and K = 3, including the logarithm of estimated probability of data (Ln Prob) values, values of proportion of membership of each pre-defined populations in each of the two or three clusters and the corresponding a values are given in [fig_ref] Table 4: Membership Proportions of each pre-defined six Adi populations in each of K... [/fig_ref]. The pattern of sub structuring among the studied subpopulations is depicted in [fig_ref] Figure 2: Bar plot estimation figures of six Adi sub-tribes, inferred from the STRUCTURE... [/fig_ref]
## Genetic affinity among the studied populations
The pattern of clustering and the genetic affinity between the six Adi sub-tribes are shown in the D A -NJ phylogenetic trees (supplementary [fig_ref] Figure 1: Map of Arunachal Pradesh showing the geographical distribution of the studied Adi... [/fig_ref] and the PCA plot [fig_ref] Figure 3: PCA plot, based on D A distance, of the 6 studied Adi... [/fig_ref]. The studied populations depict a single close cluster of four populations (Panggi, Komkar, Padam and Pasi-Lower), the remaining two populations, viz., Adi Pasi-Upper and Adi Minyong separating away from the others. The PCA plot also show a similar pattern of clustering, substantiating the pattern obtained from the dendrogram, with the exception of Adi Pasi-Lower which was distantly located from the Panggi-Komkar-Padam cluster.
Genetic relationship of Adi with other populations a) Tibeto-Burman speaking populations of India. The D A -NJ phylogenetic tree, depicting the genetic relationship of Adi subpopulations with the sixteen neighboring Tibeto-Burman speaking populations of north and north-east India, is shown in supplementary [fig_ref] Figure 2: Bar plot estimation figures of six Adi sub-tribes, inferred from the STRUCTURE... [/fig_ref] and the corresponding PCA plot is depicted in [fig_ref] Figure 4: PCA plot, based on D A distance, of the 22 Tibeto-Burman populations... [/fig_ref]. Overall, the geographically proximate Tibeto-Burman populations tend to cluster together. The phylogeny exhibits 3 distinct major clusters. Cluster I consists of two sub-clusters, where the first subcluster, Ladakh-Sikkim sub-cluster, includes 4 populations from Ladakh
# Discussion
Adi tribe comprises of several sub-tribes settled in relative geophysical isolation since several generations. They exhibit socio- . Pair-wise comparison of studied populations, at the analyzed loci, to investigate the extent of population differentiation. Significant values are in bold cultural as well as linguistic diversity coupled with wide variation in subsistence pattern (ranging from hunting-gathering to settled agriculture). There were very few sporadic biological studies among some sub-tribes of Adi [bib_ref] Mitochondrial DNA analysis reveals diverse histories of tribal populations from India, Cordaux [/bib_ref] [bib_ref] The northeast Indian passageway: a barrier or corridor for human migration?, Cordaux [/bib_ref] [bib_ref] Genetic kinship among an isolated Adi tribe of Arunachal Pradesh: isonymy in..., Maji [/bib_ref] [bib_ref] Genetic heterogeneity among three Adi tribes of Arunachal Pradesh, India, Krithika [/bib_ref] [bib_ref] Intertribal and temporal allele-frequency variation at the ABO locus among Tibeto-Burman-speaking Adi..., Krithika [/bib_ref] [bib_ref] Antiquity, geographic contiguity and genetic affinity among Tibeto-Burman populations of India: a..., Krithika [/bib_ref] [bib_ref] Genetic diversity at 15 microsatellite loci among the Adi Pasi population of..., Krithika [/bib_ref] [bib_ref] Genotype profile for fifteen tetranucleotide repeat loci in two Tibeto-Burman speaking tribal..., Krithika [/bib_ref] [bib_ref] Allele frequency distribution at 15 autosomal STR loci in Panggi, Komkar and..., Krithika [/bib_ref] [bib_ref] Blood group investigations in the Abor Tribe, Bhattacharjee [/bib_ref] [bib_ref] Blood group and secretor frequency among the Galong, Kumar [/bib_ref] and several of the isolated sub-tribes are yet to be investigated. This is perhaps the first ever comprehensive molecular genetic study attempted to investigate the genetic affinity and diversity among the sub-tribes of Adi and their relationship with other Tibeto-Burman tribes of India and the populations of East and Southeast Asia. The results of the allele frequency variation at 15 STR loci reveal the underlying microsatellite diversity among the studied sub-tribes of Adi. The extent of deviation of the studied loci from HWE, among Adi, differs at the sub-structural level that is indicative of their unique population structure. For instance, Pasi-Upper and Panggi deviate from HWE at maximum number of loci (6 and 4 respectively) whereas Minyong show deviation at only one locus and Padam shows none. This scenario in case of Pasi-Upper and Panggi populations is probably due to their small size and relative isolation in remote Upper Siang hilly regions. In contrast, least deviation in Minyong (one locus) and absence of deviation in Padam might be the resultant of their comparative large population size distributed over plain East Siang regions, in proximity to the urban area.
The least average heterozygosity value among Panggi (,74%) might be explained by their small size, strategic location and preferential marriage practices among clans prohibiting external gene flow. Strikingly, the maximum average heterozygosity value (,78%) was observed among Pasi-Upper in spite of their small population size and relative isolation. The results obtained from the exact test of population differentiation, for the 15 STR loci, also show wide diversity among the Adi sub-tribes. The least significant difference between Adi Padam and other subpopulations (at almost 7 loci) might be due to the fact that the other sub-tribes have formed from the larger Padam tribe, one of the earliest settlers of the region; however this requires further validation.
According to the folklore tradition of Adi, formation of their sub-groups was guided by fission-fusion process as a result of inter tribal war fares in the recent past. The above ethno-historical information is supported by the low average G ST value (2.34%) among Adi which is an indication of the low degree of genetic differentiation among the sub-groups. This low degree of differentiation among the sub-groups is further substantiated by the clustering pattern obtained from the PCA plot and phylogeny, where all the sub-tribes form a single close cluster.
A close cluster of Komkar and Panggi sub-tribes, observed in the PCA plot and the phylogenetic tree, corroborates with their geographic proximity and also the clustering of Padam with this group yields further support to the ethno-historical account that Komkar and Panggi sub-groups were formed from the larger Padam group. The separation of Pasi-Upper and Pasi-Lower in the phylogeny, despite belonging to the same ancestral group, could be the consequence of the migration of a few close kin-groups from their ancestral population of Adi Pasi at the Upper Siang district to the plain areas at the East Siang district. As a result of isolation, there have been changes in the marriage patterns leading to higher endogamy among the Adi Pasi-Upper as against the inter-tribal marriages and low endogamy among the Adi Pasi-Lower. AMOVA results also show least genetic differentiation among the sub-tribes of Adi. The low F SC values (around 2.7%) irrespective of ethno-historical or geographical grouping of the populations suggest that the formation of the sub-populations was a recent phenomenon and that the ethno-history and geography had less influence on the overall genetic make up of the populations. So in spite of the sociocultural, geographic and linguistic diversity, Adi sub-groups remain genetically less differentiated. However, this observation needs to be speculated as the increase in the number of samples and the microsatellite loci might contradict the above observation. The STRUCTURE analysis also support the findings of AMOVA, wherein no clear sub-structuring was observed among the Adi subpopulations (for both K = 2 and K = 3 runs). Overall, the low average G ST values, close clustering in PCA plot and phylogeny, low F ST and F SC values of AMOVA and absence of discrete substructuring among Adi sub-tribes support their recent formation from a common ancestral group.
The common migration history of these populations and the Manipur tribes. This further confirms the preliminary results of our earlier microsatellite study on Adi Pasi-Lower and other Tibeto-Burman populations of India [bib_ref] Antiquity, geographic contiguity and genetic affinity among Tibeto-Burman populations of India: a..., Krithika [/bib_ref].
The inclusion of populations from East and South-east Asia in the phylogenetic analyses reveals the clustering of the Luoba ethnic group of Tibet with the Adi groups of Arunachal Pradesh. According to the ethnologue information, Luoba Tibetan (Boga'er Luoba), categorized under the North-Assam branch of the Tibeto-Burman sub linguistic family, is also alternatively referred to as Adi/Abor and is supposed to have been derived from the 'Tani' group, the putative ancestral population of Adi. They are located in southern fringes of central Tibetan region, which is adjacent to the Upper Siang district of Arunachal Pradesh. The clustering of Luoba with Adi further supports the ethno-historical accounts of their putative common origin.
The fifty populations from East and Southeast Asian countries along with the Adi and other Indian Tibeto-Burman populations show an interesting pattern of clustering. Some Tibeto-Burman populations of India (e.g. Adi tribes of Arunachal Pradesh, populations from Ladakh, Mizoram, Sikkim, and Garo of Meghalaya) get clustered with the Tibetan populations from Tibet and China whereas some others (e.g. Drokpa, Balti of Ladakh and Bhutia of Sikkim) cluster along with Southeast Asian populations. All the morphologically similar populations, irrespective of their linguistic affiliation, cluster together possibly with respect to their geography and ethno-historical account of migration.
Overall, Adi and other Tibeto-Burman speaking populations of India are regionally well differentiated and exhibit genetic affinity with the neighboring populations of East/Southeast Asia, based on their shared ethno-history. However, a clearer picture will possibly emerge from the analysis of increased number of informative genetic markers and from the uniparental markers like mitochondrial DNA and Y chromosome. Further, to understand the genetic relationships between Adi sub-tribes and other neighboring Tibeto-Burman speaking populations, the generated autosomal STR data of Adi was compared with the published allele frequency data (for the nine common loci) of other sixteen Tibeto-Burman speaking populations from north (Ladakh) and northeast (Mizoram, Manipur, Sikkim, Nagaland and Meghalaya) India [bib_ref] Population data for nine fluorescent based STR loci among four important tribal..., Chattopadhyay [/bib_ref] [bib_ref] Concordance study on 15 STR loci in three major populations of Himalayan..., Kashyap [/bib_ref] [bib_ref] Genetic diversity and relationships among the tribes of Meghalaya compared to other..., Langstieh [/bib_ref] [bib_ref] Genetic polymorphism revealed by 13 tetrameric and 2 pentameric STR loci in..., Maity [/bib_ref] [bib_ref] Genetic variation of 13 STR loci in the four endogamous tribal populations..., Mastana [/bib_ref] [bib_ref] Genetic polymorphism at nine microsatellite loci in four high altitude Himalayan desert..., Trivedi [/bib_ref]. Also the observation that Tibeto-Burman speakers of the Indian subcontinent share similar physical features with that of the East and Southeast Asian populations instigated us to comprehend the genetic status of Tibeto-Burman speakers of India (including Adi) amidst the linguistically diverse but physically akin populations of East/Southeast Asia. So we compared the populations of north and northeast India, based on the available allele frequency data of nine common STR loci, along with that of East/Southeast Asia . Due to the unavailability of the genotype data for the studied reference populations, we had no other option but to restrict our analyses based on the available allele frequency data. Details of all the studied populations, their sample size, ethnic and linguistic affiliations, geographical locations, subsistence patterns and their literature sources are given in the supplementary [fig_ref] Table 1: Locus-wise and population-wise descriptive statistics among the studied populations, based on 15... [/fig_ref].
# Materials and methods
## Populations
## Dna isolation and microsatellite typing
High molecular weight DNA was isolated, from the collected blood samples of Adi sub-tribes, using the standard phenol/ chloroform method. One to 10 ng of individual DNA template were amplified for fifteen tetranucleotide repeat loci (D5S818, FGA, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWa, TPOX, and D18S51) on Gene-Amp PCR 9700 thermal cycler (Applied Biosystems, Foster City) by using the AmpFI STRH Identifiler kit (Applied Biosystems, Foster City) according to manufacturer's instructions. While the amplified products of Pasi and Minyong sub-tribes were separated on a 4% polyacrylamide gel using the ABI Prism 377 automated DNA sequencer (Applied Biosystems), the amplified fragments of Panggi, Komkar and Padam sub-tribes were separated and detected using the ABI PrismH 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City). The resultant data was then analyzed using GeneScan TM Analysis Software (Version 3.7) and the allele designations were done with Genotyper TM DNA Fragment Analysis Software (Version 3.7) (Applied Biosystems, Foster City). The laboratory experiments were carried out following all the quality control measures.
## Statistical analyses
The allele frequencies of the 15 STR loci were calculated, from the obtained genotype data of the Adi sub-tribes, using the DNATYPE software. The observed heterozygosity (h) at each locus and the probability of homozygosity (P) were estimated to evaluate the extent and magnitude of genetic diversity among the sub-groups of Adi. Also, likelihood ratio test (LR) and the exact test (ET) were performed to test the possible divergence of each locus from the Hardy-Weinberg Expectations (HWE) [bib_ref] Statistical power of an exact test of Hardy-Weinberg proportions of genotype data..., Chakraborty [/bib_ref] [bib_ref] Performing the exact test of Hardy-Weinberg proportion for multiple alleles, Guo [/bib_ref]. Based on the allele frequencies of the 15 autosomal STR loci of six Adi populations, the locus-wise genetic diversity (G ST ) [bib_ref] Analysis of gene diversity in subdivided populations, Nei [/bib_ref] and the population-wise average heterozygosity were estimated to understand the degree of genetic differentiation and the within-population heterogeneity respectively. Locus-wise exact test of population differentiation, using Arlequin 3.01, was performed to analyze the extent of genetic diversity at the studied loci among the six Adi sub-tribes [bib_ref] Arlequin ver. 3.01: An integrated software package for population genetics data analysis, Excoffier [/bib_ref].
To understand the genetic relatedness between the six studied Adi populations; pair-wise genetic distances using the modified Cavalli-Sfroza distance (D A ) and the standard genetic distance (D ST ) measures of Nei et al., [bib_ref] Accuracy of estimated phylogenetic trees from molecular data. II. Gene frequency data, Nei [/bib_ref] were computed using the software DISPAN. Subsequently, the conventional rectangular form of two phylogenetic trees: the unweighted pair group method with arithmetic mean (UPGMA) tree and neighborjoining (NJ) tree were constructed based on the D A and D ST distance measures by employing the software DISPAN. To check for the reliability and consistency of the clustering pattern of the obtained dendrograms, a total of 1000 and also 10,000 bootstrap replications were separately performed. In order to further explore the topology of the obtained phylogenetic trees including the positions and lengths of the branches, branching patterns as well as the cluster formation, the radiation form of the trees were also constructed using the phylogenetic software Mega v3.1 [bib_ref] MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment, Kumar [/bib_ref]. Since D A distance measure is the most efficient for obtaining correct phylogenetic trees under various evolutionary conditions and also is least affected by small size [bib_ref] Genetic distances and reconstruction of phylogenetic trees from microsatellite data, Takezaki [/bib_ref] , and because UPGMA and the NJ phylogenies depict a similar pattern of relationship between the populations, our discussions are based only on the D A -NJ trees.
To characterize the clustering trends exhibited by these studied populations, the data dimensionality was reduced by performing a covariance analysis between factors [Principle Component Analysis (PCA)]. This analysis was performed based on the D A distance matrix, of the six Adi sub-groups, using SPSS software (Version 11.0), Chicago, IL, USA. The PCA plot further substantiates the dendrogram clustering method, and especially when bootstrap values of the dendrogram are considerably low, the similar clustering in both the PCA plot and the dendrogram indicates the consistency of the results obtained.
In order to investigate the genetic variation within and between the sub-populations of Adi, Analysis of Molecular Variance (AMOVA) was performed using Arlequin 3.01 [bib_ref] Arlequin ver. 3.01: An integrated software package for population genetics data analysis, Excoffier [/bib_ref]. Also, the significance of the AMOVA values was estimated by use of 10,000 permutations. Three levels of analyses were performed, wherein at the first level the six Adi sub-groups [Pasi-Upper, Pasi-Lower, Minyong, Panggi, Komkar and Padam] were considered as a 'single group'. At the second level, the six Adi populations were categorized into 'two groups' based on their geophysical locations To obtain a vivid insight into the sub structuring among Adi sub-tribes, a model-based clustering method was employed, using genotype data consisting of unlinked markers, as implemented in Structure 2.1 program [bib_ref] Inference of population structure using multilocus genotype data, Pritchard [/bib_ref]. The program was performed by using 100,000 MCMC replications after a 20,000 burn-in length. Simulations were done with different values of K (from 1 to 5) under the assumption of admixture model and correlated allele frequencies among populations. Each run was carried out several times to ensure consistency of the results.
In addition to the above analyses performed on Adi populations, we also conducted the comparative analyses of Adi sub-groups with sixteen Tibeto-Burman speaking populations of north and northeast India and also with other neighboring East and Southeast Asian populations that share similar physical features with that of Adi. Phylogenetic analysis (as described above) as well as Principle Component Analysis, were performed on these populations, based on the available allele frequency data, to understand their underlying genetic affinity and also to obtain a better clarity of the genetic status of Adi populations with the Tibeto-Burman speaking regional populations and linguistically diverse other global populations of East/Southeast Asia.
## Supporting information
Table S1 Sample size, geographical distribution, linguistic affiliation and the subsistence pattern of the studied populations. Government of Arunachal Pradesh especially of the Siang districts. We thank Dr. Kashyap, CFSL for providing the laboratory facilities and the required materials to carry out the experiments and Dr. R Trivedi, CFSL for technical support. We also thank the research scholars of CFSL for their help in laboratory experiments. We acknowledge all the reviewers for their valuable comments which have helped us to improve our manuscript substantially.
# Author contributions
[fig] Figure 1: Map of Arunachal Pradesh showing the geographical distribution of the studied Adi sub-tribes. doi:10.1371/journal.pone.0002549.g001 [/fig]
[fig] Figure 3: PCA plot, based on D A distance, of the 6 studied Adi sub-groups. doi:10.1371/journal.pone.0002549.g003 [/fig]
[fig] Figure 2: Bar plot estimation figures of six Adi sub-tribes, inferred from the STRUCTURE analysis. doi:10.1371/journal.pone.0002549.g002 [/fig]
[fig] Figure 4: PCA plot, based on D A distance, of the 22 Tibeto-Burman populations of India. doi:10.1371/journal.pone.0002549.g004 [/fig]
[fig] [Group 1: Padam, Minyong, Pasi-Lower; Group 2: Panggi, Komkar, Pasi-Upper]. The populations of group 1 (Padam, Minyong and Pasi-Lower) are located at the lower plains of the Siang river valley and geographically separate from the populations of group 2 (Panggi, Komkar, Pasi-Upper) which, on the contrary, are isolated and settled at the higher mountain ranges. At the third level of analyses, three groups were constructed based on the ethno-historical information of the sub-groups [Group 1: Panggi and Komkar; Group 2: Padam and Minyong; Group 3: Pasi-Upper and Pasi-Lower]. [/fig]
[table] Table 1: Locus-wise and population-wise descriptive statistics among the studied populations, based on 15 STR loci [/table]
[table] Table 3: Genetic differentiation of Adi populations based on AMOVA [/table]
[table] Table 4: Membership Proportions of each pre-defined six Adi populations in each of K clusters and logarithm of estimated probability of data. [/table]
|
Immunologic monitoring in kidney transplant recipients
# Introduction
Recent advances in post kidney transplantation care, including development of new immunosuppressive drugs, have led to a dramatic improvement in short-term outcomes [bib_ref] Diagnostic techniques in the work-up of renal allograft dysfunction-an update, Chandraker [/bib_ref] [bib_ref] Long-term deterioration of kidney allograft function, Kasiske [/bib_ref] [bib_ref] Transplantation 50 years later-progress, challenges, and promises, Sayegh [/bib_ref]. Yet, recipients and clinicians who have to rely on drugs that have a limited therapeutic window are caught between the rock of rejection and the hard place of side effects such as infection, toxicity, and malignancy. The inconsistent sensitivity of individuals to immunosuppressive drugs necessitates the need for assays that can measure the immune response directly and provide more information about the recipient's immunologic status [fig_ref] Figure 1: Potential benefit and usefulness of post-transplant immunologic monitoring [/fig_ref]. For example, in order to achieve transplantation tolerance, the Holy Grail of transplantation, it is first necessary to have a reliable and reproducible method for detecting a biomarker that is able to identify recipients in whom tolerance is likely to occur. The ideal tool for clinical monitoring should be noninvasive, inexpensive, reproducible, and accessible to clinicians and patients. Here, we summarize recent findings in biomarker identification and noninvasive immunologic monitoring based on allograft recipients' peripheral blood and urine analysis.
## Monitoring of immune status
Methods for immunologic monitoring can broadly be divided into antigen-specific and non-antigen-specific assays. The advantage of antigen-specific assays is their ability to discriminate a donor-specific immune response from an immune response to third-party antigens.
## Antigen-specific assays
Being antigen-specific, these assays require the knowledge of and access to donor antigens for analysis.
## Anti-hla antibodies
The significant impact of pretransplant donor-specific crossmatch testing on transplant outcomes is well established [bib_ref] Presensitization and kidney transplant failures, Terasaki [/bib_ref] [bib_ref] Significance of the positive crossmatch test in kidney transplantation, Patel [/bib_ref]. Recent advances in solid-phase assays and flow cytometry have improved the sensitivity of the pretransplant crossmatch as well as the post-transplant monitoring of anti-HLA antibodies [bib_ref] Single human leukocyte antigen flow cytometry beads for accurate identification of human..., Pei [/bib_ref] [bib_ref] Poor graft outcome in recipients with de novo donor-specific anti-HLA antibodies after..., Li [/bib_ref]. Detection of a circulating anti-HLA antibody is now a widely used immunologic monitoring assay in the clinical setting and has been incorporated into the Banff classification system, where the presence of a donor-specific anti-HLA antibody (DSA) is considered a diagnostic criteria for antibody-mediated rejection (AMR) [bib_ref] Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff..., Sis [/bib_ref]. Microbeads with attached HLA antigens in combination with the Luminex platform are the most commonly used solid-phase assays for HLA antibody and DSA detection [bib_ref] Current methodologies for detecting sensitization to HLA antigens, Cecka [/bib_ref]. The tests are classified into three categories according to the variety and type of the beads in each set. The "screening set" is the simplest set, consisting of a pool of different beads of mixed HLA antigens of Class I and Class II. This set is good for general screening of anti-HLA antibody. The "single-antigen bead set" consists of single-antigen beads in groups [fig_ref] Figure 2: Single-antigen beads test for anti-HLA antibody [/fig_ref]. Each bead is coated with a single HLA antigen. This is the most specific set, as it identifies the type of the anti-HLA antibody. The third category is the "panel-reactive antibody set," which falls in between the other two sets in terms of specificity.
In the pretransplant setting, together with the complementdependent cytotoxic crossmatch, anti-HLA antibody testing provides additional information regarding the risk of rejection, especially in highly sensitized patients [bib_ref] Understanding crossmatch testing in organ transplantation: a case-based guide for the general..., Mulley [/bib_ref] [bib_ref] Renal transplantation in patients with pre-transplant donor-specific antibodies and negative flow cytometry..., Patel [/bib_ref] and in the monitoring of pretransplant desensitization protocols [bib_ref] Desensitization in HLA-incompatible kidney recipients and survival, Montgomery [/bib_ref] [bib_ref] Desensitization protocols and their outcome, Marfo [/bib_ref].
The emergence of post-transplant anti-HLA antibodies, either DSA or non-donor-specific (non-DSA), is associated with poorer kidney allograft outcomes [bib_ref] Poor graft outcome in recipients with de novo donor-specific anti-HLA antibodies after..., Li [/bib_ref] [bib_ref] Long-term follow up for anti-HLA donor specific antibodies postrenal transplantation: high immunogenicity..., Ntokou [/bib_ref] [bib_ref] Four-year follow-up of a prospective trial of HLA and MICA antibodies on..., Terasaki [/bib_ref]. DSAs have been shown to have more impact on graft outcomes than non-DSAs [bib_ref] Long-term follow up for anti-HLA donor specific antibodies postrenal transplantation: high immunogenicity..., Ntokou [/bib_ref]. The presence of a post-transplantation DSA can potentially guide clinicians in the evaluation of AMR. Indeed, 40% of recipients with DSA developed AMR compared to none of the recipients with non-DSA [bib_ref] Significance of qualitative and quantitative evaluations of anti-HLA antibodies in kidney transplantation, Ishida [/bib_ref]. Furthermore, the strength and breadth of the detected DSA have been correlated inconsistently with AMR risk; however, the benefit of preemptive treatment after the detection of DSA has yet to be proven.
## Mixed lymphocyte reaction and cell-mediated lymphotoxicity
In the mixed lymphocyte reaction, inactivated donor cells, acting as antigen-presenting cells, are mixed with recipient CD4 + T cells. The degree of T-cell proliferation is measured using radioactive thymidine or the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester: proliferation reflects alloreactivity mainly through recognition of Class II HLA. An assay based on a similar concept is the cellmediated lymphotoxicity assay, which measures the ability of cytotoxic T cells (CD8 + ) to kill donor cells: killing reflects alloreactivity via binding mainly to Class I HLA [fig_ref] Figure 3: Mixed lymphocyte reaction and cell-mediated lymphocytotoxicity [/fig_ref].
These assays have recently been used post-transplantation, to predict the alloimmune response and guide immunosuppressive drug adjustment [bib_ref] Tapering immunosuppression in recipients of living donor kidney transplants, Weimar [/bib_ref] [bib_ref] Donorspecific T-cell reactivity identifies kidney transplant patients in whom immunosuppressive therapy can..., Van Besouw [/bib_ref]. Because of the variation in techniques and difficulty in standardizing these assays leading to discrepant results, the clinical utility of these assays in kidney transplantation is limited [bib_ref] Cellular methods used to evaluate the immune response in transplantation, Reinsmoen [/bib_ref].
## Enzyme-linked immunosorbent spot
The enzyme-linked immunosorbent spot (ELISPOT) assay is used to detect cytokine production of alloreactive T cells. Recipient T cells are incubated with donor cells on a plate coated with an antibody specific for a certain cytokine. Donorreactive T cells, on interaction with donor-specific cells, secrete cytokine that is captured by the antibody on the plate; once T cells are washed off the plate, a labeled second cytokinespecific antibody is added, resulting in a photoreaction where each spot represents one activated T cell. Interferon gamma (IFNγ) is the most widely used cytokine for ELISPOT. In this case, recipient T cells are incubated with donor (to detect a "direct" alloimmune response) or recipient antigen-presenting cells (to detect an indirect alloimmune response) on an anti-IFNγcoated plate, and IFNγ-producing T cells (spots) are visualized on an immunospot image analyzer [bib_ref] Evolution of the enzyme-linked immunosorbent spot assay for post-transplant alloreactivity as a..., Gebauer [/bib_ref] [bib_ref] Lymphocyte markers and prediction of long-term renal allograft acceptance, Babel [/bib_ref] [fig_ref] Figure 4: Enzyme-linked immunosorbent spots [/fig_ref].
Several studies have revealed a correlation between IFNγproducing T cells detected prior to and/or after transplantation and renal transplantation outcomes [bib_ref] Evolution of the enzyme-linked immunosorbent spot assay for post-transplant alloreactivity as a..., Gebauer [/bib_ref] [bib_ref] Modified ELISPOT technique-highly significant inverse correlation of post-Tx donor-reactive IFN gamma-producing cell..., Nather [/bib_ref] [bib_ref] Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and..., Nickel [/bib_ref] [bib_ref] Enzyme-linked immunosorbent spot assay analysis of peripheral blood lymphocyte reactivity to donor..., Najafian [/bib_ref] [bib_ref] Bang BK: Pretransplant donor-specific interferon-gamma ELISPOT assay predicts acute rejection episodes in..., Kim [/bib_ref] [bib_ref] Pretransplant donor-specific and non-specific immune parameters associated with early acute rejection, Reinsmoen [/bib_ref]. A higher donorreactive T-cell response (more spots) was found in recipients who experienced acute rejection compared to recipients who had stable allograft function [bib_ref] Evolution of the enzyme-linked immunosorbent spot assay for post-transplant alloreactivity as a..., Gebauer [/bib_ref]. Bestard et al. [bib_ref] Circulating alloreactive T cells correlate with graft function in longstanding renal transplant..., Bestard [/bib_ref] studied long-term surviving living donor kidney transplant recipients and demonstrated that circulating donor-specific alloreactive T cells are associated with graft injury and are detectable long after transplantation. Interestingly, donor-specific alloreactive T cells that correlated with allograft function and rejection were primed by the direct pathway, whereas T cells that correlated with proteinuria were primed by the indirect pathway. Another study of post-transplant donor-specific ELISPOT found that antithymocyte globulin induction prolonged donor-specific hyporesponsiveness markedly compared to IL-2 receptor blocker induction. However, both induction modalities revealed similar third-party alloreactivity [bib_ref] Evaluation of alloreactivity in kidney transplant recipients treated with antithymocyte globulin versus..., Cherkassky [/bib_ref]. Based on ELISPOT, a panel-reactive T-cell assay can be performed using a panel of allogenic stimulator cells instead of donor cells. Similar to panel-reactive antibody, pretransplant panel-reactive T-cell assay can predict post-transplant acute renal allograft rejection [bib_ref] Pretransplant cellular alloimmunity as assessed by a panel of reactive T cells..., Poggio [/bib_ref].
Recently, Heidt S, Roelen DL, de Vaal YJ, Kester MG, Eijsink C, Thomas S, van Besouw NM, Volk HD, Weimar W, Claas FH, and Mulder A [bib_ref] A novel ELISPOT assay to quantify HLA-specific B cells in HLAimmunized individuals, Heidt [/bib_ref] developed an HLA-specific B-cell ELISPOT assay using recombinant HLA monomers as the target for ELISPOT. This assay allows quantification of B cells producing specific HLA antibodies. HLA-immunized healthy individuals and patients who were on the waiting list for retransplantation revealed higher numbers of HLA-specific B cells compared to nonimmunized individuals. A large-scale study of transplant recipients is needed to validate the benefit of this new tool for monitoring humoral immunity in transplant recipients.
Post-transplant monitoring of alloreactivity by ELISPOT is promising and may be able to provide information on risk stratification, including tailoring of immunosuppressive drugs. More studies are needed to validate this concept.
## Non-antigen-specific assays
## Measurement of adenosine triphosphate
The measurement of immunosuppressive drug levels does not predict directly the reactivity of T cells. Efforts to gauge T-cell responsiveness have led to the search for assays that can measure T-cell proliferation to nonspecific stimuli. The measurement of the nucleotide adenosine triphosphate (ATP) theoretically allows a direct analysis of T-cell activity and assessment of the immunosuppressive state. The Immu-Know assay was approved by the Food and Drug Administration (FDA) for detecting changes in activity of the immune system. After incubating a whole blood sample, with the nonspecific mitogen phytohemagglutinin, CD4 + T cells are isolated magnetically and lysed to release ATP, which can be detected and quantified by adding a luminescent detecting reagent [fig_ref] Figure 5: Measurement of the nucleotide ATP [/fig_ref]. A higher ATP level implies a higher degree of T-cell reactivity and, therefore, potential under immunosuppression, whereas a lower level implies over immunosuppression.
A meta-analysis of 504 solid-organ transplant recipients by Kowalski et al. [bib_ref] Assessing relative risks of infection and rejection: a meta-analysis using an immune..., Kowalski [/bib_ref] showed that low ATP values were associated with infections, whereas high ATP values were associated with acute rejection. A prospective study of ATP release in 36 renal transplant recipients by Pérez-Flores et al. [bib_ref] Intracellular ATP levels in CD4+ lymphocytes are a risk marker of rejection..., Perez-Flores [/bib_ref] showed the association between ATP values and adverse events. The recipients with a higher ATP level in the early post-transplant period were more likely to have acute rejection. The infection episodes were associated with lower ATP values. However, Huskey et al. [bib_ref] Single time point immune function assay (ImmuKnow) testing does not aid in..., Huskey [/bib_ref] did not find any association between single time point of ATP values and acute rejection or opportunistic infections in renal transplantation recipients. Furthermore, Serban et al. [bib_ref] Significance of immune cell function monitoring in renal transplantation after Thymoglobulin induction..., Serban [/bib_ref] found that in renal transplant recipients who received thymoglobulin induction therapy, only low ATP values could predict adverse events. Higher ATP values did not predict acute rejection. Interestingly, ATP levels had no correlation with CD4 + T-cell counts but tended to correlate with total white blood cell and neutrophil counts. After phytohemagglutinin stimulation and CD4 beads isolation, 22% of cells were contaminated with myeloid cells.
Another concern is the range of ATP level, as a recent metaanalysis has revealed that within the FDA-approved predictive range of this assay (225-525 ng/mL), its sensitivity and specificity are only 0.36 and 0.80 for infection, and 0.24 and 0.73 for rejection, respectively, in renal transplant recipients [bib_ref] Can immune cell function assay identify patients at risk of infection or..., Ling [/bib_ref]. By contrast, modifying the predictive range to 238-497 ng/mL was found to improve the diagnostic accuracy in a Chinese kidney transplant study [bib_ref] Assessing immunologic function through CD4 Tlymphocyte ahenosine triphosphate levels by ImmuKnow assay..., Zhou [/bib_ref].
Overall, although the ATP release assay is simple to perform, it is non-antigen specific and unable to diagnose infection or rejection in renal transplant recipients. Interpretation in parallel with clinical findings should be made with caution, and longitudinal monitoring seems to be more reliable than single time point measurements estimating the risk for infection or rejection.
## Soluble cd30
CD30 is a member of the tumor necrosis factor receptor superfamily. In T cells, it is associated with T helper 2 (Th2) cells. CD30 has been described as a marker of memory T cells and is also found on B cells, CD8 + T cells, and natural killer (NK) cells [bib_ref] Deciphering CD30 ligand biology and its role in humoral immunity, Kennedy [/bib_ref] [bib_ref] High variation of individual soluble serum CD30 levels of pre-transplantation patients: sCD30..., Altermann [/bib_ref]. After T-cell activation, soluble CD30 (sCD30) is released into the bloodstream and can easily be detected by an enzyme-linked immunosorbent assay.
An increased level of serum sCD30 has been found in Hodgkin's disease and immune diseases driven by Th2 cells. Some studies revealed that high pretransplant sCD30 values are associated with poor post-renal transplant outcomes [bib_ref] Evaluation of pretransplant immunologic status in kidney-transplant recipients by panel reactive antibody..., Cinti [/bib_ref] [bib_ref] Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high..., Süsal [/bib_ref].
Prior to transplant, potential kidney recipients have higher sCD30 values compared to healthy controls [bib_ref] High variation of individual soluble serum CD30 levels of pre-transplantation patients: sCD30..., Altermann [/bib_ref] [bib_ref] Identification of patients at risk of acute rejection by pretransplantation and posttransplantation..., Sengul [/bib_ref] [bib_ref] Plasma levels of soluble CD30 are increased in children with chronic renal..., Barbano [/bib_ref]. In the early post-transplantation period, sCD30 is useful in differentiating recipients who develop acute renal allograft rejection from those with acute tubular necrosis and without complications [bib_ref] Evaluation of posttransplantation soluble CD30 for diagnosis of acute renal allograft rejection, Pelzl [/bib_ref]. Post-transplant measurement of sCD30 to predict late outcomes has also been studied : higher sCD30 levels were associated with lower long-term renal allograft function and survival [bib_ref] Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high..., Süsal [/bib_ref] [bib_ref] Posttransplant sCD30 and neopterin as predictors of chronic allograft nephropathy: impact of..., Weimer [/bib_ref] [bib_ref] Soluble CD30 and Cd27 levels in patients undergoing HLA antibody-incompatible renal transplantation, Hamer [/bib_ref] [bib_ref] HLA antibodies and soluble CD30 are associated with poor renal graft outcome:..., Langan [/bib_ref]. Higher sCD30 levels on Day 5 and Day 7 post-transplantation were associated with later acute rejection episodes [bib_ref] Level of soluble CD30 after kidney transplantation correlates with acute rejection episodes, Yang [/bib_ref] [bib_ref] Panel reactive HLA antibodies, soluble CD30 levels, and acute rejection six months..., Domingues [/bib_ref] [bib_ref] Pre-and post-transplant monitoring of soluble CD30 levels as predictor of acute renal..., Wang [/bib_ref]. However, this association was not reported in other studies [bib_ref] Soluble CD30 in patients with antibody-mediated rejection of the kidney allograft, Slavcev [/bib_ref] [bib_ref] Soluble CD30 as a prognostic factor for outcome following renal transplantation, Platt [/bib_ref].
To apply sCD30 monitoring to clinical practice, it is important to remember that sCD30 is a large molecule (120 kDa). Its values can be affected by renal function and dialysis, as it has been reported that sCD30 can be more a marker of renal function than an immunologic biomarker [bib_ref] Plasma levels of soluble CD30 are increased in children with chronic renal..., Barbano [/bib_ref] [bib_ref] Association between serum soluble CD30 and serum creatinine before and after renal..., Lopez-Hoyos [/bib_ref] [bib_ref] Serum levels of soluble CD26 and CD30 in patients on hemodialysis, Nakao [/bib_ref]. In addition, one study in healthy children showed that sCD30 concentration was affected significantly by age [bib_ref] Age-dependent changes of serum soluble CD30 concentration in children, Chrul [/bib_ref].
More studies are needed to clarify the reliability of sCD30 in immunologic monitoring, particularly early after renal transplantation, in patients with fluctuating renal function.
## Flow cytometry-based immunologic monitoring
Flow cytometry is used for counting cells and studying the different light-scattering patterns of single cells when exposed to a light beam. This technique can widely be exploited when different cell markers are labeled with fluorescent tags.
Surface antigens, intracellular antigens, cytokines, and phosphorylated proteins can be detected using flow cytometry. Advantages of this assay not only include the small sample volume required to perform it but also the its ability to simultaneously phenotype different cell populations [bib_ref] Flow cytometry-based pharmacodynamic monitoring after organ transplantation, Dieterlen [/bib_ref]. Because different types of lymphocytes express different markers, flow cytometry can provide information regarding the immunophenotype of each sample, such as the proportion of naïve T cells, activated CD4 + T cells, memory T cells, regulatory T cells, dendritic cells, B cells, and NK lymphocytes.
Monitoring and properly balancing the inflammatory and regulatory sides of the immune system are important in transplantation. Effector/memory T cells help prevent infection and cancer, but are associated with acute rejection [bib_ref] Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following..., Pearl [/bib_ref] ; they can be detected and differentiated from naïve T cells by staining for CD25, CD45RA, CD45RO, and CD62L [bib_ref] Fazekas de St Groth B: Expression of interleukin (IL)-2 and IL-7 receptors..., Seddiki [/bib_ref] [bib_ref] Peripheral blood sampling for the detection of allograft rejection: biomarker identification and..., Heidt [/bib_ref] [bib_ref] FOXP3+ regulatory T cells in the human immune system, Sakaguchi [/bib_ref]. Regulatory T cells, which are thought to be crucial for immunoregulation and transplant tolerance, stain positive for CD4, CD25, and FoxP3 [bib_ref] FOXP3+ regulatory T cells in the human immune system, Sakaguchi [/bib_ref] [bib_ref] Regulatory T cells in transplantation tolerance, Wood [/bib_ref]. Detection of FoxP3 requires intracellular staining, fixation, and permeabilization, which impairs cell viability.
Regulatory T-Cells isolation for further study and treatment using FoxP3 as a marker thus remains limited in human studies. Staining of CD127 (IL-7 receptor) provides an alternative to intracellular FoxP3 staining. In CD4 + CD25 + cells, the CD127 expression was correlated inversely with FoxP3 expression, and indeed CD4 + CD25 + CD127 lo cells showed suppressive activity [bib_ref] Evaluation of posttransplantation soluble CD30 for diagnosis of acute renal allograft rejection, Pelzl [/bib_ref]. Furthermore, an increased number of CD4 + CD25 + CD127 hi -activated T cells and a decreased number of FoxP3 + regulatory T cells have been associated with chronic humoral rejection in renal transplant recipients [bib_ref] Monitoring of CD4 +CD25highIL-7Ralphahigh activated T cells in kidney transplant recipients, Vallotton [/bib_ref] [bib_ref] Variation in numbers of CD4+CD25highFOXP3+ T cells with normal immunoregulatory properties in..., Braudeau [/bib_ref] , whereas tolerant recipients have been found to have normal numbers of regulatory T cells similar to healthy controls.
Recently, the latency-associated peptide (LAP), the aminoterminal domain of TGF-β precursor peptide, has been identified as a novel surface marker specific for regulatory T cells [bib_ref] Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b)..., Tran [/bib_ref] [bib_ref] Latencyassociated peptide identifies a novel CD4+CD25+ regulatory T cell subset with TGFbeta-mediated..., Chen [/bib_ref]. CD4 + CD25 + LAP + T cells expressed higher levels of regulatory T-cell-associated molecules (FoxP3, glucocorticoidinduced TNFR-related gene, and CTLA-4) than CD4 + CD25 + LAPcells. These CD4 + CD25 + LAP + T cells also revealed suppressive function both in vitro and in vivo [bib_ref] Latencyassociated peptide identifies a novel CD4+CD25+ regulatory T cell subset with TGFbeta-mediated..., Chen [/bib_ref]. This novel surface marker may possibly serve as an alternative flow cytometry detection target to intracellular FoxP3 in human studies and for potential therapeutic purposes.
Immunosuppressive drugs may have different effects on T-cell subsets. Flow cytometry can be useful in detecting such differences. Many studies have shown that calcineurin inhibitors (CNIs), but not the mammalian target of rapamycin (mTor) inhibitors, are associated with lower ratios of regulatory T cells [bib_ref] Reduced numbers of blood natural regulatory T cells in stable liver transplant..., Segundo [/bib_ref] [bib_ref] Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells..., Segundo [/bib_ref] [bib_ref] Decrease of CD4+CD25+ T cells in peripheral blood after liver transplantation: association..., Demirkiran [/bib_ref] [bib_ref] Monotherapy rapamycin allows an increase of CD4 CD25 FoxP3 T cells in..., Hendrikx [/bib_ref]. Recently, a modified form of CTLA4-Ig, belatacept, which blocks B7 signaling, was introduced into clinical transplantation with promising results [bib_ref] Five-year safety and efficacy of belatacept in renal transplantation, Vincenti [/bib_ref]. In mice models, B7 signaling has been shown to be essential in homeostasis of regulatory T cells [bib_ref] B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T..., Salomon [/bib_ref]. CTLA4-Ig treatment of human renal transplant recipients has shown similar effects on CD4 + CD25 + CD127 lo regulatory T cells to those of CNIs [bib_ref] The effect of costimulatory and interleukin 2 receptor blockade on regulatory T..., Bluestone [/bib_ref] , and their levels were comparable in pre-and post-transplant.
B cells have been recognized to play an increasingly important role in immune regulation. Depletion or deficiency of B cells has been shown, in multiple mouse models, to deteriorate immunologically mediated diseases [bib_ref] Rejection and regulation: a tight balance, Ashoor [/bib_ref]. Increased numbers of total B cells, including activated B cells, memory B cells, and early memory B cells, have been found in the peripheral blood of tolerant recipients [bib_ref] Patients with drug-free long-term graft function display increased numbers of peripheral B..., Pallier [/bib_ref]. Moreover, B cells of these recipients also expressed high levels of CD1d + and CD5 + , which are considered to be regulatory phenotypes. These findings have been confirmed by larger studies conducted by Reprogramming the Immune System for Establishment of Tolerance (RISET) and Immune Tolerance Network (ITN) [bib_ref] Development of a cross-platform biomarker signature to detect renal transplant tolerance in..., Sagoo [/bib_ref] [bib_ref] Seyfert-Margolis VL: Identification of a B cell signature associated with renal transplant..., Newell [/bib_ref]. In addition, these studies found that tolerant recipients had increased levels of naïve (CD19 + CD27 -IgM + IgD + ) and transitional B-cell subsets (CD19 + CD24 + CD38 + IgD + ), which also possessed immunoregulatory function. Analysis of B-cell subsets showed transitional B cells to be the most predictive population for transplant tolerance, with a sensitivity of 83% and specificity of 75% [bib_ref] Seyfert-Margolis VL: Identification of a B cell signature associated with renal transplant..., Newell [/bib_ref].
## Gene expression monitoring
The rationale for gene expression monitoring is based on the idea that gene disruption may precede clinical or histological rejection [bib_ref] Noninvasive prediction of organ graft rejection and outcome using gene expression patterns, Anglicheau [/bib_ref]. Gene expression studies provide not only diagnostic but also predictive values [bib_ref] Noninvasive prediction of organ graft rejection and outcome using gene expression patterns, Anglicheau [/bib_ref] [bib_ref] Serial peripheral blood perforin and granzyme B gene expression measurements for prediction..., Simon [/bib_ref] [bib_ref] Cytotoxic lymphocyte gene expression in peripheral blood leukocytes correlates with rejecting renal..., Vasconcellos [/bib_ref]. Peripheral blood is an easy source for DNA isolation. Gene expression studies can be divided into a classic single-gene study and high-throughput microarrays, enabling the study of complete gene expression. Gene expression profiling with microarrays is increasingly used by transplant researchers for many purposes, including searching for gene profile patterns for specific conditions, identifying biomarkers for immunologic monitoring, and studying the mechanisms of rejection and tolerance [bib_ref] Microarrays: monitoring for transplant tolerance and mechanistic insights, Zarkhin [/bib_ref]. However, single-gene-based testing has higher sensitivity and specificity than microarrays. A candidate gene detected by a microarray should be validated further by single-gene-based tests. Another concern in gene expression study is the transcription of gene to protein. Not all expressed genes will be transcribed to proteins. This finding may lead to discrepancies between gene profiles and clinical outcomes.
Perforin and granzyme B are secreted by CD8 + T cells and NK cells to destroy target cells, including those of the allograft, during rejection. An increased expression of perforin or granzyme B is associated with both acute cellular rejection and AMR [bib_ref] Serial peripheral blood perforin and granzyme B gene expression measurements for prediction..., Simon [/bib_ref] [bib_ref] Cytotoxic lymphocyte gene expression in peripheral blood leukocytes correlates with rejecting renal..., Vasconcellos [/bib_ref] [bib_ref] Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B..., Veale [/bib_ref]. Simon T, Opelz G, Wiesel M, Ott RC, and Süsal C [bib_ref] Serial peripheral blood perforin and granzyme B gene expression measurements for prediction..., Simon [/bib_ref] serially studied perforin and granzyme B expression from renal transplant recipients. Acute rejection could be predicted 2-30 days (median 11 days) prior to making the diagnosis of acute rejection. The expression of perforin and granzyme B mRNA in the urine of renal transplant recipients was studied by Li B, Hartono C, Ding R, Sharma VK, Ramaswamy R, Qian B, Serur D, Mouradian J, Schwartz JE, and Suthanthiran M [bib_ref] Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin..., Li [/bib_ref]. Expression of urinary perforin and granzyme B was higher in recipients having biopsy-proven acute rejection than in those without acute rejection. Moreover, perforin exhibited a sensitivity of 83% and specificity of 83% in the prediction of acute rejection, and granzyme B showed a sensitivity and specificity of 79% and 77%, respectively. Interestingly, an increased expression of perforin and granzyme B was also found in recipients with BK virus or cytomegalovirus infection [bib_ref] Peripheral blood sampling for the detection of allograft rejection: biomarker identification and..., Heidt [/bib_ref].
The Fas ligand is expressed by CD8 + T cells. It can induce apoptosis by binding to Fas ligands on other cells. Fas ligand mRNA expression has been also shown to be associated with acute rejection [bib_ref] Cytotoxic lymphocyte gene expression in peripheral blood leukocytes correlates with rejecting renal..., Vasconcellos [/bib_ref] [bib_ref] Ferraz AS: Granzyme B, FAS-ligand and perforin expression during acute cellular rejection..., Netto [/bib_ref]. However, this association has not been confirmed in all studies [bib_ref] Gene expression of perforin by peripheral blood lymphocytes as a marker of..., Shin [/bib_ref] [bib_ref] Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft..., Graziotto [/bib_ref].
FoxP3, the regulatory T-cell-related gene, has been also studied in the peripheral blood from renal transplant recipients. In long-term monitoring, FoxP3 mRNA levels were shown to be lower in recipients with chronic rejection than in those with stable graft function [bib_ref] Clinical significance of regulatory T-cellrelated gene expression in peripheral blood after renal..., Iwase [/bib_ref] [bib_ref] Expression of regulatory T-cell-related molecule genes and clinical outcome in kidney transplant..., Alvarez [/bib_ref]. Not surprisingly, high FoxP3 levels were also detected in tolerant renal transplant recipients [bib_ref] GATA3 and a dominant regulatory gene expression profile discriminate operational tolerance in..., Moraes-Vieira [/bib_ref]. In addition, several studies showed that FoxP3 levels increased as a protective response in acute rejection episodes; this can be applied as a diagnostic tool during acute allograft function deterioration [bib_ref] Noninvasive diagnosis of acute rejection in kidney transplants with delayed graft function, Aquino-Dias [/bib_ref] [bib_ref] Messenger RNA: for FOXP3 in the urine of renal-allograft recipients, Muthukumar [/bib_ref]. Furthermore, in a study of recipients having delayed graft function by Aquino-Dias EC, Joelsons G, da Silva DM, Berdichevski RH, Ribeiro AR, Veronese FJ, Goncalves LF, and Manfro RC [bib_ref] Noninvasive diagnosis of acute rejection in kidney transplants with delayed graft function, Aquino-Dias [/bib_ref] , higher levels of FoxP3 mRNA were found in the peripheral blood of recipients with a diagnosis of acute rejection than in those with acute tubular necrosis. Another study by Muthukumar et al. [bib_ref] Messenger RNA: for FOXP3 in the urine of renal-allograft recipients, Muthukumar [/bib_ref] showed that urinary FoxP3 level was higher in recipients with acute rejection than in those with chronic allograft nephropathy and those with normal biopsy. Among recipients who experienced acute rejection, FoxP3 level was higher in those who had reversed rejection after treatment than in those who did not.
Studies have also identified biomarkers for tolerance: Toag-1 and the ratio of FoxP3 to α-1,2-mannosidase can potentially identify the tolerant recipients [bib_ref] Identification of gene markers for the prediction of allograft rejection or permanent..., Sawitzki [/bib_ref]. These findings have been confirmed in a larger-scale human study [bib_ref] Development of a cross-platform biomarker signature to detect renal transplant tolerance in..., Sagoo [/bib_ref]. Tolerant . Studies of sCD30 in peripheral blood of post renal transplant recipients
## References outcome measured results
Weimer et al. [bib_ref] Posttransplant sCD30 and neopterin as predictors of chronic allograft nephropathy: impact of..., Weimer [/bib_ref] Allograft function Higher sCD30 level at 1 y associated with lower allograft function at 2 y CMV disease associated with transient elevation of sCD30 level Langan et al. [bib_ref] HLA antibodies and soluble CD30 are associated with poor renal graft outcome:..., Langan [/bib_ref] Allograft survival Higher sCD30 level in 1 y associated with poorer 6-y allograft survival Wang et al. [bib_ref] Pre-and post-transplant monitoring of soluble CD30 levels as predictor of acute renal..., Wang [/bib_ref] Acute rejection Higher sCD30 level at Day 5 associated with acute rejection Slavcev et al. [bib_ref] Soluble CD30 in patients with antibody-mediated rejection of the kidney allograft, Slavcev [/bib_ref] Acute humoral rejection sCD30 level not associated with acute humoral rejection Yang et al. [bib_ref] Level of soluble CD30 after kidney transplantation correlates with acute rejection episodes, Yang [/bib_ref] Acute rejection Higher sCD30 level at Day 7 associated with acute rejection Lopez-Hoyos et al. [bib_ref] Association between serum soluble CD30 and serum creatinine before and after renal..., Lopez-Hoyos [/bib_ref] Allograft function Significant correlation between sCD30 and serum creatinine at all times of the study Hamer et al. [bib_ref] Soluble CD30 and Cd27 levels in patients undergoing HLA antibody-incompatible renal transplantation, Hamer [/bib_ref] Acute rejection Higher sCD30 level at 4-6 wks not associated with acute rejection, but associated with poorer allograft function at 1 y Allograft function Domingues et al. [bib_ref] Panel reactive HLA antibodies, soluble CD30 levels, and acute rejection six months..., Domingues [/bib_ref] Acute rejection Higher sCD30 level at Day 7 associated with acute rejection Süsal et al. [bib_ref] Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high..., Süsal [/bib_ref] Allograft survival Higher sCD30 level at Day 30 associated with poorer 3-y allograft survival CMV, cytomegalovirus; sCD30, soluble CD30.
renal transplant recipients had a higher ratio of FoxP3 to α-1, 2-mannosidase than those with steroid monotherapy, standard CNI treatment, and chronic allograft nephropathy. A great advantage of this high-throughput method was recently showed in two large studies of tolerant kidney transplant recipients conducted by ITN and RISET. Many genes were found to be more expressed in tolerant recipients and, interestingly, most of them were B-cell-specific genes. Three B-cell genes, IGKV4-1, IGLL1, and IGKV1D-13, were identified by multiplex real-time polymerase chain reaction as predictors of tolerance [bib_ref] Development of a cross-platform biomarker signature to detect renal transplant tolerance in..., Sagoo [/bib_ref] [bib_ref] Seyfert-Margolis VL: Identification of a B cell signature associated with renal transplant..., Newell [/bib_ref]. Tolerant recipients could be separated from those requiring immunosuppressive drugs and healthy controls by the expression of these B-cell-specific genes. T cells have long been recognized as an important key of allograft rejection and tolerance. Findings from these studies indicate that B cells play a role in transplantation tolerance.
## Proteomic study of biomarkers
Proteomics is a highly effective tool in the hunt for biomarkers. This new technique provides a high-throughput approach for studying complete sets of peptides/proteins expressed in diseases, including transplant immunology-related conditions such as acute or chronic rejection, and infection. The global study by "omics" should be followed by the classic singlemolecule studies to delve into candidate peptides/proteins. Mass spectrometry is the most widely used proteomics platform.
Most of the injuries within the kidney allograft take place in the tubulointerstitium where urine formation takes place, and the paucity of background proteins in the urine, unlike serum, makes it a preferred source of sample for proteomic studies.
Acute rejection is a multistep process that starts from immune activation, includes inflammation and tubulointerstitial injury, and ends up with damage or recovery. Many proteins and peptides are expressed during this process, and can be detected by proteomic study. The candidate proteins or peptides for the ideal biomarker should be detected early in the allograft rejection process, and be able to differentiate rejection from other causes of allograft dysfunction such as tubular injury or other nonspecific causes of irreversible damage.
Schaub et al. [bib_ref] Proteomic-based detection of urine proteins associated with acute renal allograft rejection, Schaub [/bib_ref] and Schaub et al. [bib_ref] Proteomic-based identification of cleaved urinary beta2-microglobulin as a potential marker for acute..., Schaub [/bib_ref] reported an increase in the amount of cleaved urinary β2-microglobulin in renal allograft recipients with rejection. However, the validation study found that cleaved β2-microglobulin was unable to differentiate recipients with subclinical tubulointerstitial rejection from those with normal tubular histology [bib_ref] Detection of subclinical tubular injury after renal transplantation: comparison of urine protein..., Schaub [/bib_ref]. Levels of β2-microglobulin were similar to those of other tubular injury markers such as neutrophil gelatinase-associated lipocalin and retinol-binding protein, in terms of tubular injury detection but not inflammation or rejection [bib_ref] Proteomics and renal transplantation: searching for novel biomarkers and therapeutic targets, Schaub [/bib_ref]. In a separate study, Schaub et al. [bib_ref] Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis, Schaub [/bib_ref] also reported that the urinary concentrations of CXCL10 and CXCL9, the CXC-receptor 3 (CXCR3) proinflammatory chemokines, were significantly higher in subclinical tubulitis than in subclinical borderline tubulitis and normal histology.
O'Riordan et al. [bib_ref] Bioinformatic analysis of the urine proteome of acute allograft rejection, O'riordan [/bib_ref] and O'Riordan et al. [bib_ref] Characterization of urinary peptide biomarkers of acute rejection in renal allografts, O'riordan [/bib_ref] have identified βdefensin-1 (a 4.7 kDa peptide) and α-1-antichymotrypsin (a 4.4 kDa peptide) as useful markers in diagnosing acute renal allograft rejection, as both are involved in the inflammatory process. Sigdel et al. [bib_ref] Shotgun proteomics identifies proteins specific for acute renal transplant rejection, Sigdel [/bib_ref] found that urine of recipients with allograft rejection have lower levels of Tamm-Horsfall protein (uromodulin) and CD44, and higher levels of pigment epithelium-derived factor compared to that of stable allograft function recipients and healthy controls. Because this study did not enroll recipients with other causes of renal allograft dysfunction, we are unable to conclude that the alteration of these proteins was solely caused by the rejection process. In some studies, other distinct peptides have also been identified in the urine of renal allograft rejection recipients [bib_ref] Characterization of renal allograft rejection by urinary proteomic analysis, Clarke [/bib_ref] [bib_ref] Proteomic research in renal transplantation, Clarke [/bib_ref] [bib_ref] Detection of acute tubulointerstitial rejection by proteomic analysis of urinary samples in..., Wittke [/bib_ref]. However, a definite protein or biomarker has yet to be identified.
## Analysis of t-cell receptor repertoire
Addition and deletion of nucleotides during the generation of a T-cell receptor (TCR) by rearrangement occur in a random manner. The complementary-determining region 3 (CDR3) of Vβ gene of the TCR is also created randomly and leads to diversity in the length of the CDR3. This random rearrangement process leads to a Gaussian distribution of CDR3 length. In transplant recipients, a deviation from the Gaussian distribution of CDR3 length indicates clonal expansion of T-cell population, which reflects T-cell activation or rejection [bib_ref] Harnessing the diversity of the human T-cell repertoire: a monitoring tool for..., Naesens [/bib_ref].
Miqueu et al. [bib_ref] Analysis of the peripheral T-cell repertoire in kidney transplant patients, Miqueu [/bib_ref] reported a large, multicenter, casecontrolled study of TCR repertoire by polymerase chain reaction in peripheral blood of renal transplant recipients using "Tc Landscape," a statistical method developed for analyzing the TCR repertoire distribution. Of the tolerant recipients, 92.8% were found to have a normal distribution pattern or mild kurtosis pattern compared to 42.8% of recipients with biopsyproven chronic humoral rejection. This demonstrates the association between TCR repertoire and allogenic immunity.
# Conclusion
In conclusion, because manipulation of the immune system is key to transplantation, monitoring of the immunological response is crucial in understanding the environment in which the allograft functions in any given individual. Currently, there is no best immunological monitoring method, but promising advancements have been achieved over the past few years. With the development of these technologies, understanding the strengths and weaknesses of each test will allow clinicians to integrate these monitoring methods with clinical assessment to achieve the best long-term outcomes in transplant recipients.
[fig] Figure 1: Potential benefit and usefulness of post-transplant immunologic monitoring. [/fig]
[fig] Figure 2: Single-antigen beads test for anti-HLA antibody. [/fig]
[fig] Figure 3: Mixed lymphocyte reaction and cell-mediated lymphocytotoxicity. APC, antigen-presenting cell. [/fig]
[fig] Figure 4: Enzyme-linked immunosorbent spots. [/fig]
[fig] Figure 5: Measurement of the nucleotide ATP. ATP, adenosine triphosphate; PHA, phytohemagglutinin. [/fig]
|
A case of HIV‐negative plasmablastic lymphoma of the bone marrow with a unique immunophenotype
We present an interesting case of plasmablastic lymphoma, which is a rare type of non-Hodgkin lymphoma that is typically diagnosed in HIV-positive patients and has an immunophenotype that overlaps with multiple myeloma. Our patient is unique because he is HIV-negative, has primary bone marrow disease, and has an atypical immunophenotype.A 62-year-old male presented to the emergency department of a Boston VA facility with fatigue and back pain. The patient stated that he has had worsening lower back and hip pain for 2 months, as well as a 45-pound weight loss. The patient denied fever, chills, night sweats, adenopathy, dyspnea, and chest pain. The patient's medical history was significant for coronary artery disease with coronary artery bypass grafting, ischemic cardiomyopathy with an ejection fraction of 35%, peripheral vascular disease, diabetes, atrial fibrillation, transient ischemic attack, and venous thromboembolism. On physical examination, the patient had no palpable lymphadenopathy, splenomegaly, or peripheral edema. Subsequent laboratory work revealed a white blood cell count of 3.37 K/lL (absolute neutrophil count of 2200), a hemoglobin of 7.5 g/dL with a mean corpuscular volume of 95 fL, and a platelet count of 189 K/lL. Lactate dehydrogenase was not elevated, and testing for HIV was negative. Serum protein electrophoresis and immunofixation were not evaluated at the time of initial diagnosis. The patient was admitted for further workup and PET-CT revealed several FDG-avid foci with lytic correlate on CT, particularly the L5 vertebral body and the left femur with an SUV Max of 6.4. Additionally, there were small FDG avid right common iliac nodes and a more pronounced FDG avid (SUV Max, 4.9) right pelvic sidewall node.The above findings prompted a bone marrow biopsy, which revealed a hypercellular bone marrow (100%) that was replaced with sheets of immature precursor-like cells with fine nuclear chromatin and prominent nucleoli. The predominant cells appear to have plasmacytic/plasmablastic differentiation(Fig.
A 62-year-old male presented to the emergency department of a Boston VA facility with fatigue and back pain. The patient stated that he has had worsening lower back and hip pain for 2 months, as well as a 45-pound weight loss. The patient denied fever, chills, night sweats, adenopathy, dyspnea, and chest pain. The patient's medical history was significant for coronary artery disease with coronary artery bypass grafting, ischemic cardiomyopathy with an ejection fraction of 35%, peripheral vascular disease, diabetes, atrial fibrillation, transient ischemic attack, and venous thromboembolism. On physical examination, the patient had no palpable lymphadenopathy, splenomegaly, or peripheral edema. Subsequent laboratory work revealed a white blood cell count of 3.37 K/lL (absolute neutrophil count of 2200), a hemoglobin of 7.5 g/dL with a mean corpuscular volume of 95 fL, and a platelet count of 189 K/lL. Lactate dehydrogenase was not elevated, and testing for HIV was negative. Serum protein electrophoresis and immunofixation were not evaluated at the time of initial diagnosis. The patient was admitted for further workup and PET-CT revealed several FDG-avid foci with lytic correlate on CT, particularly the L5 vertebral body and the left femur with an SUV Max of 6.4. Additionally, there were small FDG avid right common iliac nodes and a more pronounced FDG avid (SUV Max, 4.9) right pelvic sidewall node.
The above findings prompted a bone marrow biopsy, which revealed a hypercellular bone marrow (100%) that was replaced with sheets of immature precursor-like cells with fine nuclear chromatin and prominent nucleoli. The predominant cells appear to have plasmacytic/plasmablastic differentiation [fig_ref] Figure 1: Plasmablastic Lymphoma [/fig_ref]. Immunoblastic cells are also seen in a scattered distribution [fig_ref] Figure 1: Plasmablastic Lymphoma [/fig_ref] , short arrow). Immunohistochemistry was positive for LCA, CD117, CD79A, lambda immunoglobulin light chain, BCL2, EMA, and MYC protein expression (60-70%). Of note, the KI-67 proliferation index was 70-80%. Pertinent negative stains were as follows: CD19/20, CD38/138, CD56, CD30, CD10, PAX5, MUM1, HHV8, EBV, BCL6, ALK1, and TDT. The morphologic findings are most consistent with plasmablastic lymphoma, despite the atypical immunophenotype. The patient was initiated on dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin), and his course was complicated by multiple episodes of severe neutropenic sepsis. A bone marrow biopsy after three cycles revealed no residual lymphoma cells. Subsequently, the patient received one more cycle of DA-EPOCH and then entered a surveillance protocol due to his inability to tolerate further chemotherapy.
# Discussion
Plasmablastic lymphoma (PBL) is an aggressive and very rare type of lymphoma that displays some features of diffuse large B-cell lymphoma (DLBCL), and some features of multiple myeloma (MM) [bib_ref] Large B-cell lymphomas with plasmablastic differentiation: a biological and therapeutic challenge, Montes-Moreno [/bib_ref]. This tumor was first described in the oral cavity of HIV-positive patients in 1997 [bib_ref] Plasmablastic lymphomas of the oral cavity: a new entity associated with human..., Delecluse [/bib_ref]. Subsequently, the tumor has been described in regions other than the oral cavity, particularly in HIVnegative patients [bib_ref] HIV-negative plasmablastic lymphoma: not in the mouth, Castillo [/bib_ref]. Differences between HIV-positive and HIV-negative cases of PBL are significant. Clinically, HIV-positive patients have a stronger male predominance, younger age of onset, and classically manifest with EBVpositive oral cavity lesions. Additionally, HIV-positive patients with PBL respond to antiretroviral medications and have improved overall survival [bib_ref] Clinical and pathological differences between HIV-positive and HIVnegative patients with plasmablastic lymphoma, Castillo [/bib_ref]. In a recent review of 76 cases of HIV-negative PBL, one-third of the cases were immunosuppressed, and 89% had extranodal involvement [bib_ref] HIV-negative plasmablastic lymphoma: not in the mouth, Castillo [/bib_ref]. Extranodal disease in the bone marrow was found in 13% of patients.
Obtaining a definite diagnosis is critical. Morphologically, our patient's tumor was consistent with PBL. The immunophenotype, though, was more complicated. PBLs typically lose classic B-cell markers (CD19, CD20, and PAX5) and gain plasma cell markers (CD38, CD138, and MUM1) [bib_ref] Hematopoietic cell transplantation for plasmablastic lymphoma: a review, Al-Malki [/bib_ref]. Our patient was negative for both the traditional B-cell markers as well as the traditional plasma cell markers. To consider an aggressive B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL), the traditional B-cell markers would be positive. At the other end of the spectrum is MM, which would have negative B-cell markers, but would express plasma cell markers. Plasmablastic myeloma (PBM) is a diagnostic consideration, but there is usually some component of mature plasma cells in the bone marrow specimen. Furthermore, PBM would typically express plasma cell markers and is generally found in patients with preexisting MM [bib_ref] Large B-cell lymphomas with plasmablastic differentiation: a biological and therapeutic challenge, Montes-Moreno [/bib_ref]. MYC gene rearrangements have been described in 49% of patients with PBL according to one study [bib_ref] IG/MYC rearrangements are the main cytogenetic alteration in plasmablastic lymphomas, Valera [/bib_ref]. While our patient did not have fluorescence in-situ hybridization (FISH) testing, he was positive for MYC protein expression by immunohistochemistry. The final diagnosis of PBL was based on the morphology, atypical immunostains, and the exclusion of other hematologic malignancies.
Management of PBL is challenging, and the prognosis is dismal with <40% of patients living beyond two years [bib_ref] Large B-cell lymphomas with plasmablastic differentiation: a biological and therapeutic challenge, Montes-Moreno [/bib_ref]. The National Comprehensive Cancer Network (NCCN) guidelines state that "standard CHOP is not adequate therapy". Listed alternative regimens are CODOX-M/ IVAC (cyclophosphamide, vincristine, doxorubicin, methotrexate/ifosfamide, etoposide, and cytarabine), DA-EPOCH, and HyperCVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with methotrexate and cytarabine). HIV-positive patients should receive antiretroviral therapy. Autologous stem cell transplant may have a role as consolidation for patients with high-risk disease [bib_ref] Hematopoietic cell transplantation for plasmablastic lymphoma: a review, Al-Malki [/bib_ref]. Responses to novel agents have been described in case reports. Of interest is the proteasome inhibitor bortezomib, which is a logical agent to study because of its proven efficacy in MM. Several case reports have shown significant responses to bortezomib alone or in combination with chemotherapy [bib_ref] Bortezomib in plasmablastic lymphoma: a case report and review of the literature, Saba [/bib_ref] [bib_ref] Patient with HIV-associated plasmablastic lymphoma responding to bortezomib alone and in combination..., Bibas [/bib_ref]. One case series has described the efficacy of a novel combination regimen, bortezomib-EPOCH (V-EPOCH), in three patients [bib_ref] Bortezomib in combination with infusional doseadjusted EPOCH for the treatment of plasmablastic..., Castillo [/bib_ref]. The immunomodulating agent lenalidomide has been shown to have some efficacy in at least one case report [bib_ref] Patient with HIV-associated plasmablastic lymphoma responding to bortezomib alone and in combination..., Bibas [/bib_ref]. The suggested initial approach to PBL in a recent review article is as follows: six cycles of DA-EPOCH (with or without bortezomib) and intrathecal prophylaxis with each cycle [bib_ref] The biology and treatment of plasmablastic lymphoma, Castillo [/bib_ref]. The authors of that review also recommend consideration of autologous stem cell transplant for eligible candidates. Further studies evaluating the efficacy of novel agents targeting plasma cells, as well as other novel therapeutics, are critically needed.
[fig] 902 ª 2017: The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. [/fig]
[fig] Figure 1: Plasmablastic Lymphoma (400x). The small arrow identifies an immunoblast, and the large arrow identifies a cell with plasmablastic differentiation. ª 2017 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd. [/fig]
|
Manifestations of HbSE sickle cell disease: a systematic review
Background: Sickle cell disease (SCD) is commonly encountered in Africa and Middle Eastern countries. The causative mutation in the gene encoding the hemoglobin subunit β (HBB) leads to various genotypic variants of the disease. This results in varied phenotypes, with a spectrum of complications, from benign to fatal. Hemoglobin SS (HBSS) genotype is associated with most of these complications; hence, it is a severe form of SCD. On the other hand, rare genotypes such as hemoglobin SE (HBSE) are considered benign. There is limited literature about the clinical manifestations and characteristics of patients with HBSE. We pooled all available data describing the phenotypic manifestations of HBSE heterozygote worldwide to perform a systematic review.Methods:We performed a systematic review according to PRISMA guidelines using PubMed, SCOPUS, and Google Scholar databases. Two independent reviewers (FA and IK) evaluated studies for eligibility and extracted data. We synthesized data on demographics, manifestations, and management of HBSE disease. PROSPERO Registration Number: CRD42021229877.Results:We found 68 HBSE patients reported in the literature. 24 cases were extracted from case reports whereas 44 cases from case series and retrospective studies. Turkey reported the highest number of patients (n = 22). 32 (47%) of the patients were males. The mean age was 20.9 ± 18.26 years. The mean HBS and HBE percentages were 61.1% ± 7.25% and 32.3% ± 5.06%, respectively, whereas the mean hemoglobin was 11.64 ± 1.73 g/dl. Reported manifestations of HBSE disease included acute vaso-occlusive pain crisis (n = 22, 32.3%), splenomegaly (n = 11, 16.1%), hemolytic anemia (n = 10, 14.7%), infections (n = 8. 11.7%), bone infarction (n = 4, 5.8%), gallstones (n = 3, 4.4%), venous thromboembolism (VTE) (n = 2, 2.9%) and stroke (n = 2, 2.9%), and hematuria (n = 2, 2.9%). Death due to HBSE complications was reported in three patients.Conclusion: HBSE is a rare genotypic variant of SCD. It has been considered a benign form; however, there are multiple reports of severe complications. Severe complications observed in HBSE disease include vaso-occlusive crisis, acute chest syndrome, stroke, bone marrow embolism, and death.
# Introduction
SCD is a spectrum of hereditary hemoglobinopathies characterized by abnormal hemoglobin S (HbS) polymer. Globally, there are around 3.2 million SCD patients, and 43 million people have sickle cell trait. Out of these, around 176,000 people have fatal complications. The causative mutation in the gene encoding the hemoglobin subunit β (HBB) leads to the formation of various genotypic variants of the disease. This results in a cascade of sickling and unsickling erythrocytes, ultimately leading to hemolysis and/or vaso-occlusion. Common manifestations and complications include but are not limited to hyposthenuria, acute chest syndrome, renal papillary necrosis, painful crisis, pulmonary hypertension, priapism, lower limb ulcerations, osteonecrosis, stroke, and chronic hemolysis. Significant complications such as acute chest syndrome are mainly observed in patients having common genotypes such as HBSS.
HBSE, one of the rare genotypes of SCD, has been historically considered to have a benign clinical course in most of cases and is categorized as a mild form of SCD. However, many case reports mention severe vasoocclusive symptoms in the HBSE genotype, indicating that HBSE might be more severe than thought. Masiello et al. in their concise review described 26 cases of HBSE disease, among whom nine patients had sickling-related complications of varying severity. The review did not report any case with mortality. The authors concluded that HBSE might not be a mild disease, as evident from symptomatic cases. To date, 68 patients with HBSE disease have been reported. Many of these had severe complications, and some even died due to the disease manifestations. Management of SCD is expanding, with newly approved disease-modifying drugs such as Voxelotor (1500 mg daily) and Crizanlizumab (5 mg/ kg). Additionally, there are recent trials on gene therapy in SCD with promising effectiveness. Understanding the phenotypic manifestations of less studied SCD genotypes such as HBSE may improve our understanding of the effect of genotype and phenotype interaction on disease severity and suggest targeted therapies. The last review on HBSE was published in 2007; it was a concise review on 26 patients. There have been multiple published cases of HBSE after that, representing variable manifestations of this condition. Many of these reports describe a severe form of SCD, mandating an updated systematic review focused on demographics and manifestations of patients with HBSE disease. This review's main objective is to accumulate all the evidence to date on the demographics, phenotypic manifestations, and complications of the HBSE variant of SCD for its better classification and understanding.
# Materials and methods
## Literature search
A systematic literature search was performed for articles using PubMed, Google Scholar, and Scopus for any date up to January 10, 2021, and all articles in English were analyzed by two authors individually (FA and IK).
The following search term was used: "HBSE" OR "Hemoglobin SE. " The extracted articles were screened initially from the title and abstract, and subsequently, a detailed screening was conducted. The quality of the added cases was assessed by two reviewers independently (FA and IK) using the Joanna Briggs Institute case report appraisal checklist for inclusion in systematic reviews. In case of any dispute among the quality assessment, a third reviewer (MAY) independently analyzed the quality of disputed articles to reach a conclusion.
## Study selection
Eligible studies (N = 30) reported the HBSE genetic variant of SCD worldwide and included case reports, case series, and retrospective studies. Data of 68 HBSE patients were extracted and reported from the finalized 30 articles. Articles that were not original or reported variants of SCD other than HBSE were excluded from the review.
## Data collection
Epidemiological parameters, clinical pictures, including presenting complaints and complications, laboratory profiles, treatments employed, and outcomes, were noted in all the cases where available. Cases were categorized as mild or severe based on the presence or absence of severe sickling complications, which were infections, functional asplenia, stroke, VTE, bone marrow embolism, splenic sequestration, retinopathy, bone infarct, and acute chest syndrome and death. Severe complications included those which were potentially fatal or had end-organ damage. Data were recorded and analyzed in Microsoft Excel 2016 and SPSS 26.
# Results
We found 68 cases of HBSE in 30 publications. Most cases were extracted from case series and retrospective studies (n = 44), while some were found in case reports (n = 24). The highest number of cases were reported from Turkey (n = 22), Oman (n = 13), USA (n = 11), and India (n = 8) in descending order. Males represented 32 (47%) of our cases, whereas females represented 50% ofcases (n = 34). Gender was not mentioned in two cases. The mean age was 20.9 years (SD = 18.2), ranging from 5 months to a maximum of 70 years. Most patients had Asian ethnicity (n = 32, 47%), which included South and Southeast Asians, while African lineage (from at least one parent) was found in 8 cases (11.8%). Arabs represented 20.6% (n = 14) of our total cases. Acute vaso-occlusive pain crisis was the most common complication reported by 22 cases (32.3%), with splenomegaly (n = 11, 16.1%), hemolytic anemia (n = 10, 14.7%), infections (n = 8. 11.8%), bone infarction (n = 4, 5.9%) representing other common complications. Respiratory manifestations (thoracic pain, cough, bronchitis, dyspnea, or upper respiratory infection) were reported in five cases (7.3%). Gallstones were seen in three cases (4.4%). Venous thromboembolism (VTE) (2.9%) and stroke (2.9%) were both described in two separate cases each. Hematuria was also present in two patients (2.9%).
We did not find any report describing cardiopulmonary complications (pulmonary hypertension, myocardial infarction, cardiomyopathy), which commonly affect SCD patients. Other serious renal complications were not reported in any of the 68 cases in the literature.
Most of the reported patients had mild to moderate disease. However, mortality due to complications was documented in three cases. Out of those three patients, the first patient, a 52-year-old female, had an undiagnosed potentially uncomplicated disease and revealed only postmortem. The disease rapidly deteriorated, leading to venous thromboembolism and cardiac arrest within 26 h of admission. The cause of death was thought to be ischemia due to dehydration that led to a sickling crisis. The second death occurred in a 12-year-old male within 24 h of symptom onset due to sudden cardiopulmonary collapse due to vasoocclusion leading to ischemia. The third fatality occurred in a 7-year-old female with a history of multiple hospitalizations with pain crises, hemolytic anemia, and Parvovirus B19 infection. Although she had a prolonged and complicated course of her disease, her deterioration before death was acute, secondary to a massive marrow embolism in the pulmonary circulation. Mean hemoglobin (Hgb) was 11.64 mg/dl, slightly higher in males (11.69 mg/dl) than females (11.61 mg/dl). Mean percentages of hemoglobin S (HbS), hemoglobin E (HbE), and fetal hemoglobin (HbF) are given in. Average MCV was low (70.9 fL), with minimum and maximum reported being 57.9 fl and 82 fl, respectively.
# Discussion
This study represents an updated comprehensive systematic review on demographics and manifestations of HBSE genotypic variant of SCD. The genetic basis of HBS lies in glutamine to valine, at position six in the betaglobin gene, whereas that of HBE lies in the substitution of lysine for glutamine at amino acid number 26, in the beta-globin gene. HBSE variant occurs as a result of a combination of HBS and HBE genotypes. Hemoglobin E trait and disease are not uncommon and are second only to the HBS spectrum in global prevalence. However, the combination of two different abnormal mutations makes HBSE a rare occurrence.
The exact incidence of the HBSE genotype remains unknown. A retrospective review of 12 patients in Oman mentioned a point prevalence of HBSE heterozygotes at 0.05%. The authors also concluded that given a very small fraction of HBSE patients developing symptoms, it is a very mild condition, and severe sickling complications are rarely seen. However, in our review, we found aor younger were generally well. However, more than half of the patients older than 18 presented with sickling-related complications. They attributed this to the years of accumulating sickling vasculopathy that makes complication rate increase with aging. We analyzed the complications based on age by dividing them into below 18 and above 18 years in cases where ages were specified for the complications (22 children and 24 adults). Overall, the complication rate in adults was higher than the pediatric age group, which is in line with the previous review of 16 patients. The only significant difference in complications based on age was found in the occurrence of hemolytic anemia, which was present in 4.5% (1/22) of cases < 18 years old compared to 37.5% (9/24) of cases > 18 years of age (OR 12.6, P-value = 0.011). The low incidence of hemolytic anemia in children with HBSE can be related to the presence of fetal hemoglobin, which has a protective effect against many of the complications secondary to sickling, including death. Acute vaso-occlusive pain crisis was seen in 31.8% (7/22) children (age < 18 years) compared to 37.5% (9/24) adults. On the other hand, chronic pain was slightly more common in adults; 4.5% (1/22) of pediatric cases compared to 8.3% (2/24) of adult cases. Pain crisis was not considered a severe complication of SCD, but it is a clinically significant symptom for the patient due to the type of sensation involved. Additionally, in patients > 20 years of age, pain frequency has been described to be associated with early mortality. Those patients who presented with gallstones (n = 3), retinopathy (n = 2), functional asplenia (n = 2), and splenic sequestration (n = 1) were adults. The infection rate was comparable in both age groups; 13.6% (3/22) in children and 16.7% (4/24) in adults. Splenomegaly was present in 18.2% (4/22) of pediatric cases compared to 29.2% (7/24) of adults. Bone infarction and acute chest syndrome were present in 4.5% (1/22) of children compared to 8.3% (2/24) of adults. Bone marrow embolism was reported in one pediatric case only. VTE was present in one pediatric and one adult patient. Mortality, however, was seen more in children (two cases) compared to adults (one case).
We found a slight female predominance in the HBSE population (50%) compared to males (47%). It is to be noted that gender was not reported in two patients, which could have increased or decreased the asymmetry between both genders. Our finding, although less disproportionate, is in keeping with a previous review on 27 patients with HBSE disease, where the authors described 73.6% female cases. In their review, Mishra P et al. reported that 40.7% of the total patients were symptomatic. In our review, 35 (51.5%) of the 68 patients had no complications reported, while 48.5% suffered from mild, moderate, or severe SCD related complications. Although high levels of HBF are considered protective against SCD complications, no such correlation was found by Mishra et al. in their review. To analyze the association of HBF with the severity of complications, we divided the complications into mild-moderate and severe. The mean HBF level in those with mild to moderate complications was higher than those with severe complications, including death (2.4% versus 1.9%). However, no statistical significance was found. A better understanding of this patient-to-patient clinical variability in more extensive studies would dramatically improve care and guide the development of novel therapies. Studies of the natural history of these β-hemoglobinopathies have identified fetal hemoglobin levels and concomitant α-thalassemia as important modifiers of disease severity. Fortunately, improved knowledge of the human genome and the development of new genomic tools, such as genome-wide genotyping arrays and next-generation DNA sequencers, offer new opportunities to use genetics to understand better the causes of the many complications observed in β-hemoglobinopathy patients. Correlation of the clinical and hematological features of HbSE cases with their α-globin gene status and β-cluster haplotypes merits additional considerations in future studies to better understand phenotypes and the disease modifiers and probably novel agents. Turkey is a country with a higher prevalence of hemoglobinopathies such as HBE and HBS with significant population admixture and racial intermarriages. One retrospective study on 20 patients with HBSE disease reported a mean Hgb of 12.06 g/dl, which is slightly higher than what we found in our review (11.64 g/dl). The study reported a mean MCV of 69.9 fl, which on the other hand, is slightly lower than the one in our review, 70.9 fl. 15% of the patients in that study had sicklingrelated complications compared to 68% in our review. The increased published reports of complications related to HBSE reveals the potential morbidity and mortality related to the genotype. Similar to HBSS, HBSE genotype results in a range of phenotypes and may be fatal. There may be subclinical or long-term subtle complications of HBSE that have not yet been well described. The increasing evidence emphasizes early detection and close follow-up of HBSE patients for better patient management. Additionally, it is imperative to reclassify HBSE disease as a moderate form of SCD rather than mild.
As it is a variant of sickle cell disease, the clinical features and biochemical profile of HBSE patients are comparable to the classic HBSS SCD. However, there are some notable differences. In our review, we found the mean age at presentation with symptoms 20.9 years, whereas the median age of presentation in classic SCD is reported around 36 years in a previous study. Another difference that we appreciated is in mean HBF percentage, which is 2.2% in HBSE patients, and 5% in HBSS patients. Lastly, because management of HBSE disease is not well-established, the use of HU in HBSE patients is considerably low (4.4%) compared to HBSS patients (39%). A direct comparison of both types of SCD in a similar patient population in extensive studies can validate our findings.
Over the years, many treatment modalities have been developed for SCD, among which hydroxyurea and simple or exchange blood transfusion have an established role in treating or preventing complications. Curative management includes historical treatment such as stem cell transplantation and new and upcoming gene therapy. Some newer management drugs that have recently been approved for the treatment of SCD include Voxelotor and Crizanlizumab. In our review, blood transfusion and hydroxyurea have been used in few patients and improved their conditions.
Our review has some limitations inherent when conducting systematic reviews of rare conditions. Firstly, around 30 percent of the data was extracted from case reports. Secondly, missing data from case reports and retrospective studies limited an extensive data analysis. Thirdly, we acknowledge that publication bias may have contributed to the increased number of complications reported in recent years. Many patients with asymptomatic course who do not require medical attention would go unnoticed. The lack of systematic prospective data precludes a proper understanding of the actual rates, incidence, or timeline of the development of major or minor complications. Lastly, as it is challenging to distinguish HBSE from other variants of SCD, such as HBSC on readily available tests, a bias in reporting HBSE as HBSC (or other variants) could be present in the literature, decreasing the total number of reported HBSE cases.
This review opens the door to consider various SCD therapies in managing HBSE patients with severe or recurrent complications. More extensive studies on the HBSE patient population must formulate management guidelines to treat its moderate to severe complications. Hydroxyurea, the mainstay of treatment of sickle cell disease, has a wellestablished role in HBSS and Hb SB +. However, due to the rare occurrence of HBSE, there are no trials available for the use of HU in this variant of SCD. In our review, HU was used in 4.4% of patients, with good outcomes. Establishing a definite role of HU in symptomatic patients with HBSE might take years as the incidence of HBSE itself, and then the prevalence of symptoms in this patient population is still too low to analyze the use of HU in HBSE prospectively. Meanwhile, we suggest using HU in HBSE patients with moderate to severe VOC and acute chest syndrome symptoms on a case-to-case basis until more extensive evidence becomes available.
# Conclusion
HBSE disease has been considered as a very benign variant of SCD with minimal to no sickling symptoms. However, a growing literature on HBSE and its complications suggests that HBSE considerable number of patients have a moderate form of SCD with a wide range of significant complications, including mortality. More extensive studies with additional data can help categorize and understand HBSE disease and its manifestations better. |
Myeloid sarcoma: a report of four cases at unusual sites
Background: Myeloid sarcoma (MS) or granulocytic sarcoma is a rare tumor consisting of myeloid blasts with or without maturation occurring Fouzia Siraj 1 Manveen Kaur 1 at anatomic sites other than the bone marrow. MS can involve any organ Varsha Dalal 1 system but shows a predilection for skin, bone, and soft tissues of head and neck region.
# Introduction
Myeloid sarcoma (MS), also known as extramedullary acute myeloid leukemia (AML), granulocytic sarcoma (GS) or chloroma, is a tumor composed of myeloid cells occurring at an extramedullary site [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref] ,. The most common sites of involvement include skin, bone, lymph node and soft tissues. MS predominantly consists of cells of the myeloid lineage in varying stages of maturation, from myeloblasts to promyelocytes to neutrophils, that result in effacement of the tissue architecture. It can occur in four clinical situations: in patients with AML as a localized tissue mass, as blast crisis in patients with chronic myeloid leukemia (CML) or leukemic transformation in myelodysplastic syndrome (MDS), before AML and as an isolated neoplasm without evidence of AML. We report four cases of MS to highlight the unusual clinical presentation of this entity and the diagnostic dilemma it can pose [fig_ref] Table 1: Clinicopathologic features of the four cases [/fig_ref]. A 38-year-old male presented with complaint of gradually progressive, left sided nasal obstruction for the last three months. Subsequently there was loss of vision in the left eye. General physical and systemic examination was unremarkable. On anterior rhinoscopy, an irregular, firm mass was seen in the left nasal cavity. Hematologic profile revealed anemia (hemoglobin = 10.4 g/dl), leucocytosis (TLC = 15,000/µl) and marked eosinophilia (absolute eosinophil count = 11,550). Computed tomography (CT) scan showed an intranasal mass involving the bilateral ethmoid sinuses with destruction of the ethmoid septae and the medial wall of the left orbit along with an extension into the inferior extraconal compartment of the left orbit and superior displacement of the left eye ball [fig_ref] Figure 1: CT scan showing an intranasal mass involving the bilateral ethmoid sinuses with... [/fig_ref]. A clinical diagnosis of nasopharyngeal carcinoma was made. Biopsy from the mass showed a tumor in the sub-epithelial region invading the underlying bone. The tumor comprised variable cell population of eosinophils, neutrophils, segmented cells, and myeloid precursors admixed with large, atypical cells with hyperchromatic nuclei and prominent nucleoli [fig_ref] Figure 2: a [/fig_ref] Subsequent bone marrow examination showed myeloid preponderance with marked increase in mature eosinophils and presence of eosinophilic myelocytes and metamyelocytes. However, no blasts were seen. The patient was advised chemotherapy but was lost to follow-up subsequently.
## Case 2
An 11-year-old male child presented with proptosis of the right eye for nine months. Local examination revealed a retro orbital mass. Biopsy from the mass showed large atypical cells with vesicular nuclei, prominent nucleoli, and scant cytoplasm, admixed with myeloid precursors and lymphocytes [fig_ref] Figure 2: a [/fig_ref]. With differential diagnoses of rhabdomyosarcoma, Langerhans cell histiocytosis (LCH) and MS in mind, immunohistochemistry was applied. The atypical cells were strongly positive for MPO, CD43 and negative for CD68, S 100, CD1a, myogenin, and desmin. A diagnosis of MS was rendered and hematologic workup was advised to rule out co-existent acute leukemia. Bone marrow examination was normal. The patient was given chemo-radiation and on follow-up of 18 months, he is disease free.
## Case 3
A 49-year-old male presented with a two week history of sudden onset weakness of left lower limb followed by altered sensorium. General physical and systemic examination was unremarkable. Neurological examination revealed decreased power in the left lower limb (grade1/5) with an extensor plantar reflex. Routine hematologic and biochemical tests were within normal limits. Magnetic resonance imaging (MRI) of the head showed a homogenously enhancing multilobulated mass in the prepontine, premedullary and left cerebellopontine angle cistern region with mass effect over the brainstem and the left cerebral hemisphere. With a clinicoradiologic diagnosis of high grade glioma, surgery with decompression of the mass was done.
Microscopic examination of the tumor tissue showed a highly cellular tumor, diffusely infiltrating the meninges and surrounding fibroadipose tissue. It consisted of mixed population of large round cells, some with indented nuclei and numerous mature and immature eosinophils [fig_ref] Figure 2: a [/fig_ref]. Differential diagnoses included NHL, LCH, and myeloid sarcoma. Immunohistochemical stains for MPO and CD43 [fig_ref] Figure 2: a [/fig_ref] were strongly positive in the immature cells while a smaller number of cells were positive for CD68. Tumor cells were negative for CD1a, CD3, and CD20. A diagnosis of MS was made and bone marrow examination was advised to rule out acute myeloid leukemia. Bone marrow examination showed a cellular marrow with normal hemopoeitic elements and eosinophilic prominence. There was no increase in blasts or presence of abnormal cells. The patient was given chemotherapy but he expired 6 months after the diagnosis.
## Case 4
A 52-year-old male was diagnosed as a case of refractory AML seven months back and put on FLAG idarubicin. He presented with a soft tissue mass in the perineal region for the last 20 days. The swelling was nodular, firm and had a greenish hue. Biopsy was done and microscopic examination revealed a complete effacement of tissue architecture by a tumor showing large areas of necrosis. Tumor cells were large, atypical with irregular nuclei and small nucleoli and were seen infiltrating into the skeletal muscle fibers [fig_ref] Figure 2: a [/fig_ref]. Tumor cells were strongly positive for MPO. A diagnosis of MS secondary to AML was given.
# Discussion
MS is a localized tumor mass comprising immature granulocytic cells. These tumors are also called chloromas due to a greenish color of the tissues imparted by myeloperoxidase present in the cytoplasm of tumor cells [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref]. It was first described in orbit by Burns in 1811. The pathogenesis is believed to be an aberrant expression of homing signals for the leukemic blasts on extramedullary sites as compared to bone marrow [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref]. In a study by Faaij et al. in 2010, AML blasts were found to express chemokine receptors CCR5, CXCR4, CXCR7, and CX3CR1 not seen in blasts of bone marrow and peripheral blood. MS can occur as a localized tissue mass in a known case of AML (3-8% of patients), before occurrence of AML (in 0.6% patients) and in association with other myeloproliferative neoplasms like blast crisis in CML and MDS. Rarely, it occurs de novo with normal marrow and hematologic findings as seen in three of our cases [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref]. There is a predilection for males and last decades of life with a median age of 56 years. MS can involve any site of the body. Skin, soft tissues, bone, lymph nodes, GIT, and testis are affected more frequently [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref] ,. Occurrence in the head and neck region such as skull bones, orbit, and paranasal sinuses is unusual [bib_ref] Granulocytic sarcoma of the nasal cavity, Prades [/bib_ref]. Two of our patients presented with head and neck masses, one in the orbit (Case 1) and the other in the nasal cavity (Case 2). CNS and orbital involvement is commonly seen in the pediatric population. In the absence of overt leukemia, involvement of spinal cord is rare with only a few cases reported in the literature [bib_ref] Granulocytic sarcoma of the spine in a child without bone marrow involvement:..., Shiozawa [/bib_ref]. Thoracic spine is most commonly affected (64%) and approximately 5% cases occur in the cervical region as was seen in our case (Case 3). MS occurs in approximately 2-8% of patients with AML. Of these, 15-35% of the cases of MS can be seen concomitantly with AML, about 25% of the cases precede AML and up to 50% of the cases occur after the diagnosis of AML. It might as well be the initial manifestation of relapse in a patient who has been in remission [bib_ref] Myeloid sarcoma: current approach and therapeutic options, Avni [/bib_ref]. The fourth case in this series developed a soft tissue mass in the perineum 8 months after being diagnosed with AML. The most common microscopic form of myeloid sarcoma consists of a mixed population of myeloblasts, neutrophils, and neutrophil precursors. Based on the extent and degree of maturation, three major subtypes have been defined: blastic, immature, and differentiated. MS blastic type is composed primarily of myeloblasts with little evidence of maturation. The immature type is intermediate between blastic and differentiated forms and consists of myeloblasts, promyelocytes, and eosinophilic myelocytes. The differentiated or mature type is composed of promyelocytes and more mature cells with abundance of eosinophils. Myelomonocytic or pure monoblastic morphology is present in a large number of cases.
The diagnosis of MS is validated by the results of cytochemical and/or immunophenotypic analyses. The immunohistochemical panel includes CD68/KP1, antimyeloperoxidase (MPO), CD43, CD117, CD99, CD68/PG-M1, lysozyme, CD34,terminal deoxynucleotidyl transferase (TdT), CD56, CD30, glycophorin, and CD4. The combination of the above markers allows recognition of tumors with immature myeloid phenotype, as well as cases which show differentiation towards myelomonocytic, monoblastic, erythroid or megakaryocytic lineages. At times aberrant antigenic expression with cytokeratin (CK), B-or T-cell markers is noted. In 55% of the cases chromosomal aberrations are known to occur which can be confirmed by FISH and/or cytogenetics. These include monosomy 7, trisomy 8, MLL rearrangement, inv(16), trisomy 4, monosomy 16, 16q-, 5q-, 20q-and trisomy 11. In the absence of peripheral blood and bone marrow involvement MS can be misdiagnosed as a number of other neoplastic diseases depending on the site of involvement. The major differential diagnosis of MS is lymphoma including lymphoblastic lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. Other less common differentials are soft tissue sarcomas, small round cell tumors (especially in children) and undifferentiated carcinoma.
It is important to distinguish between these entities by cytochemical and immunohistochemical stains for prompt recognition and institution of appropriate treatment [bib_ref] Myeloid sarcomas: a histologic, immunohistochemical, and cytogenetic study, Alexiev [/bib_ref].
The prognosis of MS depends upon the initial context in which it appears. Most patients with isolated MS progress to AML within months of diagnosis. Cases that progress to AML seldom survive more than a year [bib_ref] Myeloid sarcoma is associated with superior event-free survival and overall survival compared..., Tsimberidou [/bib_ref].
Patients with primary myeloid sarcoma should be treated as AML even in the absence of clinically detectable leukemia. The treatment modalities include induction/intensification therapy similar to those with AML. The role of radiotherapy is also described by some authors [bib_ref] Comparison in treatments of nonleukemic granulocytic sarcoma: report of two cases and..., Yamauchi [/bib_ref].
# Conclusion
MS is an uncommon tumor and can occur in unusual locations with variable presentation as seen in the present series. Therefore, it should be considered in the differential diagnosis of a soft tissue mass regardless of the presence/absence of leukemia. It is important to make a correct diagnosis by using the armamentarium of morphology, histochemistry, and immunohistochemistry collectively for prompt institution of therapy.
## Notes competing interests
The authors declare that they have no competing interests.
[fig] Figure 1: CT scan showing an intranasal mass involving the bilateral ethmoid sinuses with the destruction of the ethmoid septae and the medial wall of the left orbit. [/fig]
[fig] Figure 2: a: Photomicrograph showing sheets of immature cells admixed with interspersed mature inflammatory cells including eosinophils and their precursors (H&E, x 200). b: Large atypical cells with vesicular nuclei, prominent nucleoli and scant cytoplasm, admixed with myeloid precursors and lymphocytes (H&E, x 200). c: Mixed population of large round cells, some with indented nuclei and numerous mature and immature eosinophils (H&E, x 400). d: Large cells with irregular nuclei and small nucleoli, infiltrating into the skeletal muscle fibers (H&E, x 400). e: The cells show diffuse strong positivity for myeloperoxidase (MPO) immunostain (x 400). f: CD43 is strongly positive in immature cells (x 400). g: Cells showing diffuse strong positivity for CD 117 immunostain (x 400). h: High Ki-67 proliferative index in immature precursors (x 400). [/fig]
[table] Table 1: Clinicopathologic features of the four cases [/table]
|
A New Electrochemical Sensor Based on Task-Specific Ionic Liquids-Modified Palm Shell Activated Carbon for the Determination of Mercury in Water Samples
In this study, a potentiometric sensor composed of palm shell activated carbon modified with trioctylmethylammonium thiosalicylate (TOMATS) was used for the potentiometric determination of mercury ions in water samples. The proposed potentiometric sensor has good operating characteristics towards Hg (II), including a relatively high selectivity; a Nernstian response to Hg (II) ions in a concentration range of 1.0 × 10 −9 to 1.0 × 10 −2 M, with a detection limit of 1 × 10 −10 M and a slope of 44.08 ± 1.0 mV/decade; and a fast response time (~5 s). No significant changes in electrode potential were observed when the pH was varied over the range of 3-9. Additionally, the proposed electrode was characterized by good selectivity towards Hg (II) and no significant interferences from other cationic or anionic species.
# Introduction
Mercury is a major water pollutant of high concern, producing severe ailments in living beings, including mental retardation. It is a toxic bio-accumulative environmental pollutant that affects the nervous system and it is released into the environment through industrial, agricultural and other anthropogenic processes. Interests in the determination of trace amount of mercury ions have OPEN ACCESS significantly increased during the past few years due to growing environmental concerns. Several analytical techniques, including, cold vapor atomic absorption spectrometry (CV-AAS) [bib_ref] Trace mercury determination in drinking and natural water samples by room temperature..., Martinis [/bib_ref] [bib_ref] Determination of Hg in water and wastewater samples by cv-aas following on-line..., Zavvar Mousavi [/bib_ref] [bib_ref] On-line preconcentration and determination of mercury in biological and environmental samples by..., Ferrúa [/bib_ref] , inductively coupled plasma optical emission spectrometry (ICP-OES) [bib_ref] Sensitive determination of mercury in tap water by cloud point extraction pre-concentration..., De Wuilloud [/bib_ref] [bib_ref] Evaluation of slurry preparation procedures for the determination of mercury by axial..., Santos [/bib_ref] , X-ray fluorescence spectrometry [bib_ref] Simultaneous determination of traces of iron, cobalt, nickel, copper, mercury and lead..., Lau [/bib_ref] , inductively coupled plasma mass spectrometry (ICP-MS) [bib_ref] Gas chromatography/plasma spectrometry-A important analytical tool for elemental speciation studies, Wuilloud [/bib_ref] [bib_ref] Mechanistic studies on the trapping and desorption of volatile hydrides and mercury..., Matousek [/bib_ref] and cold vapour atomic fluorescence spectrometry (CV-AFS) [bib_ref] Determination of very low levels of dissolved mercury(ii) and methylmercury in river..., Bagheri [/bib_ref] [bib_ref] Determination of mercury by electrochemical cold vapor generation atomic fluorescence spectrometry using..., Jiang [/bib_ref] [bib_ref] Determination of trace inorganic mercury in mineral water by flow injection on-line..., Zi [/bib_ref] have been applied for the determination of trace amounts of mercury in analytical samples. These methods have good sensitivity, and well controlled experimental conditions. However, they have several disadvantages, such as the use of expensive apparatus, complicated operation, high operation and maintenance costs, and the requirement of well controlled experimental conditions. Because of their advantages in terms of low cost, easy fabrication, simplicity, sensitivity and fast response time, potentiometric sensors based on ion selective electrodes have attracted much attention in electro-analytical chemistry and have been successfully used to determine trace levels of hazardous pollutants such as mercury [bib_ref] New triiodomercurate-modified carbon paste electrode for the potentiometric determination of mercury, Abbas [/bib_ref] [bib_ref] A mercury(ii) selective sensor based on n,n'-bis(salicylaldehyde)-phenylenediamine as neutral carrier for potentiometric..., Abu-Shawish [/bib_ref] [bib_ref] An ion selective electrode for mercury (ii) based on mercury (ii) complex..., Bakhtiarzadeh [/bib_ref] [bib_ref] Ion-selective carbon paste electrode based on tetraethyl thiuram disulfide for copper (ii)..., Gismera [/bib_ref] [bib_ref] A highly selective mercury electrode based on a diamine donor ligand, Gupta [/bib_ref] [bib_ref] Novel mercury(ii) ion-selective polymeric membrane sensor based on ethyl-2-benzoyl-2-phenylcarbamoyl acetate, Hassan [/bib_ref] [bib_ref] Applications of Hg (II) sensitive polyaniline Sn (iv) phosphate composite cation-exchange material..., Khan [/bib_ref] [bib_ref] Multi-walled carbon nanotubes-ionic liquid-carbon paste electrode as a super selectivity sensor: Application..., Khani [/bib_ref] [bib_ref] A mercury ion-selective electrode based on a calixarene derivative containing the thiazole..., Lu [/bib_ref] [bib_ref] A mercury (ii) ion-selective electrode based on neutral salicylaldehyde thiosemicarbazone, Mahajan [/bib_ref] [bib_ref] Highly selective potentiometric determination of mercury(ii) ions using 1-furan-2-yl-4-(4-nitrophenyl)-2-phenyl-5h-imidazole-3-oxide based membrane electrodes, Mahajan [/bib_ref] [bib_ref] Mercury (ii) ion-selective polymeric membrane sensor based on a recently synthesized schiff..., Mashhadizadeh [/bib_ref] [bib_ref] Mohammadpoor-Baltork, I. Mercury selective membrane electrodes using 2-mercaptobenzimidazole, 2-mercaptobenzothiazole, and hexathiacyclooctadecane carriers, Mazloum [/bib_ref] [bib_ref] Mercury (ii) selective membrane electrode based on 1,3-bis(2-methoxybenzene)triazene, Rofouei [/bib_ref] [bib_ref] Mercury(ii)-selective membrane electrode using tetrathia-diazacyclotetradeca-2,9-diene as neutral carrier, Singh [/bib_ref] [bib_ref] Highly selective sensing of mercury (ii) by development and characterization of a..., Yari [/bib_ref] [bib_ref] Mercury(ii)-selective polymeric membrane electrode based on the 3-[4-(dimethylamino)phenyl]-5-mercapto-1,5-diphenylpentanone, Yu [/bib_ref] [bib_ref] Flow injection analysis of mercury using 4-(dimethylamino) benzaldehyde-4-ethylthiosemicarbazone as the ionophore of..., Tahir [/bib_ref].
Ion selective electrodes (ISEs) are potentiometric sensors used to measure some of the most hazardous analytes in environmental laboratory and point-of-care analysers. Despite their easy fabrication, simple usage, and low cost, ISEs suffer from low response sensitivity, interference by a number of metal ions and short lifetimes. As a result, the development of new ISE materials that can address some of these limitations is a worthwhile and challenging topic of research. The ultimate goals of this study are to increase the sensitivity and selectivity of the proposed electrode by minimizing the previously mentioned undesirable electrode processes. Additionally, the application of plasticizer-free electrodes can eliminate the leaching of the electrode solvent and sensing components, thus improving the electrode lifetime.
Carbon is a very important electrode material and is widely used due to its low cost, easy functionalisation, great versatility, broad potential window, and chemical inertness. Various forms of carbons, such as glassy carbon, impregnated graphite, carbon fibres, carbon films, carbon nanotubes, and activated carbon, could be used as electrode materials. Palm shell, a waste product of palm kernel oil production, represents an important group of carbonaceous materials with unique mechanical, physical, and electrochemical properties [bib_ref] Palm shell activated carbon impregnated with task-specific ionic-liquids as a novel adsorbent..., Ismaiel [/bib_ref].
Room temperature ionic liquids are salts having very low melting temperature close to room temperature. Room temperature ionic liquids have become an extremely popular theme in recent electrochemical sensing research, due to their large electrochemical window, high conductivity, non-volatility, low toxicity and good electrochemical stability. Recently, new ion selective sensors based on room temperature ionic liquids have been developed [bib_ref] Multi-walled carbon nanotubes-ionic liquid-carbon paste electrode as a super selectivity sensor: Application..., Khani [/bib_ref] [bib_ref] Multi-walled carbon nanotubes (mwcnts) and room temperature ionic liquids (rtils) carbon paste..., Faridbod [/bib_ref] [bib_ref] Potentiometric response of ion-selective membranes with ionic liquids as ion-exchanger and plasticizer, Peng [/bib_ref] [bib_ref] Applications of ionic liquids in electrochemical sensors, Wei [/bib_ref].
In this work palm shell activated carbon was modified with trioctylmethylammonium thiosalicylate (TOMATS) to act as a new potentiometric sensor for determination of Hg (II) ion in water samples. It is worth mentioning that TOMATS acts as ionophore as well as plasticizer. To the best of our knowledge this is the first study reporting the use of TOMATS (structure as shown in [fig_ref] Figure 1: Chemical structure of TOMATS [/fig_ref] for the determination of mercury ions in water samples. TOMATS was previously shown to be a very good ligand for Hg (II) [bib_ref] Palm shell activated carbon impregnated with task-specific ionic-liquids as a novel adsorbent..., Ismaiel [/bib_ref] which makes it a potential ionophore in a potentiometric sensor.
## Experimental section
## Chemicals and reagents
Analytical reagent grade chemicals and distilled, de-ionized water were used to prepare all aqueous solutions. Commercially available granular palm shell activated carbon (PSAC) was provided by Bravo Green Sdn. Bhd, Malaysia. Activated carbon powder with particle sizes ˂40 µm were used throughout the potentiometric experiments. PSAC was washed with distilled water to remove fines and dirt; and was dried in an oven at 110 °C for 24 h. The pH of the solutions was adjusted by adding appropriate amounts of concentrated hydrochloric acid (1 M HCl) and/or sodium hydroxide (2 M NaOH). Metal salts were purchased from Merck (Selangor, Malaysia), and aqueous metal solutions were prepared by dissolving appropriate quantities of metal salts in de-ionized water. trioctylmethylammonium thiosalicylate (TOMATS) was purchased from Sigma-Aldrich (Kuala Lumpur, Malaysia).
## Apparatus
All potentiometric measurements were made using a pH/ion meter (Metrohm-781, Filderstadt, Germany) and pH Module (Metrohm-867), permitting real-time potential data collection using the proposed electrode in conjunction with a double junction Ag/AgCl reference electrode. The temperature of the cell holder was maintained at 25 °C and measured under constant stirring with a magnetic stirring bar at 180 rpm.
The electrochemical cell used in this study was constructed as follows:
Ag(s), AgCl(s) | KCl(3 M sat.) || Sample solution| modified palm shell activated carbon paste electrode.
Metal ion sample concentration was analyzed by inductivity coupled plasma optical emission spectrometer ICP-OES. (model ICP optima 7000DV, PerkinElmer, Waltham, MA, USA).
## Modified palm shell activated carbon paste electrode preparations and potential measurements
Modified palm shell activated carbon paste was prepared by hand mixing the determined quantities of palm shell activated carbon powder and TOMATS. The optimal paste quality was obtained by mixing 0.15 g PSAC and 0.15 g TOMATS in the ratio 1:1 (w/w). The constituents were thoroughly hand mixed in a 50 mm Petri dish to produce the optimal paste quality and then the paste was poured and packed into empty glassy carbon electrode (5 mm diameter) connected to the pH/ion meter by a thin copper wire to produce an electrical contact. The composite surface was polished on weighing paper until the surface displayed a shiny appearance. The surface was rinsed carefully with double-distilled water prior to each experiment. The electrode is stored in a desiccator when it is not in use to avoid adsorption of contaminants.
The potentiometric measurements were conducted as follows: the modified carbon paste electrode and reference electrode were placed in 50 mL of a stirred, 0.1 M Hg (II) solution until the potential reading was constant. The standard addition method was used to investigate the electrode response characteristics. Mercury salt standard solutions were added so that the mercury concentration ranged between 10 −10 and 10 −1 M. A suitable volume (0.2-100 µL) of mercury standards was pipetted into 50 mL of water in a measuring beaker and the potential measured in the appropriate way for the ion to be measured (i.e., with stirring and sufficient time for stable reading). The potential readings were recorded after each addition when stable values had been obtained. The concentration of solutions was checked by ICP.
The electrode potential of the electrochemical cell E cell is described by the following Nernst equation:
[formula] cell cons 2.303 log RT E E a zF = + (1) [/formula]
where E cons is a constant term (the sum of the standard potential and liquid junction potential), R is the ideal gas constant, T is the absolute temperature, F is the Faraday constant, z is the charge of the ion, and a is the activity of the ion. At low concentrations, the activity value a can be replaced with the concentration value C. The prelogarithmic factor 2.303
RT zF is obtained from the slope (S) of the plot of E cell versus log C, and the equation becomes:
[formula] cell cons log C E E S = +(2) [/formula]
Potentiometric selectivity of this electrode towards different cations was calculated with the matched potential method (MPM) [bib_ref] Determination of selectivity coefficients of ion-selective electrodes by a matched-potential method, Gadzekpo [/bib_ref]. In this method, the activity of Hg (II) was increased from a i = 1.0 × 10 −5 M (primary ion) to á i = 5.0 × 10 −5 M, and the corresponding potential change (ΔE) was measured. Then a solution of an interfering ion (a j ) in the concentration range of 1.0 × 10 −1 -1.0 × 10 −2 M was added to a new primary ion (á i ) until the same potential change (ΔE) was recorded. The selectivity factor, k ij pot , was calculated for each interferent using the following equation:
[formula] ( ' ) / pot ij i i j k a a a = −(3) [/formula]
# Results and discussion
## Response of the electrode
The calibration for the developed electrode over a wide range of solution Hg (II) activities is shown in [fig_ref] Figure 2: Calibration curve for a modified palm shell activated carbon paste electrode over... [/fig_ref]. The slope of the calibration curve (44.08 mV/dec) is close to that predicted theoretically by the Nernst equation (58.16 mV/dec for monovalent cations), which may be attributed to the formation of monovalent mercury complexes on the electrode surface. This finding indicates that the electrode was sensitive to Hg (II) over a wide range of Hg (II) activities (1 × 10 −9 -1 × 10 −2 M). In addition, the electrode showed a linear response over this range of activities, with a departure from linearity (i.e., loss of sensitivity) at activities lower than 10 −9 M Hg (II). The unique sensitivity and selectivity towards Hg (II) obtained for this electrode is due to the coordinate interaction between TOMATS and Hg (II) ions, which may be explained by the chelating effect of the ortho-positioned carboxylate group on the TOMATS molecule impregnated on palm shell activated carbon in addition to the known formation of metal-thiolates.
TOMATS which was used as the solvent mediator and plasticizer, have certain desirable properties and characteristics, such as high lipophilicity, high molecular weight, and low vapour pressure. Additionally, their viscosities and dielectric constants were adequate for the construction of an ion selective electrode with desirable analytical properties, such as selectivity, sensitivity, fast response, and long lifetime. The critical response characteristics of the proposed electrode were evaluated according to IUPAC recommendations [bib_ref] Recommendations for nomenclature of ion selective electrodes, Buck [/bib_ref].
## Effect of ph on electrode response
The pH of each solution was verified, and its effect on the electrode potential at various metal concentrations was studied. For this purpose, several Hg (II) concentrations (1.0 × 10 −6 M, 1.0 × 10 −4 M and 1.0 × 10 −3 M) were prepared, and the potential variations of the electrode over a pH range of 1-12 were followed. The pH was adjusted by adding small volumes of hydrochloric acid (1 M) and/or sodium hydroxide (2 M) to the sample solution.
The results, shown in , indicate that the potential remained constant in the pH range of 3-9, which can be used as the working pH range of the proposed electrode. However, outside this range, the electrode responses changed slightly. The diminished potential at pH > 9 was due to the interference of OH − on the surface. The response at pH < 3 seemed ascribable to the competitive binding of protons to the ligands on the electrode surface. . Effect of pH on the potential response of Hg (II) palm shell activated carbon paste electrode.
## Potentiometric selectivity coefficients
It is well known that the selectivity behavior of an electrode is one of the most important factors in its evaluation, which is measured in terms of the selectivity coefficient. The selectivity coefficient not only depends on ion charge and concentration, but it can also be affected by the type of interaction between the ion and the ionophore. The selectivity factor, log k pot is a measure of the preference of ion selective electrode for interfering ion relative to the primary ion to be measured. A selectivity factor log k pot below 1 indicates that the preference is for the primary ion.
The values of the selectivity coefficients, listed in [fig_ref] Table 1: Selectivity coefficient values of various interfering ions with Hg [/fig_ref] , reflect a very high selectivity of this electrode for mercury (II) ion over most of the tested species j. Ag + , Pb 2+ and Cu 2+ caused only slight interference. However, they do not cause any interference at low concentration. As shown in [fig_ref] Table 1: Selectivity coefficient values of various interfering ions with Hg [/fig_ref] , it can be observed, that the proposed electrode based on TOMATS exhibited better selectivity for mercury (II) ion over a wide variety of other metal ions.
## Dynamic response time
The response time of the electrode is one of the most important characteristics of the ion selective electrode. According to IUPAC recommendations, the response time of an ion selective electrode is defined as the time between the addition of the analyte to the sample solution and the time when limiting potential has reached its steady state value within ±1 mV. In this study, the response time of the electrode was tested by measuring the time required to achieve a steady state potential (within ±1 mV of the final equilibrium value) after successive immersion in a series of Hg (II) ions. The results, shown in [fig_ref] Figure 4: Response time of the electrode obtained by successive increase of Hg [/fig_ref] indicate that the response time of the electrode was approximately 5 s for the solution of mercury ion in the concentration range of 1 × 10 −8 -1 × 10 −4 M. This result is probably due to the fast complexation of Hg (II) ions by the TOMATS molecule dispersed in the palm shell activated carbon paste matrix.
## Electrode life time
The life time of the electrode depends on the distribution coefficient of the electrode compositions between the aqueous phase and the electrode phase. Accordingly, the life time of the electrode must depend on the electrode components.
In this work, the life time of the electrode was determined by performing periodic calibrations with standard solutions and calculating the slopes over Hg (II) ion concentration ranges of 1 × 10 −9 to 1 × 10 −2 M. The obtained results showed that the lifetime of the present electrode was over 90 days [fig_ref] Table 2: Mercury electrode response during 90 days [/fig_ref]. During this time, the detection limit of the electrode remained almost constant and the slope of the electrode response decreases from 44.08 to 42.17 mV per decade. Therefore, the electrode can be used for at least 3 months, without a considerable change in their response characteristic towards Hg (II) ions.
## Comparison of the response for the proposed hg (ii) electrode with other reported electrodes
The comparison of the performance of the proposed electrode with that of some recently developed electrodes for Hg (II) determination is given in . . Comparison of the proposed Hg electrode with previously reported electrodes. As clearly shown in this table the sensor proposed in this work has excellent performance in terms of response time, linear range, Nernstian slope, and detection limit. The proposed electrodes showed improved performance characteristics relative to conventional electrodes. This improvement presumably originates from the electrode composition.
## Analytical applications
The proposed electrode was applied for determination of Hg (II) in real drinking water sample. The results of Hg (II) content measured by proposed electrode were compared with those obtained by ICP. [fig_ref] Table 4: Potentiometric determination of Hg [/fig_ref] shows that Hg (II) concentration values obtained by proposed electrode were similar to those obtained by ICP, with deviations below 2% for all samples.
# Conclusions
The results presented herein demonstrate the utility of TOMATS as both plasticizer and ionophore in the preparation of new ion selective electrodes for the determination of mercury ions in water samples. The proposed electrode had fast response for detection of mercury ions. The proposed electrode has good operating characteristics towards Hg (II), including a relatively high selectivity; a Nernstian response to Hg (II) ions in a concentration range of 1.0 × 10 −9 to 1.0 × 10 −2 M, with a detection limit of 1 × 10 −10 M and a slope of 44.08 ± 1.0 mV/decade; and a fast response time (~5 s). No significant changes in electrode potential were observed when the pH was varied over the range of 3-9. The electrode was successfully applied for the determination of mercury content in drinking water samples. These characteristics and the typical applications presented in this work make the electrode suitable for measuring the mercury content in real samples without a significant interaction from other cationic or anionic species.
[fig] Figure 1: Chemical structure of TOMATS. [/fig]
[fig] Figure 2: Calibration curve for a modified palm shell activated carbon paste electrode over a wide range of Hg (II) activities. [/fig]
[fig] Figure 4: Response time of the electrode obtained by successive increase of Hg (II) ion. [/fig]
[table] Table 1: Selectivity coefficient values of various interfering ions with Hg (II) selective electrode using matched potential method (MPM). [/table]
[table] Table 2: Mercury electrode response during 90 days. [/table]
[table] Table 4: Potentiometric determination of Hg (II) in water samples using proposed electrode and ICP. From some ground water wells in the Gaza Strip; b Mean data for three replicate measurements. [/table]
|
Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells
Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE 2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE 2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE 2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis.
# Introduction
Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent painful oral aphthaes. The prevalence of RAS is between 10%~20% in the general population. RAS is an inflammatory condition of unknown etiology. Various cytokines such as interleukin-6 (IL-6) and IL-8 have been implicated as potential etiopathogenic agents [bib_ref] The effect of gastric Helicobacter pylori eradication on recurrent aphthous stomatitis, Karaca [/bib_ref]. RAS progression is regulated by the host response, and there is increasing evidence that non-immune cells such as human gingival fibroblasts (hGFs), participate in the host response [bib_ref] p38 MAPK signaling in oral-related diseases, Patil [/bib_ref]. Other studies have reported that hGFs express Toll-like receptors and CD14, which is consistent with the finding that these cells respond to lipopolysaccharide [bib_ref] Variable expression of Toll-like receptor in murine innate and adaptive immune cell..., Applequist [/bib_ref]. Arikan et al. demonstrated that lipid peroxidation seems to play a crucial role in the pathogenesis of recurrent aphthous stomatitis [bib_ref] Oxidant/ antioxidant status in recurrent aphthous stomatitis, Arikan [/bib_ref]. RAU has known to evoke the Immune alterations by the activation of T lymphocytes and cytokines secretion [bib_ref] Treatment of recurrent aphthous stomatitis. A literature review, Belenguer-Guallar [/bib_ref]. There have also been reported the dysfunction of enzyme superoxide dismutase (SOD), which participates in the inflammatory response of RAS [bib_ref] Assessment of salivary and serum antioxidant vitamins and lipid peroxidation in patients..., Saral [/bib_ref]. Recent studies reported that phorbol 12-myristate 13-acetate (PMA) could play an important role in the induction of cyclooxygenease-2(COX-2) expression and cytokines release [bib_ref] The antiangiogenic agent SU5416 down-regulates phorbol estermediated induction of cyclooxygenase 2 expression..., Saha [/bib_ref] [bib_ref] Resolution of PMA-induced skin inflammation involves interaction of IFNgamma and ALOX15, Zhang [/bib_ref] [bib_ref] Identification of IFN-gamma-producing CD4+ T cells following PMA stimulation, Kemp [/bib_ref]. The synthesis of prostaglandin (PGs) in particular prostaglandin E2(PGE 2 ) is regulated by main enzyme isoforms, cyclooxygenease-1(COX-1) and COX-2 [bib_ref] Innate immunity: A COX detox for the immune system?, Bordon [/bib_ref]. The PMA has known to induce the reactive oxygen species (ROS) mediated apoptosis in chronic inflammatory disease [bib_ref] Diminished macrophage apoptosis and reactive oxygen species generation after phorbol ester stimulation..., Palmer [/bib_ref]. It is well known that oxidative stress can arise through the increased production of reactive oxygen species (ROS) and/or a deficiency of antioxidant defenses. Insufficient antioxidant enzyme synthesis may in turn be due to decreased micronutrient availability [bib_ref] Oxidant/ antioxidant status in recurrent aphthous stomatitis, Arikan [/bib_ref]. Hence dietary intake of antioxidants is indispensable to protecting against oxidative-derived diseases. Increasing attention has been focused on the role of ROS produced by activated neutrophils during the inflammatory response [bib_ref] Neutrophil activation and production of reactive oxygen species in pre-eclampsia, Lee [/bib_ref]. To improve the deficient antioxidant synthesis, several clinical studies have suggested that zinc treatment could be useful to balance the redox potential [bib_ref] Steegers-Theunissen RP. The importance of folate, zinc and antioxidants in the pathogenesis..., Ebisch [/bib_ref] [bib_ref] Zinc: role in immunity, oxidative stress and chronic inflammation, Prasad [/bib_ref]. Zinc, thus not only function as an antioxidant but is also an anti-inflammatory agent. Zinc ions regulated T-and B-lymphocyte function, making it vital for the maintenance of normal immune function and resistance to infection [bib_ref] Zinc treatment prevents lipopolysaccharide-induced teratogenicity in mice, Carey [/bib_ref] [bib_ref] Thymulin and zinc (Zn2+)-mediated inhibition of endotoxin-induced production of proinflammatory cytokines and..., Haddad [/bib_ref]. Zinc supplementation has been shown to have a favorable effect on fibrosis [bib_ref] Possible inhibitory effect of oral zinc supplementation on hepatic fibrosis through downregulation..., Takahashi [/bib_ref] [bib_ref] Involvement of intracellular glutathione in zinc deficiency-induced activation of hepatic stellate cells, Kojima-Yuasa [/bib_ref] , metabolism in the thyroid gland (23), anti-oxidation [bib_ref] Zinc: role in immunity, oxidative stress and chronic inflammation, Prasad [/bib_ref] , and anti-depression. Zinc is an essential nutrient that is necessary for a broad range of biological activities. To date, more than 300 zinc-containing enzymes have been identified. Zinc deficiency causes biological dysfunction of gene expression, protein synthesis, immunity, skele-tal growth and maturation, gonadal development, pregnancy, taste perception, and appetite [bib_ref] Zinc deficiency alters DNA damage response genes in normal human prostate epithelial..., Yan [/bib_ref] [bib_ref] Development of alimentary zinc deficiency in growing rats is retarded at low..., Roth [/bib_ref] [bib_ref] Reduced growth and skeletal changes in zinc-deficient growing rats are due to..., Rossi [/bib_ref] [bib_ref] Pubertal arrest due to Zn deficiency: the effect of zinc supplementation, Karaca [/bib_ref] [bib_ref] The role of zinc in the treatment of taste disorders, Yagi [/bib_ref]. Some researchers have investigated the possible correlation of some oxidative stress parameters in Behcet's disease and reported that zinc levels were inversely correlated with the clinical manifestation index [bib_ref] Oxidative stress in patients with Behcet's disease: I correlation with severity and..., Najim [/bib_ref] [bib_ref] Predominance of Type 1 cytokines and decreased number of CD4(+)CD25(+high) T regulatory..., Lewkowicz [/bib_ref] , which demonstrated the importance of zinc as an antioxidant in inflammation-induced cells. Increasing evidence demonstrates that zinc has an anti-inflammatory effect [bib_ref] Zinc supplementation during pregnancy protects against lipopolysaccharide-induced fetal growth restriction and demise..., Chen [/bib_ref]. An earlier report showed that injection with zinc sulfate reduced LPS-induced teratogenicity in mice [bib_ref] Zinc treatment prevents lipopolysaccharide-induced teratogenicity in mice, Carey [/bib_ref] and a recent study found that injection with zinc sulfate alleviated LPS-induced neurodevelopmental damage in the fetal brain [bib_ref] Prenatal exposure to lipopolysaccharide results in neurodevelopmental damage that is ameliorated by..., Chua [/bib_ref]. Although previous studies have reported that zinc can reduce inflammation, it is unclear which cytokines are involved and reduced by zinc in the inflammatory state. The purpose of the present study was to investigate the effect of zinc on the process of wound healing in human gingival fibroblasts cells on an in vitro Phorbol-12-myristate-13-acetate (PMA)-induced inflammatory model. Also, the activation profiles of inflammatory cytokines were revealed in response to zinc treatment.
# Material and methods
-Chemicals and Reagents Phorbol-12-myristate-13-acetate (PMA), Zinc chloride (ZnCl2), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), Trisma base, sodium azide, sodium chloride and phenylmethylsulphonyl fluoride were purchased from Sigma (St. Louis, Mo, USA). Protein inhibitor cocktail were purchased from roche (roche, Mannheim, Germany). -Primary Cell Culture Human gingival fibroblast cells (hGFs) were obtained from three healthy adults visiting the Chonnam National University Hospital for a gingivectomy. The gingival tissues were finely cut with scissors and cultured in an alpha minimum essential medium (α-MEM) (Gib-coBRL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibioticantimycotic solution (Cambrex Bio Science, Baltimore, MD, USA) at 37 °C in a 5% CO 2 humidified chamber. The medium was replaced with fresh medium and the adherent hGFs were allowed to reach approximately 70% confluence. The cells were then detached using trypsine-ethylenediamine tetra acetic acid (trypsin-re TA: GibcoBRL, Rockville, MD, USA) solution and plated again (subcultured) in 6-well plates for each experiment.
-Cell viability assay The cell viability was evaluated using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) e182 (MTT; Sigma-Aldrich) assay. The cells were seeded at 5 x 10 3 cells per 200 uL of medium in 96-well plates, and cultured for 1 day at 37 °. Various concentrations (0,1,5,10,20, and 50 uM) of ZnCl 2 for a zinc donor were added in each well with phorbol 12-myristate 13-acetate (PMA, 1uM), and the cells were subsequently incubated at 37° for 24 hours. After removing the medium, 100 uL of MTT (50 ug/ml) was added to the cells. The cells were incubated at 37° for 4 hours and 30 minutes to allow color development, and the formazan product was solubilized by the addition of 50 ml dimethyl sulfoxide (Calbiochem Bio-Mol, La Jolla, CA). Optical density was measured at 570 nm and reference with 655nm using a microplate reader (Bio-Rad, Hercules, CA).
-Western blotting The hGFs were seeded at 5 x 10 4 cells per well in ά-MEM media, and cultured for 1 day. The ZnCl 2 (1,5,10 and 20 uM) was then added to each well in the presence of PMA (1uM), and the cells were incubated for 24 hours. The medium was removed and washed twice with PBS. Cell lysates were then prepared in 200 ul of cold lysis buffer (1% NP-40,50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02% sodium azide, 150 mg/ml phenylmethylsulphonyl fluoride, protein inhibitor cocktail). Thirty milligrams of cell lysates were separated in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL). The membrane was blocked with blocking solution [5% skim milk in Tris-Buffered Saline and Tween 20(TBST) (2.42 g/L Tris-HCl, 8 g/L NaCl, 0.1% Tween 20, pH 7.6)] for 30 minutes and rinsed briefly in TBST. The membrane was incubated overnight at 4° with anti-COX-1 (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-COX-2 (1:1,000; Abcam, Cambridge, UK), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:2,500; Santa Cruz). After rinsing with TBST, the membrane was incubated for 1 hour with anti-rabbit and anti-mouse horse radish peroxidase-conjugated (1:2,000) secondary antibody. Finally, the membrane was washed in TBST, and the immunoreactivities of the proteins were detected using an enhanced chemiluminescence detection kit (Amersham) and the levels were determined by densitometric analysis using Scion Image software (Scion Corp, Frederick, MD). -Enzyme-Linked Immunoassay for PGEThe PGE 2 expression level, the main metabolite of COX, was used in all subsequent experiments for this study. The amount of PGE 2 was measured in the supernatants using a commercially available enzyme immunoassay kit (R&D System, Minneapolis, MN, USA) according to the manufacturer's instruction. The hGFs were seeded at 5 x 10 4 cells per well in ά-MEM media, and cultured for 1 day. The ZnCl 2 (1, 5, 10 and 20 uM) was then treated to each well in the presence of PMA (1uM), and the cells were incubated for 24 hours. After 24 hr incubation, the absorbance for PGE 2 was measured at 586 nm by using a colorimetric microplate reader (Bio-rad, Hercules, CA, USA). -Detection of total ROS formation with flow cytometry Total reactive oxygen species (ROS) were assayed using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). DCF-DA enters cells passively, where it is enzymatically deacetylated by esterases to the non-fluorescent 2, 7-dihydrodichlorofluorescein (DCF-H). In the presence of oxidizing molecules such as O 2 -, DCF-H is converted to the highly fluorescent DCF. To measure intracellular ROS levels, the hGFs were seeded at 5 x 10 4 cells per well in media, and cultured for 1 day. The ZnCl 2 (1,5,10 and 20 uM) was then treated to each well in the presence of PMA (1uM), and the cells were incubated for 24 hours. After incubation, the cells were washed with PBS containing 10 nM glucose and treated with 10 mM H2DCF-DA for 20 minutes. The cells were detached using trypsin-EDTA solution, and ROS levels were analyzed using flow cytometry (Beckman Coulter Fullerton, CA) at 485 nm excitation and with 530 nm emission filters. To monitor the ROS formation, cells grown on cover slips were incubated with 10 nM of DCF-DA for 20 minutes. After the cells were washed with PBS containing 10 mM glucose, DCF fluorescence intensity was monitored using a confocal microscope (Carl Zeiss, Oberkochen, Germany) set at excitation and emission wavelengths of 488 and 525 nm, respectively. -Cytokine profiling To assess cytokine production profiles, the supernatant from the cultured hGFs was collected and assayed using a human inflammation antibody array (R&D Systems, Minneapolis, MN, U.S.A.) according to the instruction manual. This method uses a membrane coated with specific antibodies for each cytokine forming an array. The cytokine signal was detected using an ECL detection kit (Promega, Medison, WI, USA) and quantified by densitometric analysis using Scion Image software (Scion Corp, Frederick, MD, USA). The signal intensity of each spot was adjusted to the corresponding internal control provided for each membrane by the manufacturer (cytokine profiling). The ratio of expression was calculated by dividing the signal intensity of each cytokine by the signal intensity of the internal control for each sample.
-Statistics Data is expressed as the mean ± standard deviation. All experiments were carried out three times. The differences between the groups were evaluated using one way ANOVA. Data were considered statistically significant, at P < 0.05.
# Results
-The cell survival effects of zinc in PMA-treated hGFs To determine the effects of Zinc on cell survival in PMA-treated hGFs, cells were treated by 1μM of PMA with or without Zinc in a dose dependent manner. In the PMA-treated hGFs, cell survival was decreased to 80% as shown in [fig_ref] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE... [/fig_ref]. However, zinc could recover the cell survival to around 100% of control.
-The effects of zinc on COX protein expression and PGE 2 release in PMA-treated hGFs The effect of zinc was investigated on COX protein expression in PMA-induced hGFs using western blot analysis. Protein expression of COX-2 after PMA treatments was revealed in the hGFs [fig_ref] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE... [/fig_ref]. After the zinc treatment, COX-2 expression was significantly decreased to against PMA induced inflammatory response [fig_ref] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE... [/fig_ref]. However, PMA induced decrease of COX-1 expression, while ZnCl 2 supplementation recovered to control levels [fig_ref] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE... [/fig_ref]. The release of PGE 2 was increased to 1850 pg/mL in PMA-treated hGFs [fig_ref] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE... [/fig_ref]. On the other hand, PGE 2 release was reduced to 1600 pg/ mL in the presence of zinc (20 μM).
## -the effects of zinc on ros levels in pma-treated hgfs
To assess the effects of zinc on intracellular ROS levels in the PMA-treated hGFs, the production of intercellular ROS levels were measured using DCF-DA fluorescence. As shown in [fig_ref] Figure 2: Effect of zinc on PMA -treated ROS generation in hGFs [/fig_ref] , DCF-DA fluorescence was increased by PMA to 83.7% in hGFs. However, zinc (20μM) on PMA-treated cells decreased the DCF-DA fluorescence to 67.5%. To confirm this result, we compared to between PAM treatments only and ZnCl 2 supplemented groups by confocal microscopy [fig_ref] Figure 2: Effect of zinc on PMA -treated ROS generation in hGFs [/fig_ref]. Significant DCF fluorescence was seen in the PMAtreated hGFs, but Zinc supplemented groups did not have DCF fluourescence in the zinc (10μM & 20μM) treated with PMA.
-The effects of zinc on cytokine production in PMAtreated hGFs To assess cytokine production, supernatants for hGFs cultured with zinc in the presence of PMA were collected after 24 hr. Cytokine profiles were assessed using a cytokine antibody array that can detect 40 different basal levels of a number of cytokines [fig_ref] Figure 3: Effect of zinc on PMA -treated cytokines release in hGFs [/fig_ref]. In the results, four innate cytokines, four chemokines, and one not-distinguished (N.D.) cytokine were represented. Among these cytokines, growth-regulated oncogene- The cyclooxygenase -1/2 (COX-1/2) expressions and COX-2 expression level in hGFs were represented. The cells in each group were prepared for immunodetection with anticyclooxygenase-1 or 2 antibodies. The membranes were denuded and GAPDH was employed to load the same protein concentration. Densitometric analysis of COX-2 represents the mean ratio ± SD from three separate experiments. Significant differences are indicated as *p < 0.05 compared to PMA. (c) Released PGE2 was measured from recuperation of the culture medium from the previously mentioned condition, and these were processed for analysis by ELISA. alpha (GROα), interleukin-6 (IL-6), macrophage migration inhibitory factor (MIF), and stromal cell-derived factor-1(SDF-1) of innate cytokines were measured in the media [fig_ref] Figure 3: Effect of zinc on PMA -treated cytokines release in hGFs [/fig_ref]. GROα and SDF1 levels were increase by PMA, however, zinc supplements decrease to control levels or lower of control. IL-6 showed the greatest amount of release in response to PMA, increasing significantly to 2.4-fold of the control level. However, zinc (20 μM) led to a decrease in IL-6 release; reducing the level to 0.5-fold of the control. MIF levels were decreased by PMA. In zinc supplements with PMA, MIF levels were decrease more comparing to PMA treatments only. Of the chemokine, interleukin-8 (IL-8), regulated on activated normal T-cell expressed and secreted (RAN-TES), monocyte chemoattractant protein-1 (MCP-1), and serpin E1 were assayed [fig_ref] Figure 3: Effect of zinc on PMA -treated cytokines release in hGFs [/fig_ref]. IL-8, an important mediator of the innate immune response, was increased significantly to 2.7-fold the level of controls in PMA treated hGFs. In the PMA + zinc (20uM) group, IL-8 was significantly decreased to the half level of controls. MCP-1 and Serpine E1 were diminished to about 0.2-fold of the unchallenged controls after zinc treatment in the presence of PMA. PMA induced RAN-TES levels to 1.5-fold of control. At 20uM, zinc attenuated RANTES levels which were induced by PMA. In N.D., sCIAM-1 was significantly increased to 15-fold the level of control in PMA-treated-hGFs [fig_ref] Figure 3: Effect of zinc on PMA -treated cytokines release in hGFs [/fig_ref]. However, zinc led to a decrease in sICAM-1 release, reducing the levels to those of the control cells.
# Discussion
Although principle etiology of oral inflammatory disease such as RAS is unclear, some researchers have proposed aphthous formation as a cause due to oxidative stress. [bib_ref] Purine catabolic enzymes and nitric oxide in patients with recurrent aphthous ulceration, Gurel [/bib_ref]. Recurrent aphthous stomatitis (RAS) is characterized by the appearance of initially necrotic ulcers [bib_ref] Treatment of recurrent aphthous stomatitis. A literature review, Belenguer-Guallar [/bib_ref]. Immune alterations have been observed resulting in the activation of T lymphocytes, cytokines secretion [bib_ref] Treatment of recurrent aphthous stomatitis. A literature review, Belenguer-Guallar [/bib_ref]. Changes have also been reported in elements of the salivary defense system, such as the enzyme superoxide dismutase (SOD), which participates in the inflammatory response of RAS [bib_ref] Assessment of salivary and serum antioxidant vitamins and lipid peroxidation in patients..., Saral [/bib_ref]. In inflammatory model, PMA has the function of cell death and inflammatory response by cytokines regulation such as IL-6, IL-8 and T-cell activation including our results [bib_ref] Resolution of PMA-induced skin inflammation involves interaction of IFNgamma and ALOX15, Zhang [/bib_ref] [bib_ref] Identification of an IFN-gamma responsive region in an intron of the invariant..., Cao [/bib_ref]. We suggested that PMA initiated inflammatory response to contribute to its pathogenesis. Zinc therapy has been used extensively in clinical dentistry. Moreover, zinc is an important component of restorative materials and is also used as an active component in toothpaste and mouth-rinses. Clinical studies have shown that zinc is several beneficial effects such as antioxidant and anti-inflammatory properties in psoriasis, hair loss, wound healing, leg ulcers [bib_ref] Innovative uses for zinc in dermatology, Bae [/bib_ref] [bib_ref] The mechanisms of action of nicotinamide and zinc in inflammatory skin disease, Fivenson [/bib_ref] [bib_ref] Zinc and skin health: overview of physiology and pharmacology, Schwartz [/bib_ref]. Moreover, zinc is an essential element and a well-established antioxidant and zinc deficiency has been reported to be a potential risk factor for oral disease (39), (40). Chen and Liao et al. suggest that zinc can have either positive or negative effects on the physiology of cells depending on the local concentration, localization, and/ or state [bib_ref] Role of copper, zinc, selenium and tellurium in the cellular defense against..., Klotz [/bib_ref] [bib_ref] Neurotrophic and neurotoxic effects of zinc on neonatal cortical neurons, Chen [/bib_ref]. In this study, zinc ions supplementation could be apply oral disease by inflammatory response such as aphthous ulcer, aphthous stomatitis. In the present study, hGFs were treated with PMA to induce inflammation for an in-vitro experiment model. A inducible COX-2 expression in quiescent fibroblasts in response to stimulation with PMA is known to signal via protein kinase C (PKC) and its downstream signaling molecule, mitogen activated kinase (MAPK) [bib_ref] In vivo and in vitro anti-inflammatory activity of Mangifera indica L. extract..., Garrido [/bib_ref] [bib_ref] Quiescent fibroblasts are more active in mounting robust inflammatory responses than proliferative..., Chen [/bib_ref]. The treatment of PMA (1uM) subsequently led to the ROS generation, COX-2 expression, PGE 2 release, and cell viability decrease. The ability of zinc to reduce inflammation is known that zinc could lead the intracellular ROS scavenging and relive cellular oxidative stress. In present study, zinc treatment was shown to scavenge intracellular ROS, which mediate COX-2 expression, and inhibit the release of PGE 2 . COX protein played different roles according to the cause of inflammatory cause of inflammatory response. COX-2 appears to be the dominant source of prostaglandin formation in inflammation. Increase of COX-2 expressed on provocation of inflammatory cells. COX-2 regulation usually treated to chronic inflammatory diseases [bib_ref] Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid..., Mcadam [/bib_ref]. COX-1 is constitutively expressed in resident inflammatory cells, and there is evidence for induction of COX-1 during LPS-mediated inflammatory response and cellular differentiation [bib_ref] Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid..., Mcadam [/bib_ref]. However, the function of COX protein is not understood yet. Given the antioxidant capacity of zinc on oxidative stress, zinc might protect cells indirectly against ROS and subsequently relieve the anti-inflammatory reaction involving the COX-2 and PGE 2 pathway. Other reports confirm that serum zinc levels in patients with RAS were significantly lower than in controls [bib_ref] Effects of zinc treatment in patients with recurrent aphthous stomatitis, Orbak [/bib_ref]. Low zinc levels in our patients may be probably due to the increased consumption of zinc: (1) the continuous process of ulcer healing; (2) because of the antioxidant activity of zinc as it may act as free radical scavenger; (3) existence of completion in intestinal absorption; and the inverse Innate cytokines, (c) chemokines, and (d) not-distinguished cytokines were represented. For investigation of released cytokines, supernatants was harvested after 24 hr and then assayed using a cytokine profile array kit. Bars represent the mean ratio ± SD from the 2 experiments. Significant differences are indicated as *p < 0.05 compared to PMA. relationship between copper and zinc concentration [bib_ref] Copper/zinc and copper/ selenium ratios, and oxidative stress as biochemical markers in..., Ozturk [/bib_ref]. We suggested that aphthous ulcers were chronic inflammatory disease by COX-2 and PGE 2 . Moreover, Zinc supplementation might apply treatments of inflammatory response by ROS scavenge in zinc deficiency patients. Cytokines and chemokines are known to play a pivotal role in the immunopathology of a number of immune diseases, and release of appropriate cytokine is essential for the initiation and effector stages of immunity and inflammation [bib_ref] Cytokines in periodontal disease: where to from here?, Seymour [/bib_ref]. The level of inflammatory cytokines could reflect process and development of oral disease in patient [bib_ref] Salivary and serum interleukin 1 beta, interleukin 6 and tumor necrosis factor..., Brailo [/bib_ref]. Zinc treatment significantly decreased IL-6 and IL-8 release in PMA-treated hGFs. Their function is mediated by primary cytokines such as IL-1beta and TNF-alpha, which is secreted within minutes in response to stimuli [bib_ref] Nalps: a novel protein family involved in inflammation, Tschopp [/bib_ref]. These kinds of cytokines lead to increased vascular permeability to IL-6 and recruit the leukocytes to the region, including those expressing IL-8 [bib_ref] The host cytokine response to Porphyromonas gingivalis is modified by gingipains, Stathopoulou [/bib_ref]. The fact that IL-6 and IL-8 were increased in PMA-treated hGFs indicates that primary cytokines were effective in immediately enhancing the inflammatory response, so that the IL-6 and IL-8 levels are elevated in an autocrine manner. On the other hand, the IL-6 and IL -8 levels were decreased by zinc, resulting from autocrine suppression of the inflammatory response. IL-6 and IL-8 In this study, MCP-1 and Serpine E1 were significantly decreased by zinc treatment in the presence of PMA. Also, RANTES release was recovered from a higher level in PMA-treated hGFs. RANTES, MCP-1, and Serpin E1 are chemokines produced in response to stimulation by cytokines such as IL-6 and TNF-α, and are secreted by a variety of cells, including vascular smooth muscle cells (VSMCs), epithelial cells, T cells, macrophages, and platelets [bib_ref] A dual role for tumor-derived chemokine RANTES (CCL5), Adler [/bib_ref] [bib_ref] Tumor-derived chemokine MCP-1/CCL2 is sufficient for mediating tumor tropism of adoptively transferred..., Brown [/bib_ref]. RANTES and MCP-1 belong to a chemotactic cytokine family and are responsible for the chemotactic migration and activation of neutrophils and other cell types (such as monocytes, lymphocytes, basophils, and eosinophils) at sites of inflammation. The decrease of RANTES, MCP-1, and Serpine E1 by zinc treatment in presence of PMA is thus reasonable to suggest that zinc probably contribute to relieve the recruitment of inflammatory cells in the periodontal tissue. Previously, it has been reported that gingival fibroblasts produce the alpha chemokine, IL-8, upon stimulation by lipopolysaccharide or IL-1β. These findings would suggest that by producing RANTES/CCL5 and IL-8, human gingival fibroblasts might participate in the regulation of the local inflammatory process during both the acute and the chronic stages of inflammation during RAS [bib_ref] Peng Tumor-derived chemokine CCL5 enhances TGF-βmediated killing of CD8(+) T cells in..., Chang [/bib_ref] [bib_ref] The role of tumor necrosis factor receptor type 1 in orthodontic tooth..., Andrade [/bib_ref]. Considering the anti-inflammatory effects of zinc based on the present study, zinc reduces the ROS generation and decreases COX-2 expression and PGE 2 release in PMA-treated hGFs. Based on these result, the inflam-matory cytokine profile after zinc treatment indicates that zinc leads the decrease of the release in secondary pro-inflammatory cytokines and chemotactic cytokine family for the recruitment of inflammatory cells in the periodontal tissue. This evidence suggests that zinc mitigates inflammation and may be clinically useful as an anti-inflammatory treatment on RAS.
[fig] Figure 1: Effects of zinc on PMA-treated cell survival, COX-1/2 protein expressions and PGE 2 release in hGFs. (a) The cell viability was assessed using an MTT assay for the indicated concentrations of ZnCl 2 (0, 1, 5, 10, 20, and 50 µM) in the presence of PMA (1 µM). Data are reported as the mean ± SD (n = 3). (b) [/fig]
[fig] Figure 2: Effect of zinc on PMA -treated ROS generation in hGFs. (a) The DCF fluorescence distribution is represented by flow cytometry histograms (X axis: log of the fluorescence intensity; Y-axis: cell number). (b) Green fluorescence of DCF-DA, indicating intracellular ROS formation, was detected by confocal microscopy. All magnifications are × 200.e185 [/fig]
[fig] Figure 3: Effect of zinc on PMA -treated cytokines release in hGFs. (a) Expression of cytokine release in the presence or absence of zinc on PMAinduced hGFs. (PC, Positive Control; 1, GRO-alpha; 2, sICAM-1; 3, IL-6; 4, IL-8; 5, RANTES; 6, SDF-1; 7, MCP-1; 8, MIF, 9, SerpinE1). (b) [/fig]
|
Subgroup analyses on return to work in sick-listed employees with low back pain in a randomised trial comparing brief and multidisciplinary intervention
Background: Multidisciplinary intervention is recommended for rehabilitation of employees sick-listed for 4-12 weeks due to low back pain (LBP). However, comparison of a brief and a multidisciplinary intervention in a randomised comparative trial of sick-listed employees showed similar return to work (RTW) rates in the two groups. The aim of the present study was to identify subgroups, primarily defined by work-related baseline factors that would benefit more from the multidisciplinary intervention than from the brief intervention. Methods: A total of 351 employees sick-listed for 3-16 weeks due to LBP were recruited from their general practitioners. They received a brief or a multidisciplinary intervention. Both interventions comprised clinical examination and advice by a rehabilitation doctor and a physiotherapist. The multidisciplinary intervention also comprised assignment of a case manager, who made a rehabilitation plan in collaboration with the patient and a multidisciplinary team. Using data from a national database, we defined RTW as no sickness compensation benefit disbursement for four consecutive weeks within the first year after the intervention. At the first interview in the clinic, it was ensured that sick leave was primarily due to low back problems.Questionnaires were used to obtain data on health, disability, demographic and workplace-related factors. Cox hazard regression analyses were used with RTW as outcome measure and hazard rate ratios (HRR = HR multidisciplinary /HR brief ) were adjusted for demographic and healthrelated variables. An interaction term consisting of a baseline variable*intervention group was added to the multivariable regression model to analyse whether the effects of the interventions were moderated by the baseline factor. Subsequently, a new study was performed that included 120 patients who followed the same protocol. This group was analyzed in the same way to verify the findings from the original study group. Results: The multidisciplinary intervention group ensured a quicker RTW than the brief intervention group in a subgroup with low job satisfaction, notably when claimants were excluded. The opposite effect was seen in the subgroup with high job satisfaction. When claimants were excluded, the effect was also in favour of the multidisciplinary intervention in subgroups characterised by no influence on work planning and groups at risk of losing their job. Inversely, the effect was in favour of the brief intervention in the subgroups who were able to influence the planning of their work and who had no risk of losing their job due to current sick leave. Interaction analysis of the data in the new study displayed similar or even more pronounced differences between subgroups in relation to intervention type.
Conclusions: Multidisciplinary intervention seemed more effective than brief intervention in subgroups of patients with low job satisfaction, no influence on work planning and feeling at risk of losing their jobs due to their sick leave as compared with subgroups not fulfilling these criteria.
# Background
In Denmark, the costs of sick leave due to low back pain (LBP) reached approximately 3 billion Euro in 2007 which is equivalent to 22,500 employees being sick-listed full time for one year. In a review of reviews, the following risk factors were consistently associated with slow return to work (RTW): functional disability, sciatica, older age, poor general health, psychosocial strain, negative cognitive characteristics, heavy physical work and receiving welfare payments [bib_ref] Systematic reviews of low back pain prognosis had variable methods and results:..., Hayden [/bib_ref]. However, the natural history of LBP is benign and self-limited in the majority of cases. The prognosis is therefore good and most sicklisted employees will return to work within six weeks [bib_ref] RTW in back conditions, Zampolini [/bib_ref]. The target population for rehabilitation are those who have not returned to work within a few weeks. Long-term sick leave due to LBP is often rotted in a multitude of causes and multidisciplinary intervention is therefore recommended for rehabilitation of employees who are sick-listed for 4-12 weeks [bib_ref] Multidisciplinary biopsychosocial rehabilitation for subacute low back pain in working-age adults: a..., Karjalainen [/bib_ref] [bib_ref] Concepts of rehabilitation for the management of low back pain, Waddell [/bib_ref]. The efficacy of such interventions has not been consistently documented. Yet, intervention that involves a gradual RTW and workplace involvement has been shown to increase RTW rates in different settings and countries [bib_ref] Coordinated and tailored work rehabilitation: a randomized controlled trial with economic evaluation..., Bultmann [/bib_ref] [bib_ref] A population-based, randomized clinical trial on back pain management, Loisel [/bib_ref] [bib_ref] Multidisciplinary interventions: review of studies of return to work after rehabilitation for..., Norlund [/bib_ref].
Some studies have argued for the identification of specific sub-groups of sick-listed employees who should be offered specific interventions. In a randomised controlled trial, sub-group analysis indicated that a work place intervention had more effect on older or high-risk employees than on younger people or employees without previous sick leave. In another study, employees who had been sick-listed for at least eight weeks due to musculoskeletal pain were given scores assessing their chance of RTW [bib_ref] Is there a right treatment for a particular patient group? Comparison of..., Haldorsen [/bib_ref]. Afterwards, they were randomly allocated to three different groups: a light multidisciplinary intervention group, an extensive multidisciplinary intervention group or a control group. Employees with a good prognosis showed similar RTW rates in the three groups, whereas employees with a poor prognosis returned to work significantly earlier when offered the extensive multidisciplinary treatment. Thus, it is important to study which kind of intervention is effective for specific subgroups of sick-listed employees, also referred to as one of the "Holy Grail"-type of questions by the Cochrane Back Review Group. Successful RTW depends on factors related to the individual such as health, age, personality and family relations, but also on factors at work such as the psychosocial work environment and the interplay between individual and work-related factors. Multidisciplinary interventions typically rely on tailor-made "treatments" to facilitate RTW, which implies that job modifications or other RTW facilitation measures are only initiated if they are necessary. They often include efforts directed towards changing job demands, job control, work organisation or towards increasing support. This requires close collaboration between workplace stakeholders, the sick-listed employee, members of the multidisciplinary team andin the Danish context -also the social service centre responsible for sick leave reimbursement. However, if the employee and the workplace stakeholders agree on job modifications or other arrangements without the involvement of external stakeholders, the multidisciplinary intervention teams will not be involved. It is therefore relevant to study if self-reported work-related factors may be used to predict if the RTW process would benefit from intervention by professionals in the fields of occupational and social factors.
In a recent randomised study, we compared the effects of a multidisciplinary intervention (with a focus on occupational and social factors) with those of a brief intervention (only health professionals involved) aimed at facilitating RTW for sick-listed employees with LBP. After 12 months the two groups showed similar RTW rates, similar levels of reductions in disability, pain and fear avoidance scores, and the same improvement in general health scores. However, it may have been possible that specific sub-groups could have returned to work even earlier if they had received a particular kind of intervention. The objective of the present explorative study was to study whether particular subgroups identified on the basis of work-related factors would benefit more from the multidisciplinary than from the brief intervention.
After the original project had ended, sick-listed employees were included into a new study for another 12 months. We used the same intervention and randomisation procedures. A similar subgroup analysis was performed in the new study in order to test whether it was possible to reproduce the results from the original study group.
We hypothesized that particular subgroups defined by work-related factors would return earlier to work by a multidisciplinary than by a brief intervention if these work-related factors could be expected to influence the RTW process.
# Methods
## Study design and participants
The present paper was based on secondary analyses of prognostic factors in a randomised trial comparing a multidisciplinary and a brief intervention at 12-month followup. The inclusion criteria were sick leave for 3-16 weeks due to low-back problems, 16-60 years of age and ability to read and speak Danish. Exclusion criteria were 1) unemployment; 2) continuing or progressive symptoms of spinal nerve-root affection implicating plans for surgery; 3) surgery in the spine within the past 12 months; 4) diagnosis of specific back disease (e.g. tumour); 5) diagnosis of primary psychiatric disease; 6) pregnancy; or 7) known substance abuse.
Patients from nine municipalities in Central Denmark Region were referred by their general practitioner (GP) based on the inclusion and exclusion criteria. The criteria were re-evaluated at The Spine Centre, Region Hospital Silkeborg, where the study was performed. All patients referred to the Spine Centre who adhered to the inclusion and exclusion criteria were included in the study. The total population in these nine municipalities was approximately 280,000 citizens.
After recruitment for the original study had closed, a second 12-month study was conducted with identical procedures and interventions. The original project was carried out from November 2004 to July 2007 and the inclusion of patients for the second study period started in August 2007 and ended in July 2008 (hereafter referred to as the new study).
## Interventions
Prior to randomisation, the participants completed a baseline questionnaire and underwent a thorough clinical examination by a rehabilitation doctor and a physiotherapist. The methods used for clinical examination have been described previously. Reassuring explanations for pain and advice on a gradual increase in physical activity were provided. Subsequent randomisation was performed by a secretary on the basis of block randomisation generated by an externally located computer. At the following consultations, both participants and caregivers were aware of the result of the randomisation. Data analyses were performed by researchers outside the hospital. After two weeks, all participants were scheduled for a follow-up visit at the physiotherapist and usually also a follow-up visit at the rehabilitation doctor to inform the participant about the results of the magnetic resonance imaging (MRI) and other tests. After the first consultation, copies of the medical records were sent to the social service centre that was responsible for reimbursement of sick leave compensation All participants were free to contact the centre within the first three months.
For participants allocated to the brief intervention, care management stopped at the last visit at the physiotherapist or doctor. Treatment and rehabilitation were continued by the GP.
For participants allocated to the multidisciplinary intervention, a visit with the case manager was scheduled a couple of days after the first consultation. After a comprehensive interview covering aspects of work life and private life, a tailored rehabilitation plan was designed to facilitate the employee's RTW. The rehabilitation plan was discussed by the entire team at The Spine Centre. The team included a specialist of social medicine, a specialist of rehabilitation, a physiotherapist, a social worker and an occupational therapist. The case manager also contacted the work place and the social service centre to discuss and coordinate relevant initiatives. The case manager could arrange meetings between the participant and each of the other specialists, meetings at the work place and meetings with the social service centre, if relevant. A more complete description of the interventions is provided in a recent paper.
## Baseline variables
Work-related and basic socio-demographic variables were selected to cover well-known prognostic factors [bib_ref] Systematic reviews of low back pain prognosis had variable methods and results:..., Hayden [/bib_ref] [bib_ref] Determinants of occupational disability following a low back injury: a critical review..., Crook [/bib_ref] [bib_ref] Low Back Pain Prognosis: Structured Review of the Literature, Mcintosh [/bib_ref] [bib_ref] Early prognosis for low back disability: intervention strategies for health care providers, Shaw [/bib_ref] [bib_ref] Prognostic factors for duration of sick leave in patients sick listed with..., Steenstra [/bib_ref] [bib_ref] Predictors of chronic disability in injured workers: a systematic literature synthesis, Turner [/bib_ref]. For the purpose of the present paper, baseline variables covered three domains: 1) Socio-demographics including gender, age, marital status, parental status and education (none, brief courses, skilled or trained, less than 3 years of education, bachelor degree, master degree). 2) Work-related factors including occupation in the public sector, work pace, support from colleagues and superior, job satisfaction, influence on work planning, shift work and interest in returning to the same job. 3) Combined health and workrelated factors including feeling at risk of losing job because of current sick leave, worrying about losing job because of medical conditions, whether LBP was workrelated, work ability, permanent impairment of work ability, claim of compensation due to health problems, expectations of being back at work in six months (numeric rating scale, 0-10), self-reported expectations of work ability in a year or desire to obtain incapacity benefit.
Finally, the questionnaire contained items on health and health-related factors, among others the SF36 instrument and the LBP rating scale [bib_ref] Low Back Pain Rating scale: validation of a tool for assessment of..., Manniche [/bib_ref]. The LBP rating Scale yields a score calculated as the sum of answers to six questions on worst, average and actual pain during the preceding two weeks for back and leg pain assessed on VAS box scales from 0 to 10 (sum score: 0-60).
## Outcome
The outcome measure was RTW, which occurred when the participant had not received sick leave compensation for a period of four consecutive weeks. Data on sick leave compensation were obtained from a national database administered by the Ministry of Employment. This database includes information on all public transfer payments for all Danish citizens registered on a weekly basis since 1991. Reasons for sick leave or other health data are not available in the database, but such information was obtained at the first interview in the clinic. At this meeting, it was confirmed that sick leave was due primarily to low back problems.
## Analyses
Subgroup analyses were based on tests for interaction. Statistical tests for interaction, which directly examine the difference in treatment effects between sub-groups, have been reported as a useful approach when performing subgroup analyses [bib_ref] Subgroup analysis and other (mis)uses of baseline data in clinical trials, Assmann [/bib_ref].
First, non-dichotomous baseline variables were dichotomised to have a sufficiently large number of participants in each group. Dichotomisation was data-driven for some of the variables and sensitivity analyses were carried out afterwards to check if associations were depending on the cut-off point.
Second, associations between dichotomised baseline variables and RTW were analysed and adjusted for age, gender and intervention. Hazard rate ratios (HRR) and 95% confidence intervals (95% CI) were calculated by using Cox regression analyses. The proportional-hazards assumption was assessed graphically using log-log plots adjusted for all covariates. The criterion was not fulfilled for the variables: "support from colleagues" and "claim of compensation due to health problems" wherefore these variables were not used in the further analyses.
A possible moderating effect of each baseline variable on the effect of intervention was identified in the next step by adding the interaction term "baseline factor*intervention" to a regression model with the baseline factor and intervention group adjusted for age and gender [bib_ref] Subgroup analysis and other (mis)uses of baseline data in clinical trials, Assmann [/bib_ref]. If the interaction term was significant with a p-value < 0.2, the baseline variable was included in the multivariable regression models to adjust for more baseline factors in the following step. Multivariable regression models were calculated within each of the domains: 1) sociodemographics, 2) work-related and 3) health and workrelated factors. The interaction term was added in separate models for each baseline variable and the p-value was calculated for the interaction term. In this step, p < 0.05 was considered statistically significant. Besides the other baseline factors in the same domain, the interactions were adjusted for gender, age and health by including physical and mental component scores of SF36.
However, in the combined health and work-related domain, the models were not adjusted for subjective health status to avoid over-adjustment as the baseline variables in this domain were directly related to health status. Cases with missing values in any of the baseline variables in each model were excluded from the analysis. The number of cases included in the analysis of each model is shown together with the HRRs (95% CI), which were calculated for the effect of multidisciplinary versus brief intervention for each subgroup.
Sick-listed employees who have claimed economic compensation for their disease or injury (claimants) have been shown to return later to work or show less symptom relief than employees who have claimed no compensation [bib_ref] Long-term disability and return to work among patients who have a herniated..., Atlas [/bib_ref] [bib_ref] Poor prognosis in back pain among patients who have filed financial claims..., Rasmussen [/bib_ref]. All sick-listed employees in Denmark are entitled to receive sickness compensation during their sick leave within the first year. Sometimes, it is possible to receive additional compensation if the health problem is caused directly by factors at work, such as heavy lifting. These cases were administered elsewhere, and RTW would often be unlikely before the issue of additional economic compensation was decided. In the present study, patients with an additional claim of compensation are called claimants. The effect of the interventions could differ between non-claimants and claimants within subgroups and this could dilute or strengthen interaction effects between interventions and work-related factors. In the present study, the subgroup analyses were therefore repeated in analyses without claimants. Claim status was reported by the participants in the baseline questionnaire.
In the analyses with all participants, 258 patients returned to work which left enough power for 17 parameters to be tested for interaction. None of the models contained more than 10 parameters. After excluding claimants, 173 subjects returned to work which indicated that we would have enough statistical power if we used a maximum of 11 parameters.
The same baseline factors as those used in the original project were tested in regression models with interaction terms based on data from the new study if the subgroups consisted of more than 20 patients. Claimants were not excluded in the analyses of the new study due to the low statistical power. Also, the lower number of subjects reduced the maximum number of parameters in the regression models. These models were therefore only adjusted for gender.
The software package, STATA 11.1, was used for statistical analyses.
# Ethical approval
The study was discussed with the regional research ethics committee. Approval was not considered necessary by the committee because all participants received the best available clinical care and because no biological material was involved. The study was performed in accordance with the Helsinki Declaration. All participants signed informed consent. The study was approved by the Danish Data Protection Agency (No. 2007-41-1278). The Trial Registration Number is ISRCTN18609003
# Results
## Participants
Baseline characteristics and the flow of the patients in the original project have been described previously. In short, a total of 417 patients were referred to the study and 351 patients were included and randomised to brief (n = 175) or multidisciplinary intervention (n = 176). In the brief intervention group, 88 (50.3%) were women. In the multidisciplinary intervention group, 95 (54.0%) were women. In the two groups, the mean age was 41.9 (SD = 10.4) years and 42.1 (SD = 10.5) years, respectively. Seven participants did not continue after randomisation because of metastatic malignancy of the spine (n = 2), age (n = 1) or withdrawal (n = 4). This left 344 participants who completed the protocol. In the multidisciplinary intervention group, the case manager met the participants four times on average. Contacts with work place representatives were made in 87 cases (six times on average for each participant). The median duration of the intervention was 18 weeks in the multidisciplinary intervention group. In the brief intervention group, the participants were seen twice by the physiotherapist and once or twice by the rehabilitation doctor; a few participants were seen a few times more when needed.
Non-specific LBP was found in 191 (54%) patients. Radiculopathy was found in 112 participants (32%) and the remaining 48 participants (14%) were classified with other diagnoses (e.g. disc herniation without radiculopathy, spondylolisthesis). Later, 33 participants (9%) underwent surgery because of lack of improvement by conservative therapy (16 participants in the brief intervention group and 17 participants in the multidisciplinary intervention group). Mean pain levels on the LBP rating scale were 32.7 (SD = 12.4) in the brief and 31.6 (SD12.1) in the multidisciplinary intervention group.
## Predictors of rtw
During the first year of follow-up, 258 employees (74%) had returned to work. The remaining 93 subjects (26%) were still registered as receiving sickness benefits or other social transfer payments [fig_ref] Table 1: The original and the new study groups [/fig_ref]. None of the socio-demographic baseline variables were significantly associated with RTW [fig_ref] Table 2: Baseline predictors of RTW in original study group [/fig_ref]. Statistically significant associations with RTW were found for two of the work-related variables [fig_ref] Table 2: Baseline predictors of RTW in original study group [/fig_ref]. RTW was positively associated with willingness of colleagues to listen to their problems and influence on work planning. In the combined health and work-related domain, four variables were statistically significantly associated with RTW.
## Subgroups
In the first step, using all subjects in the analyses of univariables, we found significant interactions adjusted for age and gender (p < 0.20) between the type of intervention and marital status, job satisfaction, influence on work planning, interest in returning to the same job and feeling permanently impaired regarding work ability [fig_ref] Table 2: Baseline predictors of RTW in original study group [/fig_ref]. After exclusion of the 83 claimants, statistically significant interaction was seen for risk of losing job and concerned about losing job. In the next step in which multivariable models were adjusted for age, gender and other baseline factors, the subgroup with high job satisfaction in the brief intervention group returned earlier to work than the corresponding subgroup in the multidisciplinary intervention; and the effect was the opposite in the subgroup with low job satisfaction, especially when claimants were excluded [fig_ref] Table 3: Effect of multidisciplinary team-intervention compared with brief intervention in subgroups from the... [/fig_ref]. When claimants were excluded, the effect was also in favour of the multidisciplinary intervention in subgroups characterised by no influence on work planning and at risk of losing their job, whereas the effect was in favour of the brief intervention for the subgroups who had influence on work planning and no risk of losing job due to current sick leave [fig_ref] Table 3: Effect of multidisciplinary team-intervention compared with brief intervention in subgroups from the... [/fig_ref].
In the above-mentioned analyses, job satisfaction was dichotomised into the categories "very satisfied" and "very dissatisfied to more or less satisfied". Alternatively, if "more or less satisfied" and "very satisfied" had defined the best category, the worst category, "very to rather dissatisfied" would have shown an even stronger difference in effect of the two interventions (HRR = 3.26 (95% CI: 1.03-10.3, n = 30). Interaction effects of age were not present, either when the cut-point for dichotomisation was moved to a lower or when it was moved to a higher age. Other sensitivity analyses showed no different patterns of interaction effects for any of the baseline variables.
## Combined subgroups
The baseline variables that demonstrated interaction in relation to the interventions were correlated. Thus, a relatively high fraction of those who were not satisfied with their job or had no influence on work planning felt at risk of losing their job. For instance, among those without influence on work planning, 69% felt that they were at risk of losing their job. Among those who had influence on work planning, only 29% felt that they were at risk of losing their job. In both cases, participants with compensation claims were not included. A subgroup comprising participants with influence on work planning and not feeling at risk of losing their job returned to work earlier if they had received the brief intervention (HRR = 0.65 (95% CI: 0.45-0.95), n = 144, [fig_ref] Figure 1: Return to work [/fig_ref] than if they had received the multidisciplinary intervention. The effect of the interventions was reversed in the subgroup comprising participants without influence on work planning and/or at risk of losing their job (HRR = 1.42 (95% CI: 0.92-2.18), n = 117, [fig_ref] Figure 1: Return to work [/fig_ref] as the interaction term was statistically significant (p = 0.008).
## New study
In the new study, 120 patients were included (60 patients in each group). Women accounted for 58.3% of the participants, men for 41.7%. The women's mean age was 41.5 (SD = 10.1), the men's 39.9 (SD = 9.8) years. The mean pain level on the LBP rating scale was 35.2 (SD = 11.7) in the brief and 35.1 (SD12.1) in the multidisciplinary intervention group. The HRR of multidisciplinary versus brief intervention in the new study was 1.14 (95% CI: 0.74-1.76).
The subgroup analyses presented in [fig_ref] Table 3: Effect of multidisciplinary team-intervention compared with brief intervention in subgroups from the... [/fig_ref] were repeated in the new study except for those groups that were stratified with respect to marital status and interest in returning to the same job, since one strata of each of these two variables consisted of less than 20 subjects. Thus, five variables remained for subgroup analyses in the new study [fig_ref] Table 4: Effect of multidisciplinary team-intervention compared with brief intervention in the new study... [/fig_ref]. For the two work-related factors, the HHRs of multidisciplinary versus brief intervention were similar to those obtained in the original study, i.e. favourable effects of the multidisciplinary intervention in subgroups with low job satisfaction and no influence on job planning were supported by similar hazard ratios in the new study. Those at risk of losing their job due to current sick leave, those worrying about losing their job and those with a permanently impaired work ability showed higher HHRs of multidisciplinary versus brief intervention in the new study group, but the interaction was not statistically significant.
The combined subgroup in the new study of those with influence on work planning and no risk of losing their job also tended to return earlier to work if they received the brief intervention (HRR = 0.73 (95% CI: 0.41-1.28), n = 62, [fig_ref] Figure 2: Return to work [/fig_ref]. In the new study, the other combined subgroup comprising participants without influence on work planning and/or at risk of losing their job returned to work earlier if they received the multidisciplinary intervention (HRR = 2.16 (95% CI: 1.03-4.53), n = 56, [fig_ref] Figure 2: Return to work [/fig_ref]. The interaction term in the combined subgroup analysis was statistically significant (p = 0.025).
# Discussion
A number of predictors of RTW were found, but only one variable, "job satisfaction", significantly modified the effects of the interventions in the multivariable model in which all participants were included. When claimants were excluded, statistically significant interaction effects were found for another two variables: "influence on work planning" and "feeling at risk of losing one's job due to the present sick leave". Thus, participants with low job satisfaction, no influence on work, no interest in returning to the same job and at risk of losing their job seemed to return earlier to work when they received the multidisciplinary intervention, whereas participants without these characteristics returned to work earlier when they received the brief intervention.
The new independent study also showed no difference in RTW between the two interventions, and, furthermore, it supported most of the effects found in the subgroups of the original project. Only 120 patients participated in the new study and, our focus was therefore more on the estimates of the hazard ratios than on the statistical tests. The interaction effects were also found in the new study for the variables "job satisfaction", "influence on work planning" and "feeling at risk of losing one's job due to the present sick leave". In the last case, the hazard ratio for the subgroup "feeling at risk" was considerably higher, which indicates an even greater benefit of the multidisciplinary intervention than in the original study for this subgroup. Also for the variable "worried about losing one's job", a considerably stronger interaction estimate was found in the new study than in the original study. Thus, the quality of the interventions may have changed; or changes may have occurred on the labour market. The new study was carried out at the beginning of the global financial crisis at which time more patients were worried about losing their jobs than during the original project period which was characterized by an economic boom. The modifying effects of subgroups on intervention effects may be explained in different ways: It may have occurred either by statistical chance, or it may have reflected true causal relationships. The latter is considered more probable as the associations were confirmed in the new study. However, it should also be considered whether these associations were, indeed, plausible; that is, we must ask ourselves whether the interventions could be expected to show different effects in these subgroups. Experts in occupational and social factors were only involved in the multidisciplinary intervention, whereas health professionals were involved in both kinds of intervention and provided care and treatment for all participants according to the hospital's clinical standards. It would therefore be expedient to search for facilitation of RTW by the multidisciplinary intervention in subgroups needing assistance to perform their job, to make arrangements with their employer or other occupational issues. For instance, "those feeling at risk of losing their jobs" may have benefitted more from the collaboration between occupational or social professionals and the employer than those not at risk. Employees not at risk may feel more confident about their health and their ability to come to an agreement with their employer regarding job modification. A similar explanation may exist for the interacting effect between influence on work planning and the type of intervention. Influence on work planning may facilitate low back patients' RTW, for example if the patients are allowed to take breaks when needed, to change work tasks and to reduce heavy lifting or if they are allowed influence on other types of job modification [bib_ref] Systematic reviews of low back pain prognosis had variable methods and results:..., Hayden [/bib_ref] [bib_ref] Prognostic factors for duration of sick leave in patients sick listed with..., Steenstra [/bib_ref] [bib_ref] Predictors of chronic disability in injured workers: a systematic literature synthesis, Turner [/bib_ref] [bib_ref] Adjustment latitude and attendance requirements as determinants of sickness absence or attendance...., Johansson [/bib_ref]. For employees with low job control, this may have been achieved better through intervention by an occupational therapist of the multidisciplinary team than by the brief intervention where this was not possible. Modifying effects were also observed for job satisfaction. However, an explanation for this is less straightforward given that we have no information about the reasons for job dissatisfaction. Nevertheless, the correlation between the baseline variables indicated that low job control could contribute to low job satisfaction, which was not surprising. We also expected to see other subgroups, such as those with low social support from a supervisor or those with high work pace, benefit more from a multidisciplinary than from a brief intervention, but this was not the case. The backdrop against which the present study of a wide range of work-related factors was launched was the lack of previous studies on very specific work-related issues that should be considered by multidisciplinary teams. Some of these factors apparently affected the outcome of the multidisciplinary effort, whereas others were not important. This should be further examined in new studies. An ongoing compensation claim is a risk factor for not returning to work [bib_ref] Prognostic factors for duration of sick leave in patients sick listed with..., Steenstra [/bib_ref] [bib_ref] Poor prognosis in back pain among patients who have filed financial claims..., Rasmussen [/bib_ref]. In the present study, the associations between baseline characteristics and RTW differed for those with and without an ongoing compensation claim. Consequently, statistically significant interaction effects were not fully equivalent for the same variables before and after excluding claimants. Strong evidence is needed to obtain additional compensation and it may be necessary to document that LBP was directly caused by physical work loads. It may take several months or more than a year to come to a decision on this type of compensation. In some cases, sick-listed employees do not RTW before such a decision is made. This may delay RTW for all participants with a claim. The clearer identification of effects in subgroups without claimants than in subgroups where all participants were analysed indicated that the delay was greater in the multidisciplinary than in the brief intervention group. Thus, the HRR seemed to increase in subgroups benefitting from the multidisciplinary intervention when claimants were excluded.
Subgroups that modify the effect of biopsychosocial interventions have been analysed in a few studies. In a Norwegian study [bib_ref] Is there a right treatment for a particular patient group? Comparison of..., Haldorsen [/bib_ref] , a screening instrument was deployed to test whether patients sick-listed for at least eight weeks because of musculoskeletal pain would benefit more from a light or from an extensive multidisciplinary intervention. Those most likely benefitting from the extensive intervention were characterized by poor prognosis, such as having more complaints if work was continued, limited control on their work situation and difficulty turning down tasks at work or at home. The extensive multidisciplinary intervention included occasional workplace interventions and associations were reported similar to those found in the present paper. A light multidisciplinary intervention seemed sufficient for patients with medium or good prognosis. This intervention was similar to the brief intervention used in our study and the interventions studied elsewhere [bib_ref] Does early intervention with a light mobilization program reduce long-term sick leave..., Molde [/bib_ref]. Low-intensive back school has also been reported to have the same or a slightly better effect on return to work than high-intensive back school. The moderating effects of beliefs about reduced ability to work on the effects of intervention and control groups were observed in a study by Hagen et al.at three months follow-up. However, their intervention did not include a work place intervention and the moderating effect was not present after one year.
A moderating effect of age has previously been reported. However, using almost the same cut-off point as in the studies by Steenstra et al.and Hagen et al., we were unable to reproduce their results. The lack of a control group in our study, i.e. both groups received an intervention, may explain the lack of a moderating effect of age. The other modifying variables identified in the studies by Steenstra and Hagen, (e.g. sick leave in previous year, gastrointestinal complaints at baselinewere not analysed in our study. The dichotomisation was data-driven for some of the baseline variables. This may increase the risk of reporting spurious results or associations that occurred by chance. However, most participants used the two best categories of the baseline variables to describe their situation which left little choice for other cut-points than between these to categories. Also, the sensitivity analyses showed no patterns that would contradict the findings reported here. In fact, in the case of job satisfaction, a "lower" cut-point revealed an even stronger effect of the multidisciplinary intervention. This indicated that the different effects of the multidisciplinary and the brief interventions may be most pronounced for employees with the most negative or positive ratings of work-related factors. Another potential problem is that of multi-co-linearity between work-related factors. It is clear that it was often the same participants who assessed that they had low social support, low job control, low work ability and so forth. However, these factors do measure different aspect of working life and we were interested in identifying as many factors as possible with a modifying effect. The correlation between independent variables increased the risk of confounding, which was minimised by adjusting for other baseline factors in the multivariable regression models. The statistical power was relatively low for analyses of interaction. We therefore decided to adjust only for baseline factors within the same domain, i.e. the work-related or the health and work-related domain. This procedure should have eliminated the most likely candidates for confounding, but could not rule out that other factors confounded the results. A drawback of the attempt to identify as many factors as possible was the large number of analyses and the associated risk of reporting spurious results. Thus, 5% of all associations would be expected to be statistically significant by chance. This risk was acknowledged by the re-analysis and confirmation of results in a new sample of sick-listed employees, which indicated that the different effects in subgroups did not occur by chance.
However, the statistical power was low and the interaction effects were not statistically significant in the new study. Furthermore, the HRRs within specific subgroups were not consistently different from 1, even when the interaction was statistically significant. The test for interaction only revealed differences between the HRRs of two mutually exclusive subgroups. The subgroups composed of the combined subgroups of 1) no influence on work planning/risk of losing job and 2) influence on work planning/no risk of losing job was an example of this. Both in the original study and in the new study, the interaction of these subgroups on the intervention effect was statistically significant. However, in the original study, the HRR was significantly lower than 1 (brief intervention more effective) in the subgroup with influence on work planning/no risk of losing job, whereas the HRR was not significantly higher than 1 in the other subgroup. In the new study, the HRR was significantly higher than 1 (multidisciplinary intervention more effective) in the subgroup with no influence on work planning/at risk of losing job, whereas the HRR was not significantly lower than 1 in the other subgroup.
RTW was based on data from a national register considered to be valid [bib_ref] Register-based follow-up of social benefits and other transfer payments: accuracy and degree..., Hjollund [/bib_ref] and in which 100% follow-up is ensured. However, the register has limitations. The data on social transfer payments were estimated on a weekly basis, e.g. less than a week's payment will be displayed as a whole week's payment. This may result in an overestimation of the time until RTW. As the follow-up period was one year, it was considered a minor problem; and, moreover, one that would affect the two intervention groups equally.
Thus, the risk of reporting erroneous results due to mass significance, problems with multi-co-linearity and bias in post-hoc analyses was reduced by conducting similar analyses with similar results in a new study. However, the next and final step would be to conduct a new randomised trial with specific hypotheses and advance stratification by relevant subgroups.
# Conclusion
Multidisciplinary intervention seemed more effective than brief intervention in subgroups of patients with low job satisfaction, no influence on work planning and feeling at risk of losing their jobs due to their sick leave as compared with subgroups with high job satisfaction, influence on work planning and no perceived risk of losing their jobs. The findings were confirmed in a new subset of patients receiving similar interventions.
[fig] Figure 1: Return to work (RTW) of subgroups in the original study group. Participants with compensation claims were excluded. Fraction of participants with RTW is shown during follow-up. The first visit at the clinic is at week 0. A. Subgroup of 144 participants with influence on work planning and no risk of losing their job. B. Subgroup of 117 participants without influence on work planning and/or at risk of losing their job. [/fig]
[fig] Figure 2: Return to work (RTW) of subgroups in the new study group. Fraction of participants with RTW is shown during follow-up. The first visit at the clinic is at week 0. A. Subgroup of 62 participants with influence on work planning and no risk of losing their job. B. Subgroup of 56 participants without influence on work planning and/or at risk of losing their job. [/fig]
[table] Table 1: The original and the new study groups. [/table]
[table] Table 2: Baseline predictors of RTW in original study group. [/table]
[table] Table 3: Effect of multidisciplinary team-intervention compared with brief intervention in subgroups from the original study group [/table]
[table] Table 4: Effect of multidisciplinary team-intervention compared with brief intervention in the new study group. [/table]
|
Refractory Hypothyroidism Due to Improper Storage of Levothyroxine Tablets
Context:A not negligible part of hypothyroid patients on levothyroxine therapy do not normalize serum thyrotropin (TSH) concentrations. "Refractory hypothyroidism," i.e., a con dition characterized by persistently abnormal serum TSH levels despite adequate titration of lT4 substitution therapy, requires biochemical and instrumental investigation, but no definite etiology is found in up to 15% of cases.objective: To report patients presenting with refractory hypothyroidism with proven improper storage of levothyroxine tablets.Design: Patients on lT4 substitution therapy referred to three Italian outpatient Clinics of Endocrinology between January 2013 and December 2015 for refractory hypothy roidism were investigated for levothyroxine tablet exposure to humidity, light, and high temperature.Results:We report eight patients, accounting for approximately 1% of all hypothyroid patients and 5% of those with refractory hypothyroidism in our series. Careful anamnesis disclosed that these patients stored levothyroxine tablets inappropriately. Normalization of serum TSH concentrations was obtained in all cases by simply recommending to store the new levothyroxine tablets away from heat, light, and humidity.Conclusion: Refractory hypothyroidism linked to improper storage of lT4 tablets does exist and might be an underrecognized entity. In addition to proper modalities of ingestion of lT4 tablets, patients need to be instructed on proper modalities of storage, as well.
# Introduction
Hypothyroidism is a common disorder, with a prevalence of approximately 5% and an incidence of approximately 250/100,000 per year in the adult population, but both prevalence and incidence keep rising [bib_ref] Serum TSH, T(4), and thyroid antibodies in the United States population (1988..., Hollowell [/bib_ref]. Consequently, levothyroxine (l-T4) is one of the most prescribed medications. In the United States, l-T4 ranked first in the year 2014 list of top medicines by prescription, because 120 million prescriptions were dispensed. This represents a steady 4% annual increment compared with 103, 105, 112, and 117 million in the years 2010, 2011, 2012, and 2013, respectively. In The Netherlands, the prescriptions of l-T4 rose steadily from approximately 305,000 in the year 2005 to 465,000 in 2011 [bib_ref] Use of T4, T4 +T3, and T3 in the Dutch population in..., Jong [/bib_ref]. In the United Kingdom, the number of prescriptions has doubled from approximately 7 million in the year 1998 to 19 million in the year 2007 [bib_ref] Trends in thyroid hormone prescribing and consumption in the UK, Mitchell [/bib_ref]. Thus, it is likely that LT4 Tablets' Improper Storage Frontiers in Endocrinology | www.frontiersin.org
July 2017 | Volume 8 | Article 155 l-T4 prescriptions will increase further worldwide. Even though novel formulations (soft gel capsules, oral solution) have been launched into the marketplace, yet they are not available in all countries [bib_ref] The administration of l-thyroxine as a soft gel capsule or liquid solution, Vita [/bib_ref] , and thus the classic formulation of l-T4 for oral use is the tablet. Replacement therapy of hypothyroidism, which is prescribed by both specialists and general practitioners [bib_ref] Clinical practice guidelines for hypothyroidism for hypothyroidism in adults: cosponsored by the..., Garber [/bib_ref] , is monitored by measuring serum thyrotropin (TSH) to ensure it reaches target levels (normalization). Except for the pregnancy setting, the recommended upper limit for serum TSH concentrations is 4.12 mU/L (7), though elderly persons tend to have greater serum levels [bib_ref] Serum TSH, T(4), and thyroid antibodies in the United States population (1988..., Hollowell [/bib_ref]. Based on a recent survey among endocrinologists from all continents, of whom approximately 900 responded, almost 100% of respondents request serum TSH assay to monitor substitutive l-T4 therapy [bib_ref] A 2013 survey of clinical practice patterns in the management of primary..., Burch [/bib_ref]. However, 60% of endocrinologists also request serum-free thyroxine, 8% request free triiodothyronine, and 8% request total triiodothyronine. Once the first serum TSH check is performed (mostly between 4 and 8 weeks after initiation of therapy), rechecks are performed every 6 months by half of the respondents, but every 3 months or less by approximately 10% of the respondents [bib_ref] A 2013 survey of clinical practice patterns in the management of primary..., Burch [/bib_ref]. Approximately 15-20% of patients taking l-T4 show persistently abnormal serum TSH levels (a condition called "refractory hypothyroidism"), and this problem is frequently addressed by increasing the daily dose of l-T4 (9, 10), with associated frequent requests of the above hormone assays. Ultimately, a thorough diagnostic work-up is necessary to disclose the cause of the problem [bib_ref] When thyroid hormone replacement is ineffective?, Benvenga [/bib_ref]. However, in approximately 15% of such patients, a cause cannot be found [bib_ref] When thyroid hormone replacement is ineffective?, Benvenga [/bib_ref].
Recent guidelines recommend that "l-thyroxine should be stored per product insert at 20-25°C (68-77°F) and protected from light and moisture" [bib_ref] Clinical practice guidelines for hypothyroidism for hypothyroidism in adults: cosponsored by the..., Garber [/bib_ref]. Nevertheless, except for the above review article (9), other reviews (11), guidelines (12), or major textbooks [bib_ref] Treatment of hypothyroidism, Jonklaas [/bib_ref] do not mention inappropriate storage among the causes of increased requirement of l-thyroxine. Thus, improper storage remains an overlooked cause. Also, no case reports have appeared to support the above recommendation until the description of grossly improper storage by one of us [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref]. For this hypothyroid woman, there were multiple factors of improper storage of the T4 tablets. Tablets were removed from the blister, transferred in a transparent vial, and exposed directly to the humidity originating from a humidifier, to the sunlight, and to the high temperature originating from closeness of the tablets to the bedroom heating unit [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref]. After the patient was instructed to remove all these factors for new l-T4 tablets, serum TSH became entirely normal.
The observation of the above patient [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref] underscores the importance of careful history taking as the first step in the management of patients with refractory hypothyroidism [bib_ref] When thyroid hormone replacement is ineffective?, Benvenga [/bib_ref]. Particularly, careful history taking should start with detailed information on modalities of l-T4 tablet storage, the timing of l-T4 ingestion with respect to the timing and type of meals, the liquids used to swallow the tablet, and the use of other drugs/ dietary supplements. Upon publication of such case report [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref] , Salvatore Benvenga was alerted by the coauthors of the present article that they have observed similar cases of elevation of serum TSH, the cause of which could only be improper storage of l-T4 tablets. Indeed, in all these patients, proper storage of new tablets was ensued by normalization of serum TSH. These patients and the additional patients observed by Salvatore Benvenga are reported here.
## Background patients and methods
Between January 2013 and December 2015, we have observed eight patients with refractory hypothyroidism in whom, upon careful anamnesis, we suspected improper storage of their l-T4 tablets. Four patients live in Southern Italy (Messina, Sicily), two in Central Italy (Pisa, Tuscany), and two in Northern Italy (Modena, Emilia-Romagna).
Prior to our observation, these eight patients had been managed by their general practitioner, endocrinologist, or both. Based on the experience gained with the patient described previously [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref] , a careful anamnesis was carried out, starting from a detailed interview on the modalities of storage of the l-T4 tablets. Indeed, in the checklist of the 15 items to take care of in history taking and physical examination of patients with refractory hypothyroidism, the first is to inquire carefully about storage of l-T4 [bib_ref] When thyroid hormone replacement is ineffective?, Benvenga [/bib_ref]. The only abnormality that surfaced at anamnesis in the eight patients was an improper storage of l-T4 tables. Improper storage consisted in taking the l-T4 tablets away from their packages and/or exposing them to heating sources, light and humidity. In all eight patients, we also wished to check the expiration date of the l-T4 tablets that patients were taking, this typically being 18 months from manufacturing. In all cases, the tablets were not expired and were still valid for at least 8 months. Because we were confident that inappropriate storage was the cause of persistent TSH elevation, we immediately suggested proper storage for new packages of l-T4 tablets from the same brand in the same pharmacy, and no changes in the l-T4 daily dose were done to patients. Indeed, we advised the patients to eliminate the l-T4 tablets exposed to heat, light, and/or humidity sources, to take new l-T4 packages from the pharmacy, and to keep new l-T4 tablets within their packages-away from the above cited sources-until the intake time. A thorough diagnostic work-up (9) was planned, should this measure have failed upon the first TSH check 2 months after the beginning of therapy with properly stored new l-T4 tablets.
Three patients observed by Salvatore Benvenga were available for a temperature check of the microenvironment associated with improper storage and the microenvironment associated with proper storage in months representative of the four seasons, using a digital thermometer. External and internal temperatures in the three homes were taken with the same digital thermometer, which was provided by Salvatore Benvenga. No home had air conditioning.
statistics Continuous data are given as mean ± SD and median, while categorical data as percentage. The statistical significance of the difference in serum TSH under the proper storage conditions versus the improper storage conditions was analyzed by the Mann-Whitney test (difference between serum levels) or by the exact Fisher's text (difference between percentages of target levels). For microenvironment temperatures, the corresponding differences between storages were tested by the one-way analysis of variance (ANOVA). We considered a P value lower than 0.05 as statistically significant, and a P value comprised between 0.10 and 0.5 as borderline significant.
# Results
Data are summarized in [fig_ref] taBLe 1 |: Details on changes in thyroid function tests prior to and during lT4... [/fig_ref].
All patients were adult or elderly, and all but one were women. Seven patients were affected by the goitrous variant of Hashimoto's thyroiditis. The diagnosis of Hashimoto's thyroiditis was based on elevated antithyroid (ab anti-thyroglobulin and/or anti-thyroperoxidase) autoantibodies and diffusely hypoechoic aspect of the thyroid gland on ultrasound in all cases. One patient (case 4) had previously undergone total thyroidectomy due to a large multinodular goiter with compressive symptoms. The improper storage consisted in exposure of the l-T4 tablets to heating (case nos. 2, 5, 6), humidity (no. 3), both heating and humidity (no. 1), light (no. 7), or both light and heating (no. [bib_ref] Use of T4, T4 +T3, and T3 in the Dutch population in..., Jong [/bib_ref] [bib_ref] A 2013 survey of clinical practice patterns in the management of primary..., Burch [/bib_ref].
In particular, our patients used to store l-T4 tablets as follows:
-Patient 1 in a drug cabinet of water closet, above the heating unit, about 40 cm from both the shower and Jacuzzi; patient 2 in a drawer of the kitchen room, around 20 cm from the burners and owen; patient 3 in a water closet where mold originating from the roof and balcony was evident; patient 4 (previously undergone thyroidectomy for euthyroid benign multinodular goiter) in a transparent glass placed on the nightstand, under both the night light and the abat jour, since 6:00 p.m. until 8:00 a.m.; patient 5 in a drug cabinet of the water closet, above the heating unit; patient 6 in a drawer of the kitchen room, around 40 cm from the burners and owen; patient 7 in a transparent glass placed in the kitchen room, close to the window, since 2:00 p.m. until 6:00 a.m.; patient 8 in a transparent glass placed on the nightstand, close to the heating unit and the light from a night bulb, since 11:00 p.m. until 8:00 a.m.
Improper storage was associated with greater levels of serum TSH and, almost always, with the policy of the family physician and/or endocrinologist to deal with this problem by increasing the daily dose of l-T4. During this time of improper storage, a greater number of TSH assays were requested to monitor therapy. In brief, upon correctly storing the tablets, TSH levels were fourfold lower (P < 0.001), and more frequently on target. It should be noted that the proper storage-associated TSH levels of 4.2 mU/L (for the 65-year-old case no. 2), 4.3 mU/L (for the 70-year-old case no. 8), and 5.9 mU/L (for the 78-year-old case no. 7) correspond to the upper normal limit of 4.33 mU/L for the 60-69 year-old reference population, and 5.9 mU/L for the 70-79 year-old reference population (7.50 mU/L for the at least 80-year-old reference population) based on the NHANES III study [bib_ref] Serum TSH, T(4), and thyroid antibodies in the United States population (1988..., Hollowell [/bib_ref]. Finally, if increased prior to our observation, the daily dose of l-T4 could be decreased under proper storage conditions.
As mentioned under Section "Patients and methods, " three patients were available for a temperature check of the microenvironment associated with improper storage and the micro environment associated with proper storage in months representative of the four seasons [fig_ref] taBLe 2 |: Temperatures taken at the indicated places in three of the eight patients,... [/fig_ref]
# Discussion
Wrong storage of medications at patients' homes is not rare. Based on an interview of persons living in 49 homes, half of them kept medications in the bedroom or kitchen [bib_ref] An exploratory study on medications in Qatar homes, Kheir [/bib_ref]. Moreover, light, temperature and moisture sensitivity is not a thyroid hormonerestricted characteristic [bib_ref] Many common drugs in dermatology are light, temperature, or moisture-sensitive, Langner [/bib_ref].
To our knowledge, only one study (reported in abstract form) tested different formulations of l-T4 for their resistance to heat. Two novel formulations of l-T4, namely, the soft gel capsule and oral solution, were compared with the classic tablet formulation. The soft gel capsule is a gelatine shield pearl containing l-T4, which is solubilized in glycerol. Oral solution is l-T4 contained in predosed ampoules, with l-T4 solubilized in glycerol and ethanol. The tested l-T4 tablets were of three types: lactose-free but with calcium phosphate; lactose-free, calcium phosphate-free but containing colloidal silicon; lactosecontaining. This challenge consisted in keeping each of the five formulations in an incubator at the constant temperature of 25°C for 6 months. At 0, 3, and 6 months, T4 was extracted and measured by variable wavelength detector-high-performance liquid chromatography (HPLC). At the end of the 6 months, the two novel formulations ranked better than tablets.
Approximately 15-20% of patients receiving l-T4 have high serum TSH levels, even when their dose is increased, a condition referred to as "refractory hypothyroidism" that prompts endocrine consultation [bib_ref] Falling threshold for treatment of borderline elevated thyrotropin levels-balancing benefits and risks:..., Taylor [/bib_ref]. As detailed in the Section "Introduction, " the issue of improper storage of l-T4 is poorly known. This issue of improper storage is reminiscent of that pertaining to the improper ingestion of l-T4 tablets soon after food intake (most frequently at breakfast) or using coffee as a swallowing liquid or having coffee soon after swallowing the l-T4 tablets with water. The publication of papers reporting patients with refractory hypothyroidism caused by inappropriate intake of l-T4 tablets [bib_ref] Delayed intestinal absorption of levothyroxine, Benvenga [/bib_ref] [bib_ref] Altered intestinal absorption of l-thyroxine caused by coffee, Benvenga [/bib_ref] was of help to both patients and physicians, who have become more aware of such entity [bib_ref] Clinical practice guidelines for hypothyroidism for hypothyroidism in adults: cosponsored by the..., Garber [/bib_ref].
As suspected, the patient reported previously [bib_ref] Solution of a diagnostic problem upon visiting the patient at home and..., Benvenga [/bib_ref] was the classic tip of the iceberg. Together with the eight patients reported here, there are now nine cases described in the literature with refractory hypothyroidism that is likely attributable to inappropriate storage of the l-T4 tablets and consequent degradation of the hormone. In the same 3-year interval of time during which the eight patients came to observation , the authors of the present paper observed a total of approximately 1,500 (or 500 per year) new hypothyroid patients under l-T4 therapy. Twelve percent of these 1,500 patients (180 or 60 patients per year) have refractory hypothyroidism caused by pseudomalabsorption or true malabsorption (e.g., celiac disease, protonic pump-inhibitors, etc.) [bib_ref] When thyroid hormone replacement is ineffective?, Benvenga [/bib_ref]. Hence, in the series reported here, improper storage accounts for approximately 4.5% (8/180) of all cases of refractory hypothyroidism. Also, improper storage would occur at a frequency of approximately 1% (8/1500) of hypothyroid patients taking l-T4 tablet. While we acknowledge that our frequency cannot be universally representative, some inappropriate storage of l-T4 tablets and refractory hypothyroidism was supported by multiple measurements of serum TSH with highly statistically differences compared with the corresponding measurements under conditions of proper storage. Additional support comes from differences in temperatures in the microenvironment associated with inappropriate storage and temperatures associated with appropriate storage. One limitation of our study is not having measured FT4 by high-performance liquid chromatography (HPLC) in both improperly stored and properly stored T4 tablets. A second limitation is not having performed an oral acute loading test with l-T4 for the purpose of excluding pseudomalabsorption. A third limitation is not having tested patients for possible gene polymorphisms that impact on thyroid function tests, though existence of such polymorphisms seems unlikely (see below). Also taking into account the relative high frequency in the population and the possibility of persistent serum TSH elevation upon standard replacement doses of projection can be made. For instance, with an estimated 15 million Americans having hypothyroidism, approximately 150,000 of them would have refractory hypothyroidism that could be due to improper storage of l-T4. If their refractory hypothyroidism is managed by progressively increasing the daily dose of l-T4 and if two TSH tests at a cost of $ 50 each are requested to monitor serum TSH, this practice would result in almost $ 8 million excess costs. However, the real rate of refractory hypothyroidism due to improper storage of l-T4 tablets could be higher than 1% of the total hypothyroid patients, because the common strategy of increasing the daily dose of l-T4 could obtain normalization of serum TSH.
The strength of the study is to have collected a series of patients with refractory hypothyroidism that could be attributed to precisely described circumstances in which one or more of three environmental factors were operating: temperature, light, and humidity. Another strength is that the link between l-T4, one such polymorphism is the Ala92Thr variant of type 2 deiodinase [bib_ref] Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and..., Peeters [/bib_ref].
Thus, it is extremely unlikely that our adult or elderly patients with biochemical (elevated TPOAb and/or TgAb levels) and ultrasound features of thyroiditis had any genetic defect (24), e.g., monoallelic mutations in the TSH receptor (TSHR) gene, causing TSH-resistance and, consequently, refractory hypothyroidism. Indeed, monoallelic inactivating mutations of TSHR are associated with normal or hypoplasic thyroid, which is in contrast with the enlarged thyroid glands detected by US in all our patients. Nonetheless, the thyroidectomized patient was entirely euthyroid prior to thyroidectomy (data not shown). Finally, upon evaluating the past history of patients with Hashimoto's thyroiditis, we found that four of them had performed thyroid function some years earlier. These tests were performed when one consanguineous relative had developed Hashimotos' thyroiditis-related hypothyroidism, so they wanted to make sure they were euthyroid and thyroid autoantibody negative. While they were indeed euthyroid, the four patients had elevated levels of TPOAb and/or TgAb (data not shown).
Recently, Van Wilder and co-workers have reported three patients presenting with persistent clinical and biochemical signs of hypothyroidism despite replacement therapy with high doses of levothyroxine [bib_ref] Pseudomalabsorption of levothyroxine: a challenge for the endocrinologist in the treatment of..., Van Wilder [/bib_ref]. Although these three patients denied noncompliance, a peroral 1,000 mcg l-T4 challenge test was positive in all cases, confirming the suspicion of pseudomalabsorption. Daily ingestion of the l-T4 tablets was confirmed by relatives and/or caregivers living with the patients, suggesting the absence of non-compliance as a possibility to explain hypothyroidism in these eight cases.
Another limitation of the present study is that, for humidity, microenvironment indicators were not provided.
## Concluding remarks
Some hypothyroid patients use to store l-T4 tablets impro perly, and this habit causes failure of serum TSH to be normalized. Indeed, when these patients are instructed on the correct storage of l-T4 tablets, serum TSH concentrations normalize. Immediate recognition of l-T4 inappropriate storage avoids mismanagement with increasing daily dose and frequent checks of hormone levels, and also avoids management of patients with unnecessary work-up when patients are referred to specialists. Thus, upon prescription of l-T4 tablets, patients need to be instructed on proper modalities of either ingestion or storage.
# Ethics statement
This study was carried out with written informed consent from all subjects. All subjects gave written informed consent in accordance with the declaration of Helsinki. Because patients recruited in the study underwent the regular work-up of hypothyroid subjects and were not submitted to life-risk procedures, the study protocol was not submitted to the Ethical Committed of the authors' Institutions.
# Author contributions
All the authors contributed equally to recruited patients, and wrote the present work. SB collected data and performed statistical analysis.
[fig] .: Temperatures very close to or above 25°C were measured under conditions of improper storage Similar temperatures were observed only in summer months under conditions of proper storage With regard to pertinent weather conditions of the years 2013-2015 for three cities where patients continue to live, the annual relative humidity was 64% (Messina), 728% (Pisa), and 787% (Modena), with an average of 247 days (Messina) to 352 days of relative humidity ≥ 60% (Modena) Messina was the warmest city, with an average annual temperature of 196°C (maximum 226°C, minimum 159°C), and 94 days of values ≥ 25°C (maximum temperature, 144 days; minimum temperature, 31 days) Like most of Sicily, Messina features an annual average of 300 days and 2700 hours of sunshine compared with an average of 2000 hours in the rest of Italy (15), particularly around 2150 hours in Modena and around 2300 hours in Pisa (16, 17) In sum, Salvatore Benvenga observed twice more cases than either coauthor probably because the weather conditions of Messina mimic the environmental conditions of improper storage better than either Pisa or Modena [/fig]
[table] taBLe 1 |: Details on changes in thyroid function tests prior to and during lT4 therapy, with associated daily dosage of lT4, under conditions of improper lT4 storage (i.e., before our observation) or proper lT4 storage (after our observation and recommendations) in the eight patients with refractory hypothyroidism. a Get a new lT4 package and store it in a drawer of the bedroom commode (located about 3 m from the heating unit). Get a new lT4 package and store it into a drawer of a small room communicating with the kitchen room, but far away from burners and heating unit 175 μg/day[2.03] bw, body weight; FT4, free thyroxine; l-T4, levothyroxine; m, mean; N.A., not available; OR, odds ratio; TSH, thyrotropin.Details on improper and proper modalities of storage. In each of the eight patients, TSH levels above 4.12 mU/L are typed boldface. [/table]
[table] taBLe 2 |: Temperatures taken at the indicated places in three of the eight patients, all three from Messina, under conditions associated with improper storage (in italics) or proper storage. a [/table]
|
Staying afloat: community perspectives on health system resilience in the management of pregnancy and childbirth care during floods in Cambodia
BMJ Global Health
10.1136/bmjgh-2019-002272:e002272. 2020; BMJ Global Health , et al. Saulnier DD 2020; BMJ Global Health , et al. Saulnier DD Supplementary material BMJ Global Health
Annex 1. Topic guide in English and Khmer for focus group discussions and semi-structured interviewsFlood experiencesGive example of recent flood in the area and confirm event (when, where, length of flood). What happened during this flood?Explore: Effect on village and health, other recent floods Care seeking and management of health needs How do women take care of their pregnancy during a flood? What do women do when they give birth during a flood?Explore: Health center, private providers, spiritual healers, traditional medicine, traditional birth attendants, role of family, baby's father, boats and transport, delivering at home, community-based programs from health center during floods How do floods change the prenatal care women can get? How do floods change the delivery care women can get?Explore: Reasons for changes, change in quality of care, supplies, medicines, and providers, cost, access and availability Is the way women manage their pregnancy during floods different if you compare…? What about for deliveries? a) Women who are richer with women who are poorer, b) First child or multiple children, c) Grew up in this district or from another district, d) Complicated or uncomplicated pregnancy, e) Early in the pregnancy versus last month of the pregnancy Anticipating and coping with uncertainty When a woman is pregnant, how does she prepare for the rainy season?Supplementary material
Explore: Beginning compared to end of pregnancy, transport and boats, money, spiritual or traditional medicine practices What does a woman do if there is a problem with her pregnancy during a flood? What does she do if there is a problem during the birth and there is a flood? Explore: Care seeking by type of provider and reasons why, decision to seek care, referrals to hospital External factors influencing the health system What makes it easier for women to manage their pregnancy or birth during floods? What makes it harder? Can you describe a time when a woman was not able to get prenatal care during a flood? What happened? What about a time when a woman was not able to get delivery care? What happened then? Interaction with the community What are some reasons that women visit _____ providers for prenatal care during floods? What are some reasons that women visit ____ when she delivers a baby during a flood?
Explore: Public providers, private providers, traditional birth attendants, drug shops, traditional medicine providers, other How do you think the pregnant women feel about the prenatal care that they can get during floods? How do you think pregnant women feel about the delivery care they can get during floods?
Explore: Trust in provider, attention, convenience, and quality of services, feeling of ownership Gathering and using knowledge How do women decide what kind of prenatal care they will get during floods? How do they decide what kind of delivery care they will get? Explore: Sources of information, social media and how they communicate with others, ability to make decisions Who do women talk to about prenatal care during floods? Who do they talk to about delivery care during floods?
Explore: Interaction with providers, traditional birth attendants, spiritual healers, traditional medicine, family and community What is an example of something that all women in this village should know about managing their pregnancy during floods? …for delivering a baby? |
Bone mineralization and immediate function of six dental implants in patients with Klinefelter syndrome
Certain metabolic diseases, including Klinefelter syndrome, cause severe atrophy in the upper and lower jaw of patients, primarily males. The question then arises whether or not immediate implant loading can be performed in a single day. Due to complications with bone mineralization and implant osseointegration, and further complicated by the abnormal abundance of trabecular bone and lacuna in the maxilla, the long-term prognosis of KS patients requiring immediate implant loading in the upper jaw is limited.The chromosomal abnormality of Klinefelter syndrome, KS, is characterized in 80% of cases by 47, XXY, and in 20% by the XXXY karyotype. These genes influence the hypothalamus. The hypothalamus directs the entire endocrine system and is the most important determiner of hormones in the body. While a diagnosis of KS is often made in childhood as the result of observed behavioral problems or learning disabilities, if left undiagnosed, it will cause significant hormonal problems and interfere with the sexual maturation of patients. Untreated KS can cause dramatic physical effects and is responsible for increased morbidity and decreased life expectancy. Congenital malformations of the heart, as well as malformations in the urinary, muscular, and endocrine systems, result in hypogonadism, diabetes, and hypothyroidism. Furthermore, the low levels of testosterone associated with KS increase the risk of vascular diseases and diabetes independent of chromosomal factors. 1 Mental illnesses associated with KS also contribute to increased morbidity.2A characteristic facial morphology of individuals with KS is mandibular prognathism. A study on KS subjects showed increased flexion of the cranial base, with a greater genial angle and pronounced jaw. A shortening of the anterior cranial base has also been noted, as have abnormalities in facial height, the mandibular branch, and special dimensions of the dental crown. 3AbstractPatients with Klinefelter syndrome face many challenges in oral treatment and bone mineralization due to multiple systemic dysfunctions. This case report follows the geometrical treatment with immediate implant loading of an adult male patient with Klinefelter syndrome. Satisfactory results were demonstrated in clinical follow-up.K E Y W O R D Sbone mineralization, immediate loading, Klinefelter
The bodies of KS patients are characterized by increased fat mass and decreased lean body mass. Reduced bone mass has likewise been found in up to 48% of cases.Patients with KS also have a testosterone deficiency resulting in early osteoporosis and insufficient insulin-like factor 3 (INSL3).Reduced bone mass, combined with low testosterone levels, results in a bone mineral deficit and causes an abundance of lacuna in the bone, a major obstacle for successfully performing oral surgeries.
KS patients have a higher prevalence of class I caries. KS patients therefore tend to lose their teeth at an early age. Congenital enamel deficiency (weak, soft enamel) contributes to this early tooth loss, as do the problems with hygiene associated with the mental effects of KS. Furthermore, the regular medications taken by some patients, such as tranquilizers and anticoagulants, reduce saliva secretion, which in turn promotes the onset of caries, and, ultimately, complete tooth loss. Moreover, early tooth loss is exacerbated by the gum inflammation and periodontal diseases which accompany hormonal imbalances.The purpose of the present study is to evaluate several topics relating to Klinefelter syndrome, KS. The first is the reaction of the metabolic system to bone remodeling under high compressive stress. The stress patterns were induced by conventionally placed and tilted implants supporting a fixed prosthesis in a patient with Klinefelter syndrome. The second is to better understand the hormonal stress on bone in KS patients. And the last is to evaluate dental implant placement practices.
This case report, based on a critical discussion of available results, proposes clinical and practical recommendations for KS screening and management in order to detect, prevent, or treat the decline in bone mass in KS patients using immediate dental implant loading. 7
## | case presentation
This patient's preference for fixed implant restoration is the inclusion criteria for this study. The patient is a 38-year-old man suffering from Klinefelter syndrome, KS. Due to this syndrome, the patient suffers from other systemic cardiovascular diseases, hormonal imbalances, and mental disorders. The mental disorders are especially important to consider because they interfere with the patient's ability to follow care instructions, and thus elevate the risk of dental implant loss or bone loss.
The patient had an atrophic full edentulous maxilla. However, the patient was not willing to undergo bone augmentation procedures and instead required a dental implant. Compared to normal patients, the subject of our case has an abundance of trabecular bone with a large amount of enlarged lacuna. In this case, it is imperative to stimulate the bone. This, combined with the patient's inability to follow care instructions, prompted the decision to perform the entire procedure in a single day using immediate dental implant loading.
## | treatment
## | surgical procedures
The 3D geometry of the maxilla, consisting of cortical and trabecular bone, was reconstructed with a computerized tomography scan .
After administration of local anesthesia with 2% mepivacaine with 1:100 000 epinephrine (France), a midcrestal incision was made from the pterygoid area. A mucoperiosteal flap was elevated and all roots with granuloma were removed, as were implants with peri-implantitis. All parts of the bone were irrigated. The maxillary sinus walls were not touched. The only zones disturbed were as follows: (a) the lateral incisor area with a zero degree implant, (b) the premolar-canine implant at 35 degrees, and (c) the molar II-molar III implant at 17 degrees. The patient received six implants with the same protocols, and implant angulation was determined using the triangle of bone concept. The measurements were performed in both the buccal-oral and mesio-distal directions and repeated twice for every direction. The mean of the four measurements was then calculated. After a healing period of 3-6 months, the prosthetic rehabilitation phase could be started . Multiunit abutments were connected to the implants, and an impression was taken. If the final torque of any implant was under 50 Ncm, cover screws were connected (30 Ncm were needed in zone I and 17 Ncm in zones II and III) and prosthetic fabrication was delayed for one hour, after which all crown copings were fixed with 15 Ncm.
Bone densitometry was performed following the procedure using computer tomography. 3D scanning was used to evaluate the soft and hard tissues, the maxilla bone anterior, the maxilla bone posterior, and the maxilla pterygoid bone area . Implants were evaluated using ISQ: implant anterior-0 degrees; posterior-35 degrees; and pterygoid-maxilla area-17 degrees. All implants were measured with Osstell ISQ at the time of the immediate loading and every 3 months after for 1 year .
Prosthetic protocol: The patient's maxilla had atrophied. The patient also appeared to have an Angle Class III malocclusion. Irreversible hydrocolloid impressions were taken to obtain two casts from the residual ridge of the patient before surgery. These casts were used to mold open custom trays and record base waxes.
The fixed prosthetic was prepared one day before surgery. After the procedure, another impression was taken in order to make precise adjustments. The crowns of the temporary prosthetic are fixed to the coping with adhesive directly in the oral cavity.
For immediate loading, a 35-degree angled multiunit abutment, a 17-degree angled multiunit abutment in the posterior implant, and a straight multiunit abutment for the anterior implant were connected to the prosthesis. All straight implants were torqued 30 Ncm, and the angulated abutments were torqued 20 Ncm according to the manufacturer's recommendation. A prosthetic temporary tooth was fixed directly to the coping in the oral cavity with resin. Occlusion was adjusted, and mutually protected occlusion was established. The patient was placed on a soft diet.
Prosthetic procedures were carried out by a prosthodontist. Baseline radiographs were taken on the day of delivery, since it is inconvenient for patients with a severely resorbed mandible to hold integral films in the correct position. Panoramic radiographs, TC, were taken in order to evaluate the marginal bone level. The patient came to the clinic every week for cleaning for 3 months following the procedure. In the absence of pain or any other complications, follow-up visits were scheduled at six-and twelve-month intervals. After that, yearly visits were established in order to monitor prescriptions, CBCT, blood analyses, and hormonal levels.
# | results
The implants appear stable, with no suppuration or pain at the implant site. The prosthesis is considered stable if it functions without pain or mobility. Marginal bone levels were recorded using radiographs calibrated by the length of each implant. CT radiographs were digitalized and imported to Romexis R version 2.6 software (Planmeca, IL, USA). Marginal bone loss was defined as the distance between the implant shoulder and the first bone-implant contact, using the medial and distal aspect of each implant in millimeters. The mean value of medial and distal bone loss of each implant was insignificant. The implants were stable, and there was no implant loss.
Blood samples were evaluated before surgery and then every 3 months postsurgery for 1 year. Absolute data were expressed as the mean + one standard deviation (SD) of the mean. Comparisons between groups were performed F I G U R E 1 Hypogonadal patient, Klinefelter syndrome before surgery by a Student's t test for continuous data after the acceptance of normality of the data. P-values (two-sided) <.05 were considered as statistically significant.
The cardiovascular risk factors (CRFs) of the subject are a waist circumference >102 cm, hypertension (blood pressure >130/85 mm Hg found in three different . We found that a patient with KS had significantly higher levels of T3, PTH, and estradiol, as well as lower levels of testosterone and EPCs than the control's INR .
Postsurgical blood and hormonal analysis showed a correlation between hormonal levels, bone mineralization, and osseointegration of the implant. This correlation deserves further study .
It is possible, however, that the differences found between KS studies might be related to the bone site being examined, given the varying proportions of trabecular and cortical bone found at specific sites on the skeleton and the higher sensitivity of trabecular bone to testosterone .
# | discussion
The aim of the present study was to examine the immediate implant loading rehabilitation of edentulous jaws with four tilted implants and two straight implants in a patient with KS.In standard practice, KS patients receive an immediate provisional acrylic prosthesis, but this prosthetic usually breaks. For such patients, it is more advantageous to fix the coping to the crown in the oral cavity.A high retention rate of immediate loading has been reported for healthy patients in the literature, but this is not the case for patients with a hormonal imbalance as found in KS.Single-day geometrical placement of abutments and prosthetic teeth in KS patients provides a great opportunity for osseointegration without mental stress. Stress is an important factor to consider because stress blocks the hormones essential for osseointegration. Reduced pain is another factor as pain also acts as a hormone blocker. The findings show that T levels below 200 ng/dL result in a significant decrease in bone mineral content while KS patients with normal T levels have normal bone content.The patient in this study had normal T levels.
T cells are responsible for intercellular signaling and detecting mechanical pressure and loads. This mechanism functions to help adapt bone to mechanical forces.Osteocytes seem to coordinate the bone remodeling process by regulating both osteoclast and osteoblast activity. Bone lining cells, osteocytes, cover surfaces where there is no bone resorption or formation, but they play a role in preventing local bone resorption by inhibiting osteoclasts.In adulthood, estrogen helps maintain bone mass by inhibiting osteoclastic bone resorption. While testosterone influences trabecular bone, estrogen receptors are primarily found in cortical bone. This means cortical bone is most sensitive to estrogen changes which inhibit osteoclastic bone resorption.The effect of T cell-stimulating hormones, which in turn stimulates increased levels of bone metabolism, was found to be due to unregulated gene expression in KS patients. This significantly influences osteoclastic function, and KS patients demonstrate bone resorption similar to that of postmenopausal women. KS patients therefore essentially suffer the effects of osteoporosis.Several studies have been conducted to determine whether a decrease in bone formation or an increase in bone resorption is present in KS. There is a significant reduction in the bone formation markers osteocalcin and bone-specific alkaline phosphatase (BSAP) in both young and adult untreated KS patients.However, contradictory results on bone markers in KS have been published.Differences in populations, such as age, the duration of testosterone supplementation, the degree of obesity, and the mental state of the patient, are all possible explanations for these differences in bone markers.Whereas both mechanisms of bone formation and resorption seem to be present in adult KS patients, a study on children and adolescents with KS noted only a reduction in bone markers.Prospective and interventional investigations are needed to define if increased follicle-stimulating hormone (FSH) production in hypogonadal men is related to a decrease in bone density. It also needs to be determined whether this effect is indeed attributable to the stimulation of osteoclastic bone resorption.In many studies, KS patients tend to have lower 25 OH vitamin D levels than healthy subjects. The widespread recent interest in the ability of vitamin D to prevent bone fractures has also generated studies evaluating vitamin D and its effect on the bone composition of KS men. Undiscovered X chromosomal factors, a higher degree of adiposity, and lowered sunlight exposure may all contribute to a lower vitamin D status in KS, but these have not been investigated in previous studies and the exact cause of this deficiency remains unknown.The impaired bone mineralization around dental implants in male KS patients is probably related to several factors including hypotestosteronemia, vitamin D deficiencies, and decreased physical activity and muscle strength.Giving our patient vitamins D and B improved both the bone mineralization and bone turnover in the patient. These vitamins stimulate the intestinal absorption of calcium and phosphate and are therefore important for bone mineralization.
Most research into immediate loading dental implants is only about healthy patients. No information is available regarding patients with metabolic diseases, nor are there recommendations for treatment. In our case, testosterone levels were so low that it was unnecessary to determine the level of hypotestosteronemia in order to indicate the necessity of testosterone therapy. The same is true for determining the duration and intensity of physical activity needed in order to maintain normal bone mineral density (BMD) in late adolescence or young adulthood when interventions will be most effective. Previous studies have reported that incidences of mechanical complication are higher when a bridge contains a mix of implants and teeth.This complication was not a factor in this case as the patient's teeth were already severely damaged. Regardless, fixing a bridge containing only implants is recommended.
KS is associated with implant tooth loss, as well as significant morbidity due to vascular diseases and osteoporosis. Several factors, such as low hormonal status, vitamin D and B deficiencies, lower muscle strength, decreased activity, and impaired mentality, appear to negatively influence the process of bone mineralization in KS. Given the lack of well-performed prospective studies, evidencebased preventive actions and optimal therapeutic interventions are not yet possible.
# | conclusion
The present report shows that if a surgeon performs geometrical 3D planning and a biomechanical crown treatment is combined with a hormonal system in equilibrium, a great result can be obtained without mechanical or systemic complications.
## Conflict of interest
None declared.
## Orcid
Galina Ciobanu https://orcid.org/0000-0002-0416-0998 |
Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7
Supplementary Figure 1 | DC bias voltage and electrothermal voltage tuning of the resonance frequency. Variation of the series and the parallel resonance frequency with (a) squared of DC bias voltage (V 2 DC ) (b) electrothermal voltage (V A ). The frequency shift due to the elctrothermal voltage is greater than that due to the DC bias voltage.
## Supplementary figure 2 | demonstration of a 2-bit nor logic gate operation at v dc = 20v
and V AC = 2dBm. (a) Frequency responses of the resonator for different logic input conditions where (0,0) logic input condition (in black) shows high S 21 transmission signal (1) at 119.03 kHz and for other logic input conditions shows low S 21 signal (0). Truth table of the NOR logic gate is shown in the inset. (b) Demonstration of NOR logic operation when the frequency of the AC input signal is chosen as 119.03 kHz. Two input signals, A and B are shown in black and red, respectively, where the switch OFF/ON corresponds to the 0/1 logic input conditions. S 21 transmission signal in blue corresponds to the logic output and fulfills the NOR truth table.
[fig] Figure 3 |: Phase noise analysis. Variation in the phase with time at a constant frequency of 117.663 kHz. Supplementary Note 1: Dependence of the series and parallel resonance frequency on the DC bias voltage and the electrothermal voltage Here, we show the characteristics of the resonance frequency for an arch resonator while varying the DC bias voltage and the electrothermal voltage. The variation of the series and parallel resonance frequencies of the resonator with the square of the DC bias voltage is plotted in Supplementary Figure 1a. Electrothermal frequency modulation has an essential role in the execution of logic functions in this architecture. To see the effect of the electrothermal voltage on the resonance frequency of the resonator, the voltage across the resonator (V A ) is changed by fixing the AC actuation voltage at 2dBm and the DC bias voltage at 45V. The variation in the resonance frequency of the resonator with the electrothermal voltage is shown in Supplementary Figure 1b. For an electrothermal voltage of 0.43V, the frequency of the resonator is increased by 4.51 kHz. Supplementary Note 2: Logic operation at low DC bias voltage We have performed an experiment to demonstrate 2-bit NOR logic operation at low DC bias voltage condition on a similar device. Logic operation is performed at a pressure of 1 Torr with an actuation voltage of 2dBm and a DC bias voltage of 20V. Note that all other logic operations can be realized by properly selecting the operation frequencies. Also, by operating at low pressure, one can further reduce the voltage load and can perform all the logic operations by selectively choosing the AC driving frequency. Supplementary Figure 2a shows the operation of a 2-bit NOR logic gate at V DC = 20V and V AC =2dBm. Execution of the NOR logic is demonstrated in Supplementary Figure 2b.Supplementary Note 3: The effect of phase noise on the device performanceIn order to understand the effect of phase noise at a given temperature on the performance of the device, we estimated the frequency fluctuations due to phase noise of the resonator. The shift in the resonance frequency can be obtained through the change of phase signal measured at a constant excitation frequency. From the measured frequency response of the electrical signal, the phase slope of the linear regime around resonance is determined to be . A study of the phase evolution at a fixed frequency of 117.663 kHz has been performed, which is shown in theSupplementary Figure 3. Accordingly, the phase noise is determined to be d min =0.0614 0 , which implies a frequency shift given by Hz. It is seen that the frequency fluctuation of the resonator lies within the bandwidth of the resonator. Hence, the defined logic state would still remain intact at a set operating frequency and the device would perform the desired logic operation successfully. [/fig]
|
Luminescence signature of free exciton dissociation and liberated electron transfer across the junction of graphene/GaN hybrid structure
Large-area graphene grown on Cu foil with chemical vapor deposition was transferred onto intentionally undoped GaN epilayer to form a graphene/GaN Schottky junction. Optical spectroscopic techniques including steady-state and time-resolved photoluminescence (PL) were employed to investigate the electron transfer between graphene and n-type GaN at different temperatures. By comparing the near-band-edge excitonic emissions before and after the graphene covering, some structures in the excitonic PL spectra are found to show interesting changes. In particular, a distinct ''dip'' structure is found to develop at the center of the free exciton emission peak as the temperature goes up. A mechanism that the first dissociation of some freely moveable excitons at the interface was followed by transfer of liberated electrons over the junction barrier is proposed to interpret the appearance and development of the ''dip'' structure. The formation and evolution process of this ''dip'' structure can be well resolved from the measured time-resolved PL spectra. First-principles simulations provide clear evidence of finite electron transfer at the interface between graphene and GaN.S ince its first successful exfoliation from bulk graphite and isolated existence in nature 1 , graphene has drawn intensive research interest from scientists in the areas of physics, materials science, chemistry, engineering, etc., due to its excellent electric, mechanic and thermal properties 2,3 , and also due to numerous potential applications in optoelectronic devices, electronic devices, solar cells, phototransistor etc 4-7 . Very recently, particular effort has been devoted to the investigation of integrating graphene with semiconductors, especially GaN, for possible improvements of performance of existing devices and even exploring new schemes 6,8-11 . It is well known that as a technologically important wide band gap semiconductor, GaN has tremendous applications in various fields such as short-wavelength light emitting diodes and lasing diodes, high-temperature and high-power electronic devices etc12,13. Previous research about graphene/GaN hybrid structure has been mainly concentrated on the electric/electronic properties 8,14-16 , while few works have been devoted to the photoelectric properties of the composite 17 . In terms of the technological importance of both GaN and graphene in different areas, the optical properties of graphene/GaN hybrid structures are of broad interest due to their unique physical and chemical properties. In this article, we present a detailed study of the optical properties of graphene/GaN hybrid structure at different temperatures by using steady-state and time-resolved photoluminescence (PL) techniques. We observe the luminescence signature of free exciton dissociation and electron transfer over the Schottky barrier of graphene/GaN hybrid structure. Moreover, we find that the efficiency of this two-step carrier process is dependent on the kinetic energy of movable excitons created in the GaN side.In order to verify layer number and also homogeneousness of the graphene layer, the Raman spectra and image of the graphene/GaN hybrid structure were measured. The micro-Raman scanning images of the 2D mode of OPEN SUBJECT AREAS: ULTRAFAST PHOTONICS SURFACES, INTERFACES AND THIN FILMS
Large-area graphene grown on Cu foil with chemical vapor deposition was transferred onto intentionally undoped GaN epilayer to form a graphene/GaN Schottky junction. Optical spectroscopic techniques including steady-state and time-resolved photoluminescence (PL) were employed to investigate the electron transfer between graphene and n-type GaN at different temperatures. By comparing the near-band-edge excitonic emissions before and after the graphene covering, some structures in the excitonic PL spectra are found to show interesting changes. In particular, a distinct ''dip'' structure is found to develop at the center of the free exciton emission peak as the temperature goes up. A mechanism that the first dissociation of some freely moveable excitons at the interface was followed by transfer of liberated electrons over the junction barrier is proposed to interpret the appearance and development of the ''dip'' structure. The formation and evolution process of this ''dip'' structure can be well resolved from the measured time-resolved PL spectra. First-principles simulations provide clear evidence of finite electron transfer at the interface between graphene and GaN.
S ince its first successful exfoliation from bulk graphite and isolated existence in nature 1 , graphene has drawn intensive research interest from scientists in the areas of physics, materials science, chemistry, engineering, etc., due to its excellent electric, mechanic and thermal properties 2,3 , and also due to numerous potential applications in optoelectronic devices, electronic devices, solar cells, phototransistor etc [bib_ref] Transparent, conductive graphene electrodes for dye-sensitized solar cells, Wang [/bib_ref] [bib_ref] Roll-to-roll production of 30-inch graphene films for transparent electrodes, Bae [/bib_ref] [bib_ref] Rectification at Graphene-Semiconductor Interfaces: Zero-Gap Semiconductor-Based Diodes, Tongay [/bib_ref] [bib_ref] Graphene-Based Nanomaterials: Synthesis, Properties, and Optical and Optoelectronic Applications, Chang [/bib_ref]. Very recently, particular effort has been devoted to the investigation of integrating graphene with semiconductors, especially GaN, for possible improvements of performance of existing devices and even exploring new schemes [bib_ref] Rectification at Graphene-Semiconductor Interfaces: Zero-Gap Semiconductor-Based Diodes, Tongay [/bib_ref] [bib_ref] Interface and transport properties of GaN/graphene junction in GaN-based LEDs, Wang [/bib_ref] [bib_ref] Graphene/SiO 2 /p-GaN Diodes: An Advanced Economical Alternative for Electrically Tunable Light..., Chang [/bib_ref] [bib_ref] Improved transport properties of graphene/GaN junctions in GaN-based vertical light emitting diodes..., Wang [/bib_ref] [bib_ref] GaN-based light-emitting diodes with graphene/indium tin oxide transparent layer, Lai [/bib_ref]. It is well known that as a technologically important wide band gap semiconductor, GaN has tremendous applications in various fields such as short-wavelength light emitting diodes and lasing diodes, high-temperature and high-power electronic devices etc. Previous research about graphene/GaN hybrid structure has been mainly concentrated on the electric/electronic properties 8,14-16 , while few works have been devoted to the photoelectric properties of the composite [bib_ref] Graphene/GaN diodes for ultraviolet and visible photodetectors, Lin [/bib_ref]. In terms of the technological importance of both GaN and graphene in different areas, the optical properties of graphene/GaN hybrid structures are of broad interest due to their unique physical and chemical properties. In this article, we present a detailed study of the optical properties of graphene/GaN hybrid structure at different temperatures by using steady-state and time-resolved photoluminescence (PL) techniques. We observe the luminescence signature of free exciton dissociation and electron transfer over the Schottky barrier of graphene/GaN hybrid structure. Moreover, we find that the efficiency of this two-step carrier process is dependent on the kinetic energy of movable excitons created in the GaN side.
In order to verify layer number and also homogeneousness of the graphene layer, the Raman spectra and image of the graphene/GaN hybrid structure were measured. The micro-Raman scanning images of the 2D mode of Raman mode of GaN were shown in [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref] and 1(b), respectively. As can be seen from [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref] , the graphene studied here was reasonably uniform in terms of its 2D mode intensity. The Raman spectra extracted from a randomly selected point in the images of [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref] and 1(b) were depicted in [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref] and 1(d), respectively. The two prominent Raman modes of single-layer graphene (G band: ,1586 cm 21 and 2D band: ,2690 cm 21 ) can be well resolved in [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref]. Their intensity ratio, I 2D /I G . 2, indicates good quality of graphene layer [bib_ref] Raman Spectroscopy and Imaging of Graphene, Ni [/bib_ref] [bib_ref] Raman spectrum of graphene and graphene layers, Ferrari [/bib_ref]. Moreover, the 2D band can be fitted quite well with a Lorentzian lineshape function [bib_ref] Raman spectrum of graphene and graphene layers, Ferrari [/bib_ref] , as shown in [fig_ref] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from... [/fig_ref]. The defect-induced D band (,1350 cm 21 ) of graphene is not well resolved probably due to the significant overlapping with the second-order Raman scattering from the underlying GaN epilayer. Compared with the mechanical exfoliated graphene on SiO 2 /Si substrate, the 2D band in the graphene/GaN hybrid structure shows a slight blueshift of 8 cm 21 . Such shift could be due to the joint effects of substrate and preparation technique [bib_ref] Raman Spectroscopy and Imaging of Graphene, Ni [/bib_ref]. The Raman features of the underlying GaN layer keep unchanged except some intensity reduction, compared with the asgrown GaN epilayer (results not shown here). This is due to reflection and absorbance of some incident laser photons.
For graphene/GaN contacts, usually a Schottky junction forms due to van der Waals attraction and electron transfer [bib_ref] Rectification at Graphene-Semiconductor Interfaces: Zero-Gap Semiconductor-Based Diodes, Tongay [/bib_ref] [bib_ref] Graphene/GaN Schottky diodes: Stability at elevated temperatures, Tongay [/bib_ref] [bib_ref] Self-adaptive electronic contact between graphene and semiconductors, Zhong [/bib_ref] [bib_ref] Graphene/GaN diodes for ultraviolet and visible photodetectors, Lin [/bib_ref]. [fig_ref] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before [/fig_ref] and (b) show schematic drawings of band structures of graphene and n-type GaN before and after their contacting, respectively. qw g is the work function of graphene (,4.9 eV for CVD sample), x e is the electron affinity of GaN (,4.1 eV), qw SBH < 0.74 eV is Schottky barrier height of graphene/GaN junction [bib_ref] Graphene/GaN Schottky diodes: Stability at elevated temperatures, Tongay [/bib_ref]. V bi is the built-in voltage. E g is the band gap of GaN and E f is the Fermi energy of GaN. As mentioned earlier, the electrical properties of graphene/GaN contact have been addressed previously. In the present work, we concentrate on its optical properties, especially luminescence signatures of free exciton dissociation at the interface and transfer of liberated electrons over the junction.
PL spectra of the graphene/GaN hybrid structure and the asgrown GaN epilayer measured under the same conditions were depicted in [fig_ref] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before [/fig_ref] for a temperature range of 1.5-77 K. Since donor bound exciton (denoted by DX) line is almost unaffected by the graphene capping layer, the PL spectra were thus normalized at the intensity of DX peak to make a clear comparison. It can be seen that the I x band (a bound exciton with undetermined origin) of the GaN epilayer is significantly weakened after the graphene layer is capped. Moreover, it attenuates faster with increasing temperature in the graphene/GaN sample compared with the case of the bare GaN. In the literature 20 , a similar sharp peak was observed, which is believed to be identical to I x emission in this work. However, its origin is still a controversy issue [bib_ref] Recombination of free and bound excitons in GaN, Monemar [/bib_ref] [bib_ref] Photoluminescence and reflectance spectroscopy of excitonic transitions in high-quality homoepitaxial GaN films, Kornitzer [/bib_ref] [bib_ref] Bound exciton dynamics in GaN grown by hydride vapor-phase epitaxy, Pozina [/bib_ref] [bib_ref] Shallow donors in epitaxial, Fischer [/bib_ref]. Considering that the intensity of I x band can be significantly reduced after the top graphene covering and its fast attenuating with increasing temperatures, we could tentatively ascribe I x to the excitons bound at a surface acceptor-like defect which has strong sensitivity to surface environment change.
In addition to the significant reduction and quick thermal quenching of I x line, more interesting behavior was observed for free exciton emissions. As can be seen from [fig_ref] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before [/fig_ref] and [fig_ref] Figure 3 |: Temperature-dependent PL spectra of the as-grown GaN [/fig_ref] , free exciton A (FX A ) bands of the two samples behave very differently as the temperature increases. For the as-grown GaN epilayer, FX A peak gradually becomes dominant due to thermal liberation of bound excitons into free excitons as previously observed [bib_ref] Direct determination of free exciton binding energy from phonon-assisted luminescence spectra in..., Xu [/bib_ref]. Its lineshape keeps unchanged except broadening due to phonon scattering effect. As for the graphene/GaN hybrid structure, however, the lineshape of the FX A peak shows a remarkable change when temperature is beyond 30 K. A dip at peak center develops and becomes noticeable for temperatures above 77 K. Furthermore, free exciton B (FX B ) emission band also exhibits similar behavior although its intensity is relatively much weaker. These results reveal that the graphene capping layer has a strong influence on free excitons in the GaN underlying layer.
In order to understand this phenomenon, electronic coupling and charge transfer between graphene and GaN need to be considered [bib_ref] Rectification at Graphene-Semiconductor Interfaces: Zero-Gap Semiconductor-Based Diodes, Tongay [/bib_ref] [bib_ref] Self-adaptive electronic contact between graphene and semiconductors, Zhong [/bib_ref]. Owing to its zero band gap, graphene is usually regarded as a semimetal and once in touch with semiconductors would exhibit metallike functions in metal/semiconductor junction. For graphene/GaN, a Schottky junction usually forms at the interface with a Schottky barrier height (qw SBH ) [bib_ref] Rectification at Graphene-Semiconductor Interfaces: Zero-Gap Semiconductor-Based Diodes, Tongay [/bib_ref]. Before contact, neutral single-layer graphene (idealized) has the Fermi level aligned with Dirac points where the occupied branch (bottom) and unoccupied branch (upper) touch to one another, as schematically displayed in [fig_ref] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before [/fig_ref]. Besides, the density of states (DOS) in graphene shows a low and linear dispersion with zero DOS at Dirac points. After paving onto GaN, graphene sheet may take electrons from GaN since as-grown intentionally undoped GaN is n-type conductivity. The electron flow-in could raise graphene's Fermi level during the pinning of the Fermi level in the entire hybrid structure. As a result, a Schottky contact with rectifying characteristics forms, as schematically shown in [fig_ref] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before [/fig_ref]. Consequently, a narrow depletion layer emerges in the side of GaN layer near the interface with the establishment of a build-in field V bi . Such contact, in particular the built-in electric field may have a strong impact on the surface related properties of GaN, such as freely moveable excitons and surface defects. Since the laser wavelength 325 nm used in this study is in ultraviolet region, efficient photon absorption occurs within only a very shallow range of about hundred nm underneath the GaN surface due to the large absorption coefficient. In other words, the efficient photon absorption mainly takes place near and within the thin depletion layer in which a built-in electric field already established due to the electron transfer from GaN to graphene in the hybrid structure. In such circumstances, excitons generated in the ,100 nm thick layer of GaN near the interface may be significantly affected by graphene and some optical signatures of the effects could be observed.
It is known that free and bound excitons form chronologically within a short time after the excitation in wide band gap semiconductors at low temperatures [bib_ref] Formation dynamics of excitons and temporal behaviors of Fano resonance due to..., Zheng [/bib_ref]. For free excitons, they are movable and have high probability to arrive at crystal/air interface (surface) where they can annihilate in ways of either radiative recombination or destroyed by surface defects/states. In the former process, light emission occurs. The latter is non-radiative process, and may lead to some reduction in emission intensity or particular features in spectral lineshape by competing with the former process. Therefore, free excitons could be sensitive to the surface environment variation. For bound excitons, such as excitons bound at impurities inside crystal, they are fixed at particular locations, especially at low temperatures. They are thus insensitive to the surface circumstance change except the case of excitons bound at surface defects. Bound excitons usually have much larger oscillator strength than free excitons and thus their emission band is usually dominant over free excitons at low temperatures. Furthermore, they randomly distribute over a much wider interior region in crystal, for example, in GaN.
In the graphene/GaN hybrid structure studied in the present work, a particular attention will be thus paid on free excitons and their PL spectral features. As observed in the PL spectra at different temperatures, on one hand, free exciton emission line such as FX A quickly becomes dominant in the PL spectra with increasing temperature due to the efficient thermalization of donor bound excitons into free excitons. On the other hand, kinetic energy of free excitons will increase with increasing temperature. Without doubt, increase in kinetic energy can raise the arriving probability of free excitons at the interface where they may recombine radiatively or get dissociated. Owing to the existence of a graphene layer, some of free excitons arriving at the interface may be dissociated since the semi-metallic graphene selectively captures electrons. Of course, those broken excitons will no longer contribute to the light emission. It is known that in light emission of excitons, conservation principles of both energy and momentum should be obeyed. Compulsorily restricted by principle of momentum conservation, only free excitons with almost zero momentum (i.e., located in the vicinity of the zone center in k space.) are ''bright'' excitons contributing to emission due to extremely small momentum of photons. Some of these free excitons with almost zero momentum arrive at the interface and non-radiatively decomposed into electrons and holes, causing the observed ''dip'' structures at the spectral centers of FX A and FX B bands.
In order to obtain a deeper insight into the formation of ''dip'' structures, time-resolved PL spectra of the graphene/GaN hybrid sample and the as-grown GaN were measured. The results at 77 K were depicted in [fig_ref] Figure 4 |: Time-resolved PL results of the graphene/GaN [/fig_ref]. It is perfectly clear that appearance and evolution of the dip structures can be seen. Before 180 ps delay time after the femtosecond pulse excitation, the ''dip'' structures don't emerge, suggesting that transport of free excitons to the interface and dissociation there don't efficiently occur within 180 ps. When the delay time was 293 ps the dip structure can be clearly seen. The average transport 1 dissociation time of free excitons is thus estimated to be 110 ps. As the delay time further increases, the dip structures develop and become more noticeable. In sharp contrast to the case of the hybrid structure, the bare as-grown GaN sample shows normal time-resolved PL spectra. Regarding the peak intensity evolution of FX A band with the delay time, luminescence time constants show a significant reduction for the hybrid structure, i.e., from ,300 ps of the bare sample to ,150 ps of the hybrid structure.
At last, the first-principles simulations on electronic structures of bare GaN and graphene/GaN heterostructure were performed by using the pseudopotential-based code VASP within the Perdew-Burke-Ernzerhof generalized gradient approximation [bib_ref] Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis..., Kresse [/bib_ref] [bib_ref] Generalized Gradient Approximation Made Simple, Perdew [/bib_ref] , where we use a substrate GaN supercell of size 2 3 2 3 8. Throughout the theoretical calculations, a 400 eV cutoff in the plane wave expansion and a 6 3 6 3 1 Gamma k grid are chosen to ensure the calculation with an accuracy of 10 25 eV. The lattice constants are taken from the experimental values, while the internal atomic positions are optimized until the largest force on each atom was 0.005 eV/Å . As shown in the left panel of , the carbon atoms of graphene couple tightly with the outermost layer of nitrogen atoms so that finite electron transfer may occur in between graphene and GaN. The right panel of depicts the calculated total density of states (DOS) of bare GaN and graphene/GaN hybrid structure, respectively. Clearly, a significant change in total DOS can be seen before and after graphene contacting with GaN, especially in the free exciton (,3.495 eV) luminescence region. The value of charges transferred from graphene to substrate GaN is estimated to be 0.02e per carbon atom. Our theoretical results show good agreement with experiment and confirm the picture of free exciton dissociation and transfer of liberated electrons.
To conclude, an interesting ''dip'' structure was observed on the PL spectra of free excitons in graphene/GaN hybrid structure. Its development was also found to be dependent on temperature. Such dip structure is interpreted as a result of free exciton dissociation and transfer of liberated electrons at the interface between graphene and GaN. Time evolution of the ''dip'' structure was clearly seen via measuring time-resolved PL spectra. The first-principles calculations confirm the physical picture of free exciton dissociation and electron transfer at the interface between graphene and GaN.
## Experimental section
Intentionally undoped GaN epilayer (,4 mm) was grown on sapphire substrate by metal-organic chemical vapor deposition (CVD). The carrier concentration in this n-type GaN was estimated as 5.02 3 10 15 cm 23 from the room-temperature Hall measurement. The GaN epilayer was cut into a few pieces (with size of a few millimeters). One piece served as a control sample while others were covered with graphene. Large-area single-layer graphene sheets were synthesized by CVD method on Cu foils (diameter: 25 mm) at 1000uC with a flow of H 2 (40 sccm) and CH 4 (60 sccm) for 15 min under the pressure of 160 Pa. After 15 min of growth, the sample was cooled down to room temperature in H 2 atmosphere. After the growth of graphene, the Cu foils were covered with a layer of poly (methyl methacrylate, PMMA). The Cu substrates were then etched in an aqueous iron www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 7687 | DOI: 10.1038/srep07687 chloride (FeCl 3 ) solution, leaving the graphene/PMMA film which was washed in deionized water for three times to achieve an aqueous environment with negligible ions for preparation of transfer onto GaN surface. Before the transfer step, the GaN epilayer pieces were cleaned using typical surface-cleaning techniques. It should be mentioned that new graphene growth routes have been very recently developed, which did not require metal catalyst etching or hazardous chemicals at lower temperature [bib_ref] Plasma nanoscience: from nanosolids in plasmas to nano-plasmas in solids, Ostrikova [/bib_ref] [bib_ref] Multifunctional Three-Dimensional T-Junction Graphene Micro-Wells: Energy-Efficient, Plasma-Enabled Growth and Instant Water-Based Transfer..., Kumar [/bib_ref].
Optical spectral measurements were carried out on the graphene/ GaN hybrid structure and the as-grown GaN epilayer piece for comparison. The Raman scattering measurements were taken under the back-scattering geometric configuration on a WITec-Alpha confocal Raman microscope system using the 514.5 nm Ar 1 laser with an excitation power of ,1 mW as the excitation source. In the variable-temperature photoluminescence (PL) measurements, the samples were loaded on the cold finger of the cryostat of a 7 Tesla Spectromag system (Oxford Instruments Nanoscience) with varying temperature from 1.46 K to 300 K. The samples were excited by the 325 nm line from a He-Cd Laser (Kimmon) with output power of 35 mW. The PL signals were dispersed by an ultra-high-resolution monochromator (McPHERSON 20621) equipped with a photomultiplier. Standard lock-in amplifier technique was used to improve the signal-to-noise ratio. In the time-resolved PL (TRPL) measurements, the samples were loaded on the cold finger of a closed cryostat providing a varying temperature from 20 K to 300 K. The excitation source was the 266.7 nm laser pulses (pulse width ,100 fs) generated from a frequency tripled mode-locked Ti: sapphire oscillator with a repetition rate of 76 MHz. The details of the experimental arrangements have been described elsewhere [bib_ref] Formation dynamics of excitons and temporal behaviors of Fano resonance due to..., Zheng [/bib_ref].
[fig] Figure 1 |: Micro-Raman scanning images with an area of 5 3 5 measured from the graphene/GaN hybrid structure. (a) Integrated from the intensity of 2D Raman mode of graphene. (b) Integrated from the intensity of E high 2 Raman mode of GaN. (c) and (d) Representative Raman spectra extracted from 1(a) and 1(b), respectively. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 7687 | DOI: 10.1038/srep07687 [/fig]
[fig] Figure 2 |: Schematic drawings of energy band structures of graphene and n-type GaN before (a) and after the contacting (b). Temperature-dependent PL spectra of the as-grown GaN (black solid line) and graphene/GaN (red solid line) are depicted in semi-logarithmic scale (c). All spectra are normalized at DX peak intensity. DX, I x , FX A , and FX B lines can be well resolved and indicated by different color vertical arrows.www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 7687 | DOI: 10.1038/srep07687 [/fig]
[fig] Figure 3 |: Temperature-dependent PL spectra of the as-grown GaN (black) and graphene/GaN hybrid structure (red) from 77 K to 200 K. Dashed blue and magenta lines align with the centers of FX A and FX B bands, respectively. [/fig]
[fig] Figure 4 |: Time-resolved PL results of the graphene/GaN (left) and as-grown GaN (right) at 77 K: Decay curves of FX A center (top), time-resolved PL images (mid) and time evolutional PL spectra (bottom). The two vertical lines mark the spectral center positions of FX A and DX, respectively. In the images (mid), the emission signal intensity increases as the image color goes from blue, green, to red. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 7687 | DOI: 10.1038/srep07687 [/fig]
|
Folinic Acid, Fluorouracil, and Oxaliplatin Therapy for Recurrent Esophageal Cancer with Syndrome of Inadequate Antidiuretic Hormone Secretion (SIADH) After Preoperative Cisplatin/5-Fluorouracil Therapy
# Background
Cisplatin/5-fluorouracil therapy is the standard therapy for unresectable and recurrent esophageal cancer [bib_ref] Phase Ⅱ evaluation of cisplatin and 5-fluorouracil in advanced squamous cell carcinoma..., Lizuka [/bib_ref]. Cisplatin-based chemotherapy often causes adverse effects, such as nausea, vomiting, and renal dysfunction, which may necessitate dose modification or treatment prolongation [bib_ref] A phase Ⅱ study of nedaplatin and 5-fluorouracil in metastatic squamous cell..., Kato [/bib_ref]. In general, esophageal cancer patients have a lower performance status and poorer nutrition than patients with other cancer types. Therefore, novel combination therapies are urgently needed to improve the efficacy and overcome drug toxicity in this setting. Some patients may benefit from switching to folinic acid, fluorouracil, and oxaliplatin (FOLFOX) therapy, especially those with impaired renal function or challenges with high-volume infusions [bib_ref] Definitive chemoradiotherapy with FOLFOX versus fluorouracil and cisplatin in patients with oesophageal..., Conroy [/bib_ref]. A rare adverse effect of cisplatin is syndrome of inadequate antidiuretic hormone secretion (SIADH) [bib_ref] Syndrome of inappropriate antidiuretic hormone secretion following dis-dichlorodiammineplatinum II in a patient..., Levin [/bib_ref]. Here, we present a case of SIADH in a patient with esophageal squamous cell cancer who was receiving cisplatin/5-fluorouracil therapy and later successfully continued treatment with FOLFOX therapy.
## Case report
A 77-year-old man presented to the clinic with pharyngeal discomfort over the previous 2 months. An upper gastrointestinal endoscopy revealed an esophageal tumor in his thoracic esophagus. The patient was diagnosed with squamous cell carcinoma after a biopsy and was referred to our hospital. His medical history included myocardial infarction, stroke, and diabetes. His medications included aspirin, azilsartan, amlodipine besilate, indapamide, carvedilol, rosuvastatin calcium, glimepiride, sitagliptin phosphate hydrate, and voglibose. The patient did not use diuretics. Imaging studies showed no distant metastases, and a final diagnosis of cT3N0M0 stage II cancer was made. The standard care for esophageal cancer [bib_ref] A randomized trial of postoperative adjuvant chemotherapy versus neoadjuvant chemotherapy with cisplatin..., Igaki [/bib_ref] is to operate after neoadjuvant chemotherapy (NAC). Cisplatin/5-fluorouracil therapy (cisplatin 80 mg/m 2 , 5-fluorouracil 800 mg/m 2 ) was commenced as NAC. Hyperhydration (3000 mL/day) with mannitol and magnesium sulfate support were provided before he received cisplatin on day 1. The patient was given 200 ml of 20% mannitol injection on day 1 only. On day 8 of administration, the patient had lightheadedness, diaphoresis, and nausea and became unconscious; blood test results showed a decrease in serum sodium levels from 134 mEq/L to 106 mEq/L (135-146 mEq/L). Urine osmolality was 612 mOsm/L (50-1300 mOsm/kg), serum osmolality was 229 mOsm/L (275-290 mEq/L), fractional excretion of sodium (FENa) was 1.1%, urine Na concentration was 79.7 mEq/L, serum arginine vasopressin (AVP) was 1.2 pg/mL, and serum uric acid level was 2.6 mg/dL (3.6-7.0 mg/dL). The results of other investigations are shown in . We diagnosed the patient with cisplatin-induced SIADH as there were no abnormalities on head and chest computed tomography (CT). There was also no evidence of adrenal, thyroid, cardiac, renal, or liver dysfunction or signs of dehydration. Subsequently, water restriction was started, and treatment with a salt-added diet and 3% hypertonic saline infusion was initiated. The patient was discharged with gradual improvement in serum sodium levels to 130 mEq/L on day 16 of administration. endoscopy showed a marked reduction in the size of the esophageal lesion after NAC. Positron emission tomography-CT (PET-CT) also showed a decrease in fluorine-18-deoxyglucose (FDG) accumulation (from 22.33 to 18.69 times the standard uptake value [SUV]), which we considered to be a minor clinical response. It was determined that continuing NAC was not possible because of marked hyponatremia; therefore, 1 month later, the patient underwent thoracoscopic esophagectomy, laparoscopic gastrointestinal reconstruction, and 2-field lymph node dissection with a pathological diagnosis of stage III T3N2M0 well-to-moderately differentiated squamous cell carcinoma. Four months after the surgery, a thoracoabdominal contrast-enhanced CT showed a soft tissue shadow extending from the cervical anastomosis to the superior mediastinum. PET-CT showed an accumulation of FDG at the esophageal anastomosis (19.67 times the SUV) and the cervical lymph nodes , as well as at the mediastinal lymph nodes, right pleura, and 10 th rib. The patient was diagnosed with recurrent esophageal cancer, right pleural dissemination, mediastinal lymph node metastasis, and rib metastasis. We decided to start the FOLFOX therapy (folinic acid 200 mg/m 2 , fluorouracil 2800 mg/m 2 , and oxaliplatin 85 mg/m 2 ) as the patient developed marked hyponatremia with cisplatin/5-fluorouracil therapy preoperatively. The serum sodium concentration was maintained above 130 mEq/L after 1 course, and the patient was able to continue treatment without developing SIADH. Serum sodium concentration and creatinine levels before and after chemotherapy are shown in . The patient did not develop SIADH, but contrast-enhanced CT after 4 courses of chemotherapy showed an enlarged lesion, which indicated disease progression. Chemotherapy was discontinued, and the patient received best supportive care.
# Discussion
SIADH associated with advanced cancer is a common electrolyte abnormality. The frequency of SIADH (Na <130 mEq/L) has been reported to be 3.7% in patients receiving chemotherapy [bib_ref] A prospective study on hyponatraemia in medical cancer patients: Epidemiology, aetiology and..., Berghmans [/bib_ref] [bib_ref] Hyponatremia related to medical anticancer treatment, Berghmans [/bib_ref]. SIADH is thought to be mainly induced by platinumbased drugs, such as cisplatin, or by electrolyte abnormalities caused by vomiting, diarrhea, and massive infusions commonly associated with chemotherapy [bib_ref] A prospective study on hyponatraemia in medical cancer patients: Epidemiology, aetiology and..., Berghmans [/bib_ref] [bib_ref] Hyponatremia related to medical anticancer treatment, Berghmans [/bib_ref]. There are 2 possible pathophysiological mechanisms for cisplatin-induced hyponatremia -SIADH and renal salt wasting syndrome (RSWS) due to dehydration and renal dysfunction. The pathogenesis of RSWS is thought to be a tubular disorder caused by cisplatin, but the exact mechanism of action is not clear and there are no established diagnostic criteria. It is characterized by obvious signs of dehydration and renal dysfunction, resulting in marked sodium diuresis [bib_ref] Cisplatinum-induced renal salt wasting, Kurtzberg [/bib_ref]. SIADH is thought to be caused by impaired electrolyte absorption at Henle's loop, increased hydration to prevent renal damage during cisplatin administration, and accelerated antidiuretic hormone secretion (ADH) secretion due to vomiting and pain.
## The concept of siadh was proposed by schwartz et al in 1957.
It is a syndrome in which abnormal secretion of ADH causes water retention, resulting in hyponatremia [bib_ref] A syndrome of renal sodium loss and hyponatremia probably resulting from inappropriate..., Schwartz [/bib_ref]. There have been reports of SIADH caused by vincristine, cyclophosphamide, and cisplatin as well as other antineoplastic agents [bib_ref] Syndrome of inappropriate antidiuretic hormone secretion following dis-dichlorodiammineplatinum II in a patient..., Levin [/bib_ref]. The SIADH diagnostic criteria include: 1) preserved adrenal, thyroid, renal, and cardiac function, with no cirrhosis, diuretics, and dehydration; 2) urine osmolality >100 mOsm/L; 3) serum osmolality <280 mOsm/L; 4) serum sodium concentration <135 mEq/L; 5) FENa >0.5%; and 6) urine sodium >30 mEq/L [bib_ref] Clinical laboratory evaluation of the syndrome of inappropriate secretion of antidiuretic hormone, Decaux [/bib_ref]. In some cases, the diagnostic criteria include a serum ADH concentration greater than the measurement sensitivity [bib_ref] Onset of syndrome of inappropriate secretion of antidiuretic hormone in a gastric..., Goto [/bib_ref]. However, an abnormal serum ADH concentration is not characteristic of SIADH alone and thus has no diagnostic value. In this case, we investigated the possibility of both RSWS and SIADH as causes of hyponatremia. The patient had no apparent features of dehydration or renal dysfunction. Hyponatremia improved after treatment with water restriction and sodium correction. Therefore, the main reasons for the development of SIADH could have been excessive hydration to prevent renal dysfunction during cisplatin administration and the use of oral diuretics.
The usefulness of cisplatin/5-fluorouracil therapy as NAC for stage II and III advanced esophageal cancers has been previously reported [bib_ref] A randomized trial of postoperative adjuvant chemotherapy versus neoadjuvant chemotherapy with cisplatin..., Igaki [/bib_ref]. In this case, cisplatin/5-fluorouracil therapy was administered as NAC and caused marked SIADH. Therefore, NAC was discontinued after 1 course and the patient underwent surgical treatment. Although chemotherapy was indicated for early postoperative recurrence, the risk of cisplatin-related SIADH recurrence was considered high. There is a case of SIADH after treatment with cisplatin in which the patient was able to continue treatment with carboplatin instead of cisplatin [bib_ref] Syndrome of inappropriate secretion of ADH (SIADH) following cisplatin administration in a..., Kagawa [/bib_ref]. With regard to the available chemotherapy regimen for esophageal cancer, the PRODIGE5/ACCORD17 trial compared FOLFOX-radiotherapy (RT) and cisplatin/5-fluorouracil-RT therapy in patients diagnosed with stage I-IVA cancer and eligible for radical chemoradiotherapy and concluded that progression-free survival was similar with both regimens, but FOLFOX might be more convenient [bib_ref] Definitive chemoradiotherapy with FOLFOX versus fluorouracil and cisplatin in patients with oesophageal..., Conroy [/bib_ref]. That case study also suggested that FOLFOX-RT may be an alternative to cisplatin/5-fluorouracil-RT. In addition, the E-DIS trial, performed for patients with unresectable or recurrent esophageal squamous cell carcinoma, showed comparable results between the FOLFOX and cisplatin/5-fluorouracil therapy [bib_ref] Continuation versus discontinuation of first-line chemotherapy in patients with metastatic squamous cell..., Adenis [/bib_ref]. In a study that compared the cisplatin/TS-1 and oxaliplatin/TS-1 therapy in patients with unresectable advanced or recurrent gastric cancer, 13.4% and 4.4% of patients in the cisplatin/TS-1 and oxaliplatin/TS-1 groups had hyponatremia, respectively . Cisplatin-induced SIADH is considered to be caused by impaired electrolyte reabsorption at Henle's loop and water overload due to large amounts of hydration [bib_ref] Cisplatinum-induced renal salt wasting, Kurtzberg [/bib_ref] , and it seems to occur less frequently with oxaliplatin since it does not require large amounts of hydration. However, even when using oxaliplatin, careful attention should be paid, as there have been reports of SIADH even with this regimen [bib_ref] Onset of syndrome of inappropriate secretion of antidiuretic hormone in a gastric..., Goto [/bib_ref]. 5-fluorouracil and platinum-based regimens were considered preferable in this case because of the response to the preoperative cisplatin/5-fluorouracil therapy. Switching from cisplatin/5-fluorouracil therapy with cisplatin to FOLFOX therapy with oxaliplatin allowed our patient to continue treatment without subsequent development of SIADH. Oxaliplatin has several advantages over cisplatin, including that it does not require large amounts of hydration and can be performed on an outpatient basis. However, there is currently little evidence regarding the treatment of esophageal cancer, and great caution is required when using this drug.
# Conclusions
The cisplatin/5-fluorouracil therapy is currently the standard chemotherapy regimen for esophageal cancer; however, SIADH is a known adverse effect when using cisplatin, as seen in the present case. To the best of our knowledge, there is no report of SIADH caused by cisplatin/5-fluorouracil therapy wherein the patient could continue chemotherapy without SIADH by switching to FOLFOX therapy with oxaliplatin. Oxaliplatin appears to have a lower risk of SIADH than cisplatin, suggesting that oxaliplatin can be a therapeutic option for patients with esophageal cancer who are at high risk of SIADH.
# Data availability statement
All data generated or analyzed during this study are included in this article and its supplementary material files. Further enquiries can be directed to the corresponding author.
## Declaration of figures' authenticity
All figures submitted have been created by the authors who confirm that the images are original with no duplication and have not been previously published in whole or in part.
[fig] Figure 1: (A) Thoracic and abdominal contrast-enhanced computed tomography (CT) before neoadjuvant chemotherapy (NAC). Tumor is indicated by arrows. (B) Fluorine-18-deoxyglucose (FDG) accumulation was 22.33 times the standard uptake value on positron emission tomography (PET)-CT before NAC. Tumor is indicated by arrows. (C) Thoracic and abdominal contrastenhanced CT after NAC showing a marked reduction in the size of the esophageal lesion. Tumor accumulation is indicated by arrows. (D) FDG accumulation decreased to 18.69 on PET-CT after NAC. Tumor accumulation is indicated by arrows. [/fig]
[fig] Figure 2, Figure 3: (A) Fluorine-18-deoxyglucose (FDG) accumulation at the esophageal anastomosis (19.67). Tumor accumulation is indicated by arrows. (B) FDG accumulation at the cervical lymph nodes. Tumor accumulation is indicated by arrows. Serum sodium concentration and creatinine levels before and after chemotherapy. [/fig]
|
Long-term lead elimination from plasma and whole blood after poisoning
Objective Blood lead (B-Pb), one of the most used toxicological biomarker all kind, has serious limitations. Thus, the objective is to evaluate whether plasma lead (P-Pb) is more adequate. Methods A long-term follow-up study of five cases of lead poisoning. P-Pb was analysed by inductively coupled plasma mass spectrometry. Kinetics after end of exposure was modelled. Results P-Pb at severe poisoning was about 20 lg/L; haematological effects at about 5 lg/L. Biological halftime of P-Pb was about 1 month; B-Pb decay was much slower. Conclusion P-Pb is a valuable biomarker of exposure to and risk, particularly at high exposure.
# Introduction
The lead (Pb) concentration in whole blood (B-Pb) is probably-next to ethanol in blood-the most widely used biomarker for assessment of toxic exposure and risk. However, it has clear limitations, in particular because there is saturation with increasing exposure, in particular at B-Pbs [ 700 lg/L [bib_ref] Lead in plasma and whole blood from lead-exposed children, Bergdahl [/bib_ref] , and because Pb induces anaemia, which will make the use of B-Pb problematic, because Pb is mainly present in erythrocytes, the volume of which will decrease. Pb in plasma (P-Pb) or serum is an attractive alternative, which would avoid these problems [bib_ref] Measurement by ICP-MS of lead in plasma and whole blood of lead..., Schütz [/bib_ref] [bib_ref] Whole blood, serum, and saliva lead concentrations in 6-to 8-year-old children, Costa De Almeida [/bib_ref] [bib_ref] A polymorphism in the delta-aminolevulinic acid dehydratase gene modifies plasma/whole blood lead..., Montenegro [/bib_ref] [bib_ref] Correlation between lead in plasma and other indicators of lead exposure among..., Hirata [/bib_ref]. The concentrations are very low, but the developments in analytical technique now allow adequate determination. However, P-Pb has up to now been used only occasionally.
There are indications that the toxicokinetics of Pb are affected by genetic polymorphism in the enzyme d-aminolevulinic acid dehydratase (ALAD), which is the main binding site for Pb in erythrocytes, and inhibition of which is at least partly responsible for the anaemic effect of Pb.
In spite of centuries of preventive attempts, Pb is still a major health problem. It induces clinical poisoning in occupational settings, but sometimes also by other exposures.
The aim of this study was to scrutinise the usability of P-Pb as a biomarker in cases of clinical Pb poisoning.
# Subjects and methods
## Cases
We evaluated data from five cases of clinical Pb poisoning, four non-occupational and one occupational [fig_ref] Table 1: Histories of five cases of lead poisoning [/fig_ref]. They had been exposed to Pb for 1 month-12 years. The intakes of Pb were estimated by self-reported consumption of tablets or drink, and the measured contents of Pb in those media. Four had anaemia. They were followed for 21-316 months. In all subjects, the symptoms and signs disappeared during the initial part of the follow-up.
Blood and urine for Pb and haemoglobin (B-Hb) determinations were sampled daily during the first week(s), later on weekly, monthly or more rarely.
All cases gave written informed consent for the use of their data for this study. Because of uncertainty in the diagnosis, and whether the exposure had ceased, frequent sampling was made initially.
## Analyses
## Lead
Cubital venous blood was collected in evacuated metal-free heparinised tubes. To obtain plasma, the tubes were centrifuged at 2,000g for 10 min. Samples with haemolysis at inspection were deleted. In connection with most blood sampling occasions, spot urine samples were collected in 10 mL polypropylene tubes the same day or the day before.
All samples, but those from case 5, were analysed by inductively coupled plasma-mass spectrometry (ICP-MS; [bib_ref] Relationships between trace element concentrations in human blood and serum, Barany [/bib_ref] ; for the samples from case 5, electro thermal atomic absorption spectrometry (ETA-AAS) was used. All samples were prepared in duplicate.
Quality control was strict, especially at method changes (ETA-AAS vs. ICP-MS, r = 0.98, n = 29; [bib_ref] Yearly measurements of blood lead in Swedish children since 1978: the declining..., Strömberg [/bib_ref]. The analytical accuracy was checked against reference material, (Seronorm, SERO AS, Billingstad, Norway) with the recommended values for lead in blood, plasma and urine being 393, 0.9 and 40 lg/L, respectively. Our results for the lead determination in the reference samples during the follow-up time were 98, 106 and 101% for blood, plasma and urine, respectively. Our laboratory is accredited for lead analysis in blood according to the Swedish Board for Accreditation and Conformity Assessment (SWEDAC), and during the period, we also produced good results in the UK National External Quality Assessment Service (Birmingham, UK); our mean accuracy was 96% with coefficient of variation \5%.
## Other
Blood haemoglobin (B-Hb) was analysed by a standard clinical method. Creatinine (crea) was analysed in urine samples by a modified kinetic Jaffé method (Roche Diagnostics, Mannheim, Germany). Both B-Hb and crea were analysed by an accredited clinical laboratory with scrupulous quality control. Detection limit for crea was 0.1 mmol/L and total imprecision 1.6%. For B-Hb measurements, the imprecision within the working range of 1-250 g/L was B1.0%. (1). The first exponential term describes the fast elimination phase, the second one, the slow elimination of Pb.
[formula] C Pb ðtÞ ¼ C 1 à e ðÀR 1 ÃtÞ þ C 2 à e ðÀ0:000146ÃtÞð1Þ [/formula]
C Pb (t), lead concentration at a given time (lg/L), t, time (days), C 1 , constant (concentration of the fast phase at t = 0), C 2 , constant (concentration of slow phase at t = 0), R 1 , elimination constant of phase 1 (days -1 ). The follow-up time was not sufficient to calculate the half-time in the slow phase. [bib_ref] Kinetics of lead in bone and blood after end of occupational exposure, Nilsson [/bib_ref] found it to be 13 years in a long-term study of Pb workers. Therefore, that value was used. The sum of C 1 and C 2 describes the modelled Pb content at the end of exposure (t = 0). The half-time of Pb in the fast phase has been calculated according to Eq. (2).
[formula] T 1=2 ¼ ln 2 = R 1ð2Þ [/formula]
For three cases, there were sufficient data to describe the relationship between B-Hb and P-Pb after end of exposure. Inspection of the curves indicated that one component did not give a satisfactory fit. Regression lines were calculated on the left and right sides of the division line (x = 5 lg P-Pb/L), and statistical significance of the difference between the pairs of slopes was examined. After that the threshold between the two components was calculated as the crossing point of the two lines.
Genotyping DNA was isolated from whole blood by minicolumn purification (E.Z.N.A DNA extraction kit, Omega Bio-Tek, Norcross, GA, USA) and diluted to a concentration of 5 ng/lL. The ALAD G379C polymorphism (Asn59Lys, rs1800435) was genotyped by Taqman allelic discrimination assay (ABI 7000 instrument, Applied Biosystems, Foster City, CA, USA), using the following primers and probes for ALAD: primers forward: 5 0 -TGC CTT CCT TCA ACC CCT CTA-3 0 and reverse: 5 0 -CCA AGG GCC TCA GCA TCT C-3 0 ; MGB-probes: ALAD-G: 5 0 -Fam-TGT GAA GCG GCT GG-3 0 and ALAD-C: 5 0 -Vic-TGT GAA CCG GCT GG-3 0 . The total volume of the PCR was 25 lL. Primers were added to a final concentration of 0.05 lM, probes to 0.9 lM in an 80% concentrated TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The PCR samples were incubated at 50°C for 2 min and at 95°C for 10 min. The samples underwent 40 cycles of 15 s at 95°C and 1 min at 60°C. Controls for each genotype as well as blanks were included in each run. Samples were analysed in duplicate and concordance rate was 100%.
# Results
The median P-Pb at first sampling (median 5, range 1-74 days after end of exposure) was 17 (range 2-42) lg/L [fig_ref] Figure 1: Lead elimination from plasma [/fig_ref]. The modelled median value for P-Pb (C 1 ? C 2 ) was 23 (range 3-38) lg/L at time t = 0. In Cases 1-4, the median of C 2 was 0.65 (range 0.6-0.8) lg/L, in Case 5 1.6 lg/L. In the two-compartment model, the median biological T 1/2 of the fast P-Pb phase was 27 (23-69) days [fig_ref] Table 2: Two-compartment modelling of lead in plasma and whole blood after end of... [/fig_ref].
The median B-Pb at first sampling was 790 (520-1,600) lg/L [fig_ref] Figure 1: Lead elimination from plasma [/fig_ref]. The modelled median value for B-Pb (C 1 ? C 2 ) was 840 (range 790-1,300) lg/L at time t = 0. In Cases 1-4, the median of C 2 was 155 (range 83-230) lg/L and in Case 5, it was 290 lg/L.
Median T 1/2 for the fast B-Pb component was 77 (58-120) days [fig_ref] Table 2: Two-compartment modelling of lead in plasma and whole blood after end of... [/fig_ref].
The relationship between B-Pb and P-Pb was approximately linear at low levels (ratio about 100); at P-Pbs above about 5 lg/L, the B-Pb levelled off . In Cases 1 and 2, the ratio at the highest P-Pbs was about 40, in Case 5, it was about 60.
There seemed to be a rectilinear relationship between U-Pb and P-Pb; the former expressed as lg/g crea was 22 times higher than the latter (R 2 linear = 0.5; p \ 0.001), expressed as lg/L .
The median B-Hb rose after end of exposure, from a median of 108 (range 92-139) g/L [fig_ref] Table 1: Histories of five cases of lead poisoning [/fig_ref] In three cases, there was sufficient information for a meaningful study of the relationship between B-Hb and P-Pb . The association seemed to have two components, an initial fast increase at relatively low P-Pbs, and a slower one at high ones (all Ps for pairs of regression lines B 0.01). The threshold P-Pb between the two components was calculated at 4.3, 6.6 and 5.0 lg/L, in Cases 1, 2 and 5, respectively.
Case 5, who was the only heterozygote for ALAD G379C (earlier denoted as ALAD 1-2; [fig_ref] Table 1: Histories of five cases of lead poisoning [/fig_ref] , had the longest T 1/2 for B-Pb, as compared to the others, who were homozygotes for the more common G-allele, while he did not differ from the others in P-Pb kinetics [fig_ref] Table 2: Two-compartment modelling of lead in plasma and whole blood after end of... [/fig_ref]. Also, he had higher initial both B-Pb and P-Pb [fig_ref] Figure 1: Lead elimination from plasma [/fig_ref] , and a higher B-Pb/P-Pb ratio .
# Discussion
The most important finding was that P-Pb at poisoning was about 20 lg/L. Biological half-time of P-Pb was about
## B-hb (g/l)
Case 1 Case 2 Case 5 Relationship between haemoglobin levels in blood (B-Hb) and lead in plasma (P-Pb) in sequential samples from three cases of poisoning 1 month; decay in B-Pb was much slower. P-Pb displayed a non-linear relationship with B-Pb, but rectilinear with U-Pb. The number of cases was small; in particular, we had only three cases with valid information on long-term B-Hb, which must be taken into consideration when drawing conclusions.
Since Pb content in red blood cells is much higher than in plasma, there is a risk that even a rather limited haemolysis, which may occur because of the haemolytic tendency at high Pb exposure, may contaminate the P-Pb. We eliminated the few plasma samples with haemolysis. A very slight red colour occurs before there is a serious problem of Pb carryover.
The present determination of P-Pb by ICP-MS was accurate. However, there is still uncertainty, which is reflected in the large confidence intervals in the estimates of kinetic parameters for P-Pb, which is wider than for B-Pb. In particular, Case 5 was studied before development of that method. Hence, ETA-AAS was used for P-Pb analyses, which was less sensitive. This is also obvious from the much greater variation of his data points in the elimination and B-Pb/P-Pb, U-Pb/P-Pb and B-Hb/P-Pb curves. This also explains why his first and third measurements are higher than the modelled C 1 ? C 2 . However, it is most unlikely that the analytical method explains his higher P-Pbs in general, which are more likely due to his greater skeletal Pb pool.
The elimination of Pb from both plasma and whole blood displayed at least two phases. The fast one reflects the soft tissues, the slow one the skeleton [bib_ref] Kinetics of lead in blood after the end of occupational exposure, Schütz [/bib_ref] [bib_ref] Kinetics of lead in bone and blood after end of occupational exposure, Nilsson [/bib_ref]. In spite of the long follow-up times, they did still not allow accurate estimates of the slow phase. Thus, we choose to use the better value obtained in our earlier study. The present P-Pbs are much higher than those in Swedes with no particular exposure (0.1-0.3 lg/L [bib_ref] Measurement by ICP-MS of lead in plasma and whole blood of lead..., Schütz [/bib_ref] [bib_ref] Lead in plasma and whole blood from lead-exposed children, Bergdahl [/bib_ref] , and remained so long after end of exposure. Therefore, we did not subtract a level in Swedish subjects without excessive exposure.
The method for determination of P-Pb with ICP-MS has been further developed. Hence, at our laboratory, the limit of detection is now 0.02 lg/L and the precision 6%. Hence, it is possible to use P-Pb as a biomarker in environmental health.
The number of cases is small, in particular we had only three cases with valid information on long-term B-Hb, which must be taken into consideration when drawing conclusions. In addition, the time of exposure and the total amount of Pb absorbed varied between the individuals; in particular, Case 5 differed. Hence, the body burden (mainly the skeletal content) of Pb differed, which will affect the elimination pattern after end of exposure [bib_ref] Kinetics of lead in bone and blood after end of occupational exposure, Nilsson [/bib_ref]. This was accounted for by the use of a twocomponent elimination model on an individual basis.
The relationship between the initial levels of the two components will vary depending upon the bone pool versus recent exposure. The pattern of P-Pb fits better with exposure data than B-Pb, which may be because it better reflects uptake and body burden, especially at these high uptakes. Only after careful comparison of the patterns did we merge the information into combined conclusions.
The T 1/2 for P-Pb of about 1 month is much longer than that reported after intravenous injection of Pb salt [bib_ref] Kinetics of lead following intravenous administration in man, Campbell [/bib_ref].
The present T 1/2 s for B-Pb are longer than previously reported [bib_ref] Effect of a short, heavy exposure to lead dust upon blood lead..., Schütz [/bib_ref] [bib_ref] Kinetic analysis of lead metabolism in healthy humans, Rabinowitz [/bib_ref] [bib_ref] Kinetics of lead in blood after the end of occupational exposure, Schütz [/bib_ref]. This is certainly because the present cases had B-Pbs much higher than in the earlier studies. Thus, our subjects initially had anaemia, with an attenuation of the rate of B-Pb decline when the effect on the blood cell formation and survival decreases as the body burden decays. Further -and more important -the curvilinear relationship between B-Pb and P-Pb, at the initial decrease of the body burden, will not be reflected in a simultaneous decay of B-Pb. Hence, our T 1/2 s of B-Pbs are fully compatible with both the earlier reports on B-Pb and our T 1/2 s for P-Pb. Also, the non-linear B-Pb/P-Pb relationship means that the B-Pb/P-Pb ratio will differ between individuals and over time.
In spite of the time to diagnosis being long in some of the cases, the modelling resulted in estimates of both the B-Pb and the P-Pb content at t = 0, which marked the actual end of exposure.
Poisoning with gastrointestinal, neurological and haematological symptoms and signs occurred at P-Pbs of about 20 lg/L as indicated by extrapolation of the P-Pb curves. In one of the cases (no. 4), the P-Pb at diagnosis was much lower. However, we are less certain of the relevance, since the symptoms and signs were less convincing for intoxication.
The present data clearly show the well-known anaemic effect of Pb exposure [bib_ref] Plasma-lead concentration: investigations into its usefulness for biological monitoring of occupational lead..., Bergdahl [/bib_ref]. Previous authors have described the relationship between exposure and B-Hb by use of B-Pb as a biomarker [bib_ref] Assessment of thyroid, testes, kidney and autonomic nervous system function in leadexposed..., Gennart [/bib_ref]. However, this may lead to spurious results, because the effect causes a decrease of the assumed indicator of exposure/risk, caused by the anaemia-induced decrease of binding possibilities for Pb in blood, and the saturation of binding sites. Our data clearly show the usefulness of P-Pb as an indicator of the risk of haematological effects. The shape of the B-Hb/P-Pb seemed to have at least two components. This is probably because, as said above, Pb has several different modes of action: inhibition of haem synthesis, inhibition of nucleotide synthesis and haemolysis. The present data does not allow allocation of these mechanisms to the B-Hb/P-Pb curve, but it is obvious that there is a dramatic effect at a P-Pb of about 5 lg/L. Interestingly, Case 5, who was the only heterozygote for ALAD G379C, had the longest T 1/2 for B-Pb, as compared to the others, who were homozygote for the C-allele, while he did not differ from the others in P-Pb kinetics. Also, he had a higher B-Pb/P-Pb ratio and higher initial B-Pb, which is in accordance with earlier findings [bib_ref] Delta-aminolevulinic acid dehydratase polymorphism: influence on lead levels and kidney function in..., Bergdahl [/bib_ref] [bib_ref] Effect of the delta-aminolevulinate dehydratase polymorphism on the accumulation of lead in..., Fleming [/bib_ref] [bib_ref] Associations of blood lead, dimercaptosuccinic acid-chelatable lead, and tibia lead with polymorphisms..., Schwartz [/bib_ref] [bib_ref] A polymorphism in the delta-aminolevulinic acid dehydratase gene modifies plasma/whole blood lead..., Montenegro [/bib_ref]. However, the high B-Pb observed may be due to a higher exposure, compared to the other cases.
# Conclusions
The present B-Pbs at onset of poisoning are high, well above occupational and other biological exposure limits. However, the present results are still relevant for evaluation of cases of poisoning. It is then important to consider that B-Pb, despite being one of the most used toxicological biomarkers all kind, has serious limitations because of the saturation at high exposure. Then, P-Pb is a more adequate biomarker of Pb exposure and risk than B-Pb, which is in accordance with a closer association between P-Pb and markers of haem synthesis, as compared to B-Pb, especially at high exposure [bib_ref] Correlation between lead in plasma and other indicators of lead exposure among..., Hirata [/bib_ref]. P-Pb at severe poisoning was about 20 lg/L. Biological half-time of P-Pb was about 1 month; whole blood decay was much slower. The ALAD genotype seemed to modify the toxicokinetics (higher level and slower elimination in whole blood), though only one of our cases was a heterozygote.
[fig] M: ale, F Female. ? to ??? denotes severity of clinical symptoms/signs, -lack of such a Based on intake of and level in juice eluted for 8 h. In standard procedure with 2% acetic acid for 24 h were the levels 150- [/fig]
[fig] Figure 1: Lead elimination from plasma (P-Pb; a) and whole blood (B-Pb; b) during the first 800 days after end of exposure in five cases [/fig]
[fig] CFigure 2, Figure 3 1: and C 2 are concentrations at t = 0 for the fast and slow components. T 1/2 half-time. CI 95% confidence interval Relationship between lead levels in whole blood (B-Pb) and plasma (P-Pb) in sequential samples from five cases of poisoning P-Pb (µg/L) Relationship between lead levels in urine (U-Pb) and plasma (P-Pb) in sequential samples from five cases of poisoning. The association between U-Pb and P-Pb follows the equation U-Pb = 12 ? 22*P [/fig]
[table] Table 1: Histories of five cases of lead poisoning [/table]
[table] Table 2: Two-compartment modelling of lead in plasma and whole blood after end of exposure in five cases of lead poisoning [/table]
|
Hurler–Scheie syndrome in Niger: a case series
Background: Hurler-Scheie syndrome is an intermediate form of mucopolysaccharidosis type I which is a rare lysosomal storage disorder caused by the deficiency or complete absence of enzyme alpha-L-iduronidase activity. We report the first documented cases of Hurler-Scheie syndrome observed in Niger in a Touareg family. Case presentation: We studied the case of two 12-year-old twin Touareg boys and their 10-year-old Touareg sister whose parents are first-degree cousins, and there was no history of similar cases in their previous generations. The diagnosis of Hurler-Scheie syndrome was considered in these patients on the basis of clinical and radiological arguments, with the highlighting of a deficiency of enzyme alpha-L-iduronidase in serum and leukocytes. The twins had presented the first symptoms at the age of 24 months and the diagnosis of Hurler-Scheie syndrome was made at the age of 12 years. In their younger sister, the first symptoms were observed at the age of 3 years and the diagnosis was made at the age of 10 years. The three probands were born after a normal full-term pregnancy and a spontaneous vaginal delivery according to their parents. Their birth weight, height, and head circumference were within normal limits according to their parents. The three probands were brought in for consultation for stunted growth, joint stiffness with gait disorders, deformities of the thoracolumbar spine, recurrent otitis media, decreased hearing, increased abdominal volume, snoring during sleep, and facial dysmorphism. Conclusions: Even in countries with limited access to diagnostic means, a good knowledge of the clinical manifestations of the disease can help to guide the diagnosis of mucopolysaccharidosis type I.
# Background
Hurler-Scheie syndrome is an intermediate form of mucopolysaccharidosis type I (MPS I) which is a rare autosomal recessive lysosomal storage disorder caused by mutations in the alpha-L-iduronidase gene, responsible for a deficiency or complete absence of enzyme alpha-L-iduronidase activity [bib_ref] The MPS I registry: design, methodology, and early findings of a global..., Pastores [/bib_ref] [bib_ref] The mucopolysaccharidoses: a success of molecular medicine, Clarke [/bib_ref]. It results, therefore, in a progressive intracellular accumulation of non-metabolized glycosaminoglycans (dermatan-sulfate and heparan-sulfate), which is responsible for the multiorganic damage. The estimated prevalence of attenuated forms of MPS I (Hurler-Scheie and Scheie syndromes) is 1 in every 500,000 live births. We report the first documented cases of Hurler-Scheie syndrome observed in Niger in a Touareg family.
## Case presentation
The probands are two 12-year-old Touareg boys who are twins (patient IV/3 and patient IV/4, [fig_ref] Figure 1: Family [/fig_ref] and their 10-year-old Touareg sister (patient IV/5, [fig_ref] Figure 1: Family [/fig_ref]. The three probands were born after a normal full-term pregnancy and a spontaneous vaginal delivery according to their parents who are first-degree cousins. Their birth weight, height, and head circumference were within normal limits according to their parents. The patients IV/3 and IV/4 were brought in for a consultation by their parents for stunted growth, joint stiffness with gait disorders, and deformities of the thoracolumbar spine. The first symptoms were increased abdominal volume, recurrent otitis media, umbilical hernia, and bronchial congestion with snoring during sleep, observed at the age of 24 months. Over time, the parents also noticed increased head volume, stunted growth, deformity of the spine, and decreased vision and hearing. Both patients also suffered from joint stiffness with a limitation of knee and shoulder movements and difficulty walking. They had a significant decrease in their walking perimeter due to joint stiffness and dyspnea of effort. Patient IV/3 had lost the ability to walk at the age of 11 years. Both patients did not have language disorders. They had slight intellectual disturbances. Their clinical examination found a short size of 103 cm, a facial dysmorphism with a short neck and micrognathia. They also had thoracolumbar kyphosis, and a prominent abdomen with hepatosplenomegaly and umbilical hernia. They had stiff and painful joints with limitation of active movements of abduction and antepulsion of the shoulders. Walking was impossible in patient IV/3 without human help. Patient IV/4 had paralysis of the right external oculomotor nerve. An audiogram found in both twins mixed bilateral deafness. An ophthalmological examination revealed a moderate decrease in visual acuity with corneal clouding in both twins. Cardiac auscultation revealed a mitral systolic murmur in patient IV/3. An electrocardiogram showed sinus tachycardia with signs of ventricular and atrial hypertrophy. Echocardiography showed mitral and aortic insufficiency in patient IV/3 and mitral insufficiency in patient IV/4. A chest X-ray showed cardiomegaly in both twins. An abdominopelvic ultrasound showed homogeneous splenomegaly and hepatomegaly in both twins. Skeletal X-rays showed anterosuperior hypoplasia of the vertebrae D12, L1, and L2 with kyphosis, and a conical aspect of the distal ends of the phalanges [fig_ref] Figure 2: Skeletal X-rays showing anterosuperior hypoplasia of the vertebrae D12, L1, and L2... [/fig_ref]. A cerebral computed tomography (CT) scan and brain magnetic resonance imaging (MRI) showed quadriventricular hydrocephalus with leukoaraiosis in both twins and an occipital arachnoid cyst in patient IV/4. Spinal cord MRI showed no particular abnormality. In the patient IV/5, the first symptoms were observed at the age of 3 years. She was brought in for a consultation at the age of 10 years at our request for a clinical evaluation. Her clinical history is similar to that of her older brothers but of less severity. Her clinical examination found a small size at 113 cm, a short neck, facial dysmorphism, prominent abdomen with hepatosplenomegaly, and umbilical hernia, without deformity of the spine. She did not have intellectual disturbances or language disorders. An ophthalmological examination found corneal clouding.
Considering the clinical history, clinical examination, and paraclinical examinations of our patients, a diagnosis of MPS I was suspected. Measurement of enzyme alpha-Liduronidase activity (realized in France) highlighted a deep deficiency of enzyme alpha-L-iduronidase in serum and leukocytes. The search for mutations or deletions in the alpha-L-iduronidase gene has not been performed in our patients, as well as the measurement of urinary glycosaminoglycans. At the end of all examinations, the diagnosis of Hurler-Scheie syndrome was considered.
# Discussion
In this case series, we report the first documented cases of MPS observed in Niger in a Touareg family from the Agadez region, which is the northern part of Niger. The clinical history, radiological abnormalities of the skeleton, and deficiency of enzyme alpha-L-iduronidase allowed us to consider in our patients the diagnosis of the intermediate form of MPS I (Hurler-Scheie syndrome).
The attenuated forms of MPS I such as Hurler-Scheie and Scheie syndromes, are characterized clinically by later onset of symptoms, longer life expectancy, and mild or no central nervous system involvement. In Hurler-Scheie syndrome, the first symptoms of the disease usually appear after the age of 2 years with a median age of diagnosis from 4 years [bib_ref] The natural history of MPS I: global perspectives from the MPS I..., Beck [/bib_ref]. In our case series, the age at symptoms onset was 2 years in the twins (probands IV/3 and IV/4) and 3 years in their younger sister (proband IV/5) with the diagnosis age of 12 years in the twins and 10 years in proband IV/5. The clinical manifestations of Hurler-Scheie syndrome are diverse and include musculoskeletal manifestations (kyphosis, scoliosis, kyphoscoliosis, back pain, dysostosis multiplex, joint stiffness, valgus and varus deformities, and so on), respiratory and pulmonary manifestations (obstructive sleep apnea, asthma, snoring, recurrent bronchitis, and so on), coarse facial features, short neck, stunted growth of variable degree, macrocephaly, hepatosplenomegaly of variable degree, umbilical and inguinal hernias, ophthalmologic manifestations (glaucoma, optic atrophy, retinal degeneration, corneal clouding, blindness, and so on), cardiac manifestations (valvular disease, coronary artery disease, congestive heart failure, cardiomyopathy, myocardial infarction, and so on), otorhinolaryngological manifestations (chronic recurrent rhinitis, chronic recurrent otitis media, chronic sinus infections, hearing loss of variable degree, and so on), and neurological manifestations (myelopathy, hydrocephalus, carpal tunnel syndrome, and so on) [bib_ref] The natural history of MPS I: global perspectives from the MPS I..., Beck [/bib_ref] [bib_ref] Childhood onset of Scheie syndrome, the attenuated form of mucopolysaccharidosis I, Thomas [/bib_ref]. In Hurler-Scheie syndrome, the intellect is normal or nearly normal, as in the case of our patients. The diagnosis of MPS I should be suspected in any person with the above-mentioned clinical manifestations. In addition, coarse facial features and inguinal or umbilical hernia are early manifestations and the most prevalent symptoms in patients with Hurler and Hurler-Scheie phenotypes and should be considered early signs of a potential MPS I diagnosis [bib_ref] The natural history of MPS I: global perspectives from the MPS I..., Beck [/bib_ref]. Any suggestive clinical picture of MPS I must motivate the practitioner to practice the analysis of urinary glycosaminoglycans (heparan-sulfate and dermatan-sulfate) and the measurement of the lysosomal enzyme alpha-L-iduronidase activity. Thus, the diagnosis of MPS I is established in a proband with the suggestive clinical and excessive urinary excretion of glycosaminoglycans and either detection of deficient activity of the lysosomal enzyme alpha-L-iduronidase or identification of mutations or deletions in the alpha-L-iduronidase gene on molecular genetic testing [bib_ref] The MPS I registry: design, methodology, and early findings of a global..., Pastores [/bib_ref] [bib_ref] Diagnosis and treatment trends in mucopolysaccharidosis I: findings from the MPS I..., D'aco [/bib_ref]. The management of patients with MPS I includes both preventive treatment and the treatment of severe manifestations. Preventive therapy includes enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT). These treatments allow patients affected with MPS I to obtain substantial clinical benefit for many disease manifestations, such as hepatosplenomegaly, upper airway obstruction, sleep apnea, cardiac symptoms, and coarse facial features [bib_ref] The natural history of MPS I: global perspectives from the MPS I..., Beck [/bib_ref] [bib_ref] Childhood onset of Scheie syndrome, the attenuated form of mucopolysaccharidosis I, Thomas [/bib_ref] [bib_ref] Diagnosis and treatment trends in mucopolysaccharidosis I: findings from the MPS I..., D'aco [/bib_ref]. The therapeutic choice must be determined individually for each patient with MPS I. It should take into account the patient's age, disease phenotype, developmental quotient (DQ), severity of clinical disease, and potential for growth. shows the treatment algorithm for patients with a diagnosis of MPS I. However, these treatments are effective only when they are initiated prior to the onset of irreversible organic lesions [bib_ref] The natural history of MPS I: global perspectives from the MPS I..., Beck [/bib_ref] [bib_ref] Childhood onset of Scheie syndrome, the attenuated form of mucopolysaccharidosis I, Thomas [/bib_ref]. In our case series, ERT would have been proposed in our patients, but, unfortunately, this treatment could not be realized because of the limited means of the parents of the patients and the unavailability of this treatment in Niger. The treatment of severe manifestations consists of the medico-surgical management of musculoskeletal manifestations (median nerve decompression, reparation of deformities of the spine, reparation of varus and valgus deformities, and so on), neurological manifestations (ventriculoperitoneal shunting for hydrocephalus with headache or loss of vision, spinal cord decompression in patients with cervical subluxation, and so on), respiratory and pulmonary manifestations (tonsillectomy for snoring or coarse breathing, continuous positive airway pressure for sleep apnea, and so on), cardiac manifestations (valve replacement, medical treatment of congestive heart failure, and so on), repair for hernias, and so on [bib_ref] Mucopolysaccharidosis type-IS presenting with onset of carpal tunnel syndrome at adolescence, Bahadir [/bib_ref] [bib_ref] Airway-related symptoms and surgeries in patients with mucopolysaccharidosis I, Arn [/bib_ref] [bib_ref] Urgent resection of a giant left atrial appendage aneurysm and mitral valve..., Brazier [/bib_ref].
# Conclusions
The clinical manifestations of MPS I usually appear in childhood in Hurler-Scheie syndrome, and their early recognition can lead to earlier diagnosis and early initiation of treatment, which may in turn lead to better patient outcomes.
# Funding
This study did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
## Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions HA designed the study and he conducted the recruitment and clinical evaluation of the patients. MTD conducted a literature search and he wrote the manuscript in its entirety. HB performed for all patients an echocardiogram and has been involved in revising the manuscript for important intellectual content. HA and FHD critically revised the manuscript. All authors approved the final version of the manuscript.
## Ethics approval and consent to participate
The current study respected the ethical principles depicted in the Declaration of Helsinki, and it was approved by the Institutional Review Board of the Faculty of Medicine of Abdou Moumouni University of Niamey (Niger). Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. And this was also approved by the Institutional Review Board. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
## Consent for publication
Written informed consent was obtained from the patients' next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
[fig] Figure 1: Family [/fig]
[fig] Figure 2: Skeletal X-rays showing anterosuperior hypoplasia of the vertebrae D12, L1, and L2 with kyphosis (a), and conical aspect of the distal ends of the phalanges (b) [/fig]
|
A Prospective Pilot Study to Validate the Management Protocol for Patients Presenting with Acute Urinary Retention: A Community-Based, Nonhospitalised Protocol
Acute urinary retention (AUR) in males is managed conventionally by hospital admission, alpha-adrenergic therapy, and trial without catheter. To reduce inpatient bed pressures, we set up a protocol to manage such patients in the community. We review our results in this paper.We performed a prospective study of male patients presenting to our acute admissions ward and Accident and Emergency department over 6 months. Patients with chronic urinary retention, macroscopic haematuria, sepsis, urinary tract infection, and/or serum creatinine >130 mmol/l were excluded from the study. Those enrolled were catheterised, commenced on alfuzosin (10 mg nocte), and discharged to the community. A trial without catheter (TWOC) was performed 5-7 days later. QoL/IPSS, peak flow rate, and residual volume assessment were performed following successful TWOC 3 months later.Thirty-one male patients with a median age of 69 years were studied and the median residual volume following catheterisation was 900 ml. The aetiology of AUR was benign prostatic hyperplasia (BPH) in 29 patients and constipation in the remaining 2 patients. TWOC was successful in 19 patients (61.3%) following first TWOC, 26 (83.9%) following second trial of voiding. The mean peak flow rate was 6.5 ml/sec and postvoid scan 165 ml, following an immediate TWOC. At 3 months follow-up, mean peak flow rate was 13.2 ml/sec, postvoid scan 26.5 ml, IPSS 4.5, and QoL score was 2.This study has shown that AUR can be managed safely and effectively in the community. Effective communication with the nurse urology specialist, general practitioner, and emergency department are crucial for the successful implementation of the protocol. . (2006) A prospective pilot study to validate the management protocol for patients presenting with acute urinary retention: a community-based, nonhospitalised protocol.
# Introduction
Benign prostatic hyperplasia (BPH) and lower urinary tract symptoms are common problems faced by aging men and affect up to 70% of men older than 60 years [bib_ref] Urodynamic assessment of patients with acute urinary retention: is treatment failure after..., Djavan [/bib_ref]. Clinical progression of BPH is defined by the development of acute urinary retention (AUR), renal insufficiency, recurrent urinary tract infection, incontinence, or an increase from baseline in the American Urological Association (AUA) symptom score index of 4 or more points. AUR is a common event in the natural history of BPH and may be the first manifestation of bladder outflow obstruction in up to 25% [bib_ref] Urodynamic assessment of patients with acute urinary retention: is treatment failure after..., Djavan [/bib_ref]. In Lytton's series of patients undergoing surgery, approximately 10-15% presented in acute retention [bib_ref] The incidence of benign prostatic hypertrophy, Lytton [/bib_ref]. AUR is both a significant public health issue and of consequence to the affected individual. The aetiology and pathogenesis of AUR are multifactorial. Several processes that are thought to initiate a sequence of events resulting in AUR include prostatic infarction, alpha-adrenergic overactivity, and neurotransmitter modulation [bib_ref] Natural history of prostatism. Risk factors for acute urinary retention, Jacobsen [/bib_ref]. Prostatitis, bladder overdistention, excessive fluid intake, medication, and debility have been identified as contributing factors [bib_ref] Acute urinary retention, Choong [/bib_ref]. Studies of the dynamics of AUR have shown that progressive stretching of smooth muscle fibre initially increases the contractile power and then, beyond a critical point, decreases it [bib_ref] Observations on the dynamics of acute urinary retention in man, Lawson [/bib_ref]. This is a known physiological property of smooth muscle. In AUR due to BPH, intravesical pressure falls and the pressure in the intraprostatic segment of the urethra is increased. Sympathetic activity is increased when overdistention of the bladder occurs. The administration of an alpha blocker reduces the pressure along the entire posterior urethra. Several studies have been published that emphasise the most effective way to treat AUR following initial catheterisation [bib_ref] Acute urinary retention, Choong [/bib_ref] [bib_ref] Trial without catheter following AUR urine, Taube [/bib_ref] [bib_ref] Sustained release of alfuzosin and trial without catheter after AUR. A prospective..., Mcneill [/bib_ref] , but these patients have been managed for several decades as inpatients in many centres, even with the recent trend of the use of alpha-adrenergic blocker therapy followed by a trial without catheter (TWOC). The use of alpha blockers followed by successful TWOC as an inpatient has proven benefits in reducing peri-and postoperative morbidity and mortality [bib_ref] Does prolonged catheter drainage improve the chance of recovering voluntary voiding after..., Djavan [/bib_ref] [bib_ref] Factors predisposing to bacteriuria during indwelling urethral catheterisation, Garibaldi [/bib_ref].
Following a review of the pathophysiology of BPH and AUR, we set up a prospective study to manage male patients presenting to the Accident and Emergency department (A&E) and acute admissions unit with first-time AUR. Our goals were to manage such patients in the community safely and effectively, to reduce inpatient bed pressures, and to suggest that alpha-blocker therapy and catheterisation for a sufficient period could improve the successful outcome of TWOC.
# Patients and methods
We performed a prospective study of patients presenting with AUR to our A&E department and acute admissions ward between June 2004 and December 2004. They were managed according to a protocol devised by the authors [fig_ref] FIGURE 1 successful: TWOC [/fig_ref]. Patients with chronic retention, macroscopic haematuria, sepsis, urinary tract infection, and abnormal serum creatinine (age/sex matched to the laboratory values) were excluded from the study. Those enrolled were catheterised, commenced on an alpha blocker (alfuzosin XL 10 mg nocte), and discharged home following an initial assessment including digital rectal examination (DRE) by the urologist on call. Verbal consent was obtained from all patients and they were evaluated by the nursing staff for safe discharge from the acute admissions unit or A&E. To ensure that the nurse practitioner and the general practioner (GP) received adequate information, the case notes from the A&E and the acute admissions ward were forwarded accordingly. The GP was advised to prescribe an alternative alpha blocker if the patient was intolerant to alfuzosin. The urology nurse specialist performed a TWOC 5-7 days later in the urology assessment unit. The successful outcome of TWOC was decided by an experienced urology nurse specialist who had obtained a degree in nursing/urological studies and was qualified by years of experience in conducting the nurse-led clinics for lower urinary tract symptoms. The patients who failed their first TWOC were either recatheterised or taught clean intermittent selfcatheterisation (CISC) prior to the second trial of voiding. An appointment for the second trial of voiding was given in 2-4 weeks. Flow rate and postvoid residual volume assessment were performed following
## Protocol for acute urinary retention in men
## First-time acute urinary retention in men
Catheterise with 14-16 Ch 2 way urethral catheter. Note residual volume and DRE finding in case notes. If urine analysis positive, send for dip slide. Inform district nurse and GP. Ward or A&E to bleep urology nurse specialist between 9 am and 5 pm or fax details if referral is out of hours. Appointment for trial of voiding to be organised between 5 and 7 days at nurse urology clinic.
## Criteria for immediate discharge
Trial Without Catheter followed by flow rate and postvoid scan. Repeat DRE if necessary. Serum PSA to be requested if appropriate at a later date.
## Successful unsuccessful
Review at outpatient clinic in 3 months. Repeat flow test, postvoid scan, IPSS and QoL score.
To contact the on-call urology team for further advice and management.
# Results
This prospective study over a 6-month period involved 31 male patients.The aetiology of AUR was BPH in 29 patients and constipation in the remaining 2 patients. Median age was 69 years (range 44-85 years). The median residual volume was 900 ml (range 300-1100 ml). Following commencement of alpha blocker (alfuzosin XL 10 mg nocte), TWOC was successful in 19 of the 31 patients (61.3%) following the first TWOC. The second flow test with a successful void rose to a further 26 of the 31 patients (83.8%) [fig_ref] TABLE 2 Immediate: Outcome of TWOC [/fig_ref]. Following the first TWOC, median peak flow rate (Qmax) was 6.5 ml/sec (range 3-25); median postvoid residual was 165 ml (range 21-500 ml). At 3 months follow-up, IPSS was 4.5 (range 2-11), QoL was 2 (range 1-2), median peak flow increased to 13.2 (range 4-27), postvoid residual reduced to 26.5 mls (range 0-450) . Of the remaining five patients who were unable to have a successful void, one patient had significant comorbidity and patient preference resulted in long-term catheter, one patient preferred to be managed by CISC for incomplete bladder emptying, and three patients underwent bladder outlet surgery within 3 months.
## Table 3 3 months follow-up data of the 26 patients who had a successful voiding
None of the patients needed overnight hospital admission. Patient compliance with alpha blocker was good with no side effects reported. None of the patients encountered difficulty in managing the catheter and leg bag (see [fig_ref] TABLE 4: Complications and Outcomes of Unsuccessful TWOC [/fig_ref].1100
Long-term catheter
# Discussion
The paper focuses on managing AUR in the community. Studies have shown that catheter trial performed immediately had a success rate of 44%, 48 h later recorded a 51% success, and patients benefited the most with a TWOC after 1 week (62%). Younger age group, residual volume of less than 1 l, and increased period of drainage [bib_ref] Trial without catheter following AUR urine, Taube [/bib_ref] [bib_ref] Does prolonged catheter drainage improve the chance of recovering voluntary voiding after..., Djavan [/bib_ref] were associated with a better outcome of TWOC. A long-term follow-up and benefits related to alpha blocker and TWOC has not been observed.
Our study showed that none of the patients needed overnight hospital admission. None came back for readmission with problems during the study period. The study highlights the importance of a multidisciplinary approach and effective communication with patient, district nurse, and GPs in helping to keep patients presenting with uncomplicated AUR out of the hospital. We followed a strict protocol in our study and with sufficient period of bladder drainage with alpha blocker, the percentage of successful TWOC has been encouraging. It also indicates that a second trial of voiding has proved beneficial for a few patients and the outcome has deferred or delayed surgery in 83.8% of the men who presented with AUR. This has facilitated preoperative assessment of the patient and thereby reduced the morbidity associated with the surgery performed on patients with indwelling catheter [bib_ref] Factors predisposing to bacteriuria during indwelling urethral catheterisation, Garibaldi [/bib_ref] [bib_ref] Risk factors in prostatectomy bleeding: preoperative urinary infection is the only reversible..., Elmalik [/bib_ref].
In conclusion, this study has shown that AUR can be managed in the community safely and effectively. The protocol has also paved the way to reduce inpatient bed pressure. The outcome of TWOC
[fig] FIGURE 1 successful: TWOC. Three months later, a repeat flow rate, postvoid scan, DRE, and Quality of Life (QoL)/International Prostate Symptom Score (IPSS) were performed. Serum prostate specific antigen (PSA) was measured, if indicated, at the 3 months follow-up. The urology team was contacted for further advice and management if the patient had an unsuccessful TWOC. [/fig]
[table] TABLE 2 Immediate: Outcome of TWOC [/table]
[table] TABLE 4: Complications and Outcomes of Unsuccessful TWOC [/table]
[table] TABLE 5: Unsuccessful TWOC in Relation to Age and Postcatheterisation Volume [/table]
|
Use of low-dose oral theophylline as an adjunct to inhaled corticosteroids in preventing exacerbations of chronic obstructive pulmonary disease: study protocol for a randomised controlled trial
Background: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and health-care costs. An incomplete response to the anti-inflammatory effects of inhaled corticosteroids is present in COPD. Preclinical work indicates that 'low dose' theophylline improves steroid responsiveness. The Theophylline With Inhaled Corticosteroids (TWICS) trial investigates whether the addition of 'low dose' theophylline to inhaled corticosteroids has clinical and cost-effective benefits in COPD.Discussion:The demonstration that 'low dose' theophylline increases the efficacy of inhaled corticosteroids in COPD by reducing the incidence of exacerbations is relevant not only to patients and clinicians but also to health-care providers, both in the UK and globally.BackgroundChronic obstructive pulmonary disease (COPD) is a lung disease characterised by progressive airflow obstruction that is not fully reversible and does not change markedly over several months[1]. COPD is common, is caused predominantly by cigarette smoking, and is usually diagnosed from the age of 50 years onwards. In the UK, there arenearly one million diagnosed cases, and COPD accounts for 5-6 % of all deaths (about 28,000 deaths in 2012) [2]. COPD is typically associated with increasing breathlessness on exertion, disability, work absence, premature retirement, morbidity, psychological co-morbidities, reduced quality of life, and premature mortality[1,3,4]. COPD is associated with high healthcare expenditure: in the UK, National Health Service (NHS) expenditure is about £1 billion per year; for each patient with COPD, the average annual NHS direct costs are £819 (more than £1,300 in severe COPD)[5].Exacerbations are an important clinical feature of COPD. These are usually precipitated by viral infection or air pollution and are characterised by increasing dyspnoea, cough, sputum expectoration, and malaise. Exacerbations are associated with accelerated lung function decline, reduced physical activity, reduced quality of life, and increased mortality[6][7][8][9]. Approximately 15 % of patients with COPD are hospitalised with exacerbations each year. Exacerbations are the second leading cause for emergency hospital admission and account for 60 % of the total direct costs of COPD to the NHS [1, 5]. Typically, 12 % of patients with COPD die in the year following hospitalisation with an exacerbation[9].Most COPD management guidelines recommend the use of inhaled corticosteroids usually in combination with inhaled long-acting β 2 agonists to reduce COPD exacerbation rates[1,10]. However, the responses observed in COPD are not as marked as in asthma; inhaled corticosteroids do not fully suppress airway inflammation in COPD, and patients continue to have exacerbations despite high inhaled corticosteroid doses[11]. Although inhaled corticosteroids are beneficial in COPD, the airway inflammation in COPD is relatively insensitive to their anti-inflammatory effects even at high doses[12][13][14].Oral theophylline has been used conventionally as a bronchodilator in COPD for over 70 years; however, to achieve modest clinical effects, relatively high blood
Method/Design: TWICS is a randomised double-blind placebo-controlled trial conducted in primary and secondary care sites in the UK. The inclusion criteria are the following: an established predominant respiratory diagnosis of COPD (post-bronchodilator forced expiratory volume in first second/forced vital capacity [FEV 1 /FVC] of less than 0.7), age of at least 40 years, smoking history of at least 10 pack-years, current inhaled corticosteroid use, and history of at least two exacerbations requiring treatment with antibiotics or oral corticosteroids in the previous year. A computerised randomisation system will stratify 1424 participants by region and recruitment setting (primary and secondary) and then randomly assign with equal probability to intervention or control arms. Participants will receive either 'low dose' theophylline (Uniphyllin MR 200 mg tablets) or placebo for 52 weeks. Dosing is based on pharmacokinetic modelling to achieve a steady-state serum theophylline of 1-5 mg/l. A dose of theophylline MR 200 mg once daily (or placebo once daily) will be taken by participants who do not smoke or participants who smoke but have an ideal body weight (IBW) of not more than 60 kg. A dose of theophylline MR 200 mg twice daily (or placebo twice daily) will be taken by participants who smoke and have an IBW of more than 60 kg. Participants will be reviewed at recruitment and after 6 and 12 months. The primary outcome is the total number of participant-reported COPD exacerbations requiring oral corticosteroids or antibiotics during the 52-week treatment period.
(Continued on next page) levels (10-20 mg/l) with clinical monitoring are required. Unfortunately, the therapeutic index of 'high dose' theophylline is narrow. Prior use at 'high dose' as a bronchodilator frequently resulted in drug concentrations close to those where side effects were encountered, namely nausea, gastrointestinal upset, cardiac arrhythmias, and malaise. Not surprisingly, use of 'high dose' theophylline has declined in recent years and is being replaced by better-tolerated inhaled bronchodilator therapies. Recently, however, preclinical studies have demonstrated that theophylline at 'low dose' (plasma concentration of 1-5 mg/l) increases the sensitivity of COPD airway inflammation to the anti-inflammatory effects of inhaled corticosteroids [bib_ref] Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation..., Ito [/bib_ref] [bib_ref] Decreased histone deacetylase activity in chronic obstructive pulmonary disease, Ito [/bib_ref] [bib_ref] Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression, Ito [/bib_ref] [bib_ref] Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary..., Barnes [/bib_ref] [bib_ref] Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice, Marwick [/bib_ref] [bib_ref] A molecular mechanism of action of theophylline: Induction of histone deacetylase activity..., Ito [/bib_ref] [bib_ref] Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages, Cosio [/bib_ref] [bib_ref] Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease, To [/bib_ref]. The concept that 'low dose' theophylline may produce a beneficial synergistic effect by increasing the corticosteroid sensitivity in COPD is supported by two small randomised controlled trials with inflammatory indices as primary outcomes and a Canadian health administration database study [bib_ref] Low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of COPD, Cosio [/bib_ref] [bib_ref] Treatment effects of low-dose theophylline combined with an inhaled corticosteroid in COPD, Ford [/bib_ref] [bib_ref] Effect of theophylline on the rate of moderate to severe exacerbations among..., Cyr [/bib_ref]. The potential benefit of using 'low dose' theophylline to increase corticosteroid responsiveness in COPD is that, when used in combination with inhaled corticosteroids, it should reduce exacerbation rates. Moreover, 'low dose' theophylline is inexpensive and is anticipated to avoid the side effects encountered with conventional 'high dose' theophylline, making blood monitoring unnecessary.
Here, we describe the Theophylline With Inhaled Corticosteroids (TWICS) study, a randomised double-blind placebo-controlled trial that will test the hypothesis that in patients with COPD established on a treatment regimen including an inhaled corticosteroid, the addition of oral 'low dose' theophylline will reduce the rate of exacerbation. The full protocol is available as Additional file 1.
# Methods/design
Trial design TWICS is a double-blind randomised, placebo-controlled, UK multicentre clinical trial comparing the addition of 'low dose' theophylline or placebo for 52 weeks with current COPD therapy that includes inhaled corticosteroids in COPD patients who in the previous year have had two or more exacerbations of COPD treated with oral corticosteroids or antibiotics. [fig_ref] Figure 1: Flow diagram of study design and schedule [/fig_ref] provides a schematic representation of study design and schedule.
The trial is set in primary and secondary care sites in the UK. In primary care, some general practices act as recruitment sites, whereas others act as Participant Identification Centres with identified participants being evaluated in other primary or secondary care recruitment sites. The first subject was randomly assigned on Feb. 6, 2014. In total, 1424 eligible people with COPD will be randomly assigned to receive visually identical 'low dose' theophylline (Uniphyllin MR 200 mg once or twice daily) or placebo for 52 weeks. The trial is approved by Scotland A Research Ethics Committee (#13/SS/0081, /FVC] of less than 0.7), age of at least 40 years, a smoking history of at least 10 pack-years, current use of inhaled corticosteroid therapy, a history of at least two exacerbations requiring treatment with antibiotics or oral corticosteroids in the previous year (based on patient report). In addition, participants must be clinically stable at the time of recruitment (no COPD exacerbation for at least 4 weeks).
The main exclusion criteria are current use of theophylline, hypersensitivity to theophylline, or use of drugs known to interact with theophylline or increase serum theophylline(for full list, see Additional file 1). Further exclusion criteria are a predominant respiratory disease other than COPD, unstable ischaemic heart disease, or any other clinically important disease/disorder which, in the investigator's opinion, either puts the participant at risk because of study participation or may influence the results of the study or the participant's ability to take part in the study. For women, current pregnancy or breast-feeding and planned pregnancy during the study are exclusion criteria.
## Identification, recruitment, and randomisation
Potential participants are recruited from both primary and secondary care across the UK with the aim of recruiting the majority of participants (more than 50 %) from primary care. Recruitment strategies differ between centres, depending on local geographic and NHS organisational factors.
In primary care, potential participants are identified from searches of general-practice databases. In some centres, COPD community matrons and other community-based intermediate care services for patients with COPD are available and are used to identify potential participants. In secondary care, potential participants are identified from those patients who are attending hospital respiratory out-patient clinics, spirometry services, or smoking cessation services. Some trial centres also have access to volunteer databases/registries. Potential participants are sent an invitation letter and a patient information leaflet informing them of the trial aims and level of involvement required. The letter provides a variety of methods for interested potential participants to contact the local trial team.
At the recruitment/baseline visit at the local trial centre, eligibility is established, the trial explained, questions addressed, and informed written consent provided by the participant.
Participants are randomly assigned by using a computerised randomisation system available as both an interactive voice response telephone system and an internet-based application. The randomisation service is administered by the Centre for Healthcare Randomised Trials (CHaRT), University of Aberdeen. Consenting patients will be stratified by region of trial centre and recruitment setting (primary and secondary) and then randomly assigned with equal probability to the intervention and control arms.
## Intervention
The active intervention is Uniphyllin MR 200 mg tablets and a visually identical control placebo. The packaging and labelling of active and placebo interventions are identical. Intervention is for 52 weeks of therapy.
'Low dose' theophylline dosing is based on pharmacokinetic modelling of theophylline and incorporates the major determinants of theophylline steady-state concentration (i.e., weight, smoking status, and theophylline clearance [bib_ref] Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials, Barr [/bib_ref] [bib_ref] Theophylline disposition in acutely ill hospitalized patients. The effect of smoking, heart..., Powell [/bib_ref] and is designed to achieve a steadystate serum theophylline level of 1-5 mg/l and certainly less than 10 mg/l (more than 10 mg is the level associated with 'high dose' theophylline, possible side effects, and augmentation of corticosteroid insensitivity) (Appendix 1 of the Additional file 1). Dosing is determined by the participant's ideal body weight (IBW) and self-reported smoking status. IBW is computed by using the Devine formulae: IBW female = 45 + 0.9 (height in cm -152) kg, and IBW male = 50 + 0.9 (height in cm -152) kg [bib_ref] Comparison of ideal body weight equations and published height-weight tables with body..., Shah [/bib_ref].
A dose of theophylline MR 200 mg once daily (one placebo once daily) is taken by participants who do not smoke or participants who smoke but have an IBW of not more than 60 kg. A dose of theophylline MR 200 mg twice daily (one placebo twice daily) is taken by participants who smoke and have an IBW of more than 60 kg.
To be classed as a "non-smoker", a participant must have abstained from smoking for at least 12 weeks. If less than the IBW, the actual body weight is used to determine dose. When informed of their patient's participation in the trial, general practitioners are advised to manage their patient for exacerbations as in normal clinical practice but to assume that the participant is taking low-dose theophylline, and the prescription of interacting drugs that increase serum theophylline levels should be avoided. In the event that drugs that interact to increase theophylline concentration have to be prescribed for 3 weeks or less, patients are asked to suspend taking study medication and recommence their study medication after the course of interacting drug has been completed. If the interacting drug is prescribed for more than 3 weeks, participants discontinue the study medication but remain in the study and are followed up in accordance with the trial protocol.
In secondary care trial sites, the first pack of 4-week study medication is dispensed from the local Clinical Trials Pharmacy. In primary care trial sites, the first pack of study medication is couriered to the participant's home from the lead Clinical Trials Pharmacy in Aberdeen. Subsequent packs containing a 24-week drug supply are delivered to the participants' homes at around weeks 3 and 27 via a signed-for delivery service operated by a third party. Receipt of trial medication is confirmed by signature on delivery. Written informed consent to pass on a participant's name and address to the third-party distributer is obtained at recruitment.
## Primary outcome variables
The primary outcome measure is the total number of exacerbations of COPD necessitating changes in management (minimum management change-use of oral corticosteroids or antibiotics) during the 52-week treatment period, as reported by the participant. The primary economic outcome measure is cost per quality-adjusted life year (QALY) gained during the 52-week treatment period.
## Secondary outcome variables
During the 52-week treatment period, the secondary outcomes are the following: number of participant-reported COPD exacerbations requiring hospital admission, number of episodes of pneumonia, number of emergency hospital admissions (all causes), postbronchodilator lung function (FEV 1 FVC), all-cause and respiratory mortality, serious adverse events, adverse reactions, total dose of inhaled corticosteroid, utilisation of primary or secondary health care for respiratory events, disease-specific health status (COPD Assessment Test, or CAT), Medical Research Council (MRC) dyspnoea scale [bib_ref] Standardised questionnaire on respiratory symptoms: a statement prepared and approved by the..., Fletcher [/bib_ref] , generic health-related quality of life (EQ-5D), and modelled lifetime incremental cost per QALY.
## Follow-up and data collection
Participants are reviewed at face-to-face assessments at recruitment and after 6 and 12 months of treatment. Approximately 2 weeks after enrolment, participants are contacted by telephone to ascertain whether they are tolerating the study intervention.
In the event that a participant is unable to attend a scheduled follow-up assessment visit because of an acute illness (e.g., exacerbation of COPD) or other reasons, the visit can be postponed, ideally within 4 weeks of the scheduled assessment visit. Participants unable to attend a face-to-face assessment at 6 and 12 months are followed up by telephone or a home visit or sent the questionnaire to complete at home. The schedule for data collection within the study is outlined in [fig_ref] Table 1: Schedule of study assessments X boxes represent which aspect of the assessment... [/fig_ref].
The following data are collected:
## Drug history
Regular use of prescription drugs is recorded at recruitment as free text and at the 6-and 12-month assessments.
## Smoking history
Smoking history (age commenced, age ceased, and average cigarettes smoked per day) is recorded at recruitment and at the 6-and 12-month assessments. Packyear consumption is computed at recruitment.
## Height and weight
Height and weight are measured at recruitment, and weight is measured at the 6-and 12-month assessments.
## Number of chronic obstructive pulmonary disease exacerbations
The primary outcome measure of the total number of COPD exacerbations requiring antibiotics/oral corticosteroids whilst on study medication is ascertained at the 6-and 12-month assessments. The total number of participant-reported COPD exacerbations will be validated for approximately 20 % of randomly identified participants by examination of primary care records after the study. Concordance between participant-recorded and primary care records will be computed by using percentage agreement, and more than 80 % will be deemed acceptable [bib_ref] Predictive accuracy of patient-reported exacerbation frequency in COPD, Quint [/bib_ref]. The American Thoracic Society/European Respiratory Society guideline definition of COPD exacerbation is used: a worsening of patient's dyspnoea, cough, or sputum beyond day-to-day variability sufficient to warrant a change in [bib_ref] Standards for the diagnosis and treatment of patients with COPD: a summary..., Celli [/bib_ref]. The minimum management change will be treatment with antibiotics or oral corticosteroids. A minimum of 2 weeks between consecutive hospitalisations/ start of new therapy is necessary to consider events as separate. A modified American Thoracic Society/European Respiratory Society operational classification of exacerbation severity will be used for each exacerbation: level I, increased use of their short-acting β 2 agonist; level II, use of oral corticosteroids or antibiotics; level III, care by services to prevent hospitalisation; level IV, admitted to hospital [bib_ref] Standards for the diagnosis and treatment of patients with COPD: a summary..., Celli [/bib_ref].
## Hospital admissions
The number of emergency hospital admissions whilst on study medication is ascertained at the 6-and 12-month assessments. COPD-associated emergency admissions are also identified. The number of participant-reported hospital admissions will be validated for a randomly identified sample of 20 % of participants by examination of primary and secondary care records after the study. Concordance between participant-recorded and primary/secondary care records will be computed by using percentage agreement, and more than 80 % will be deemed acceptable [bib_ref] Predictive accuracy of patient-reported exacerbation frequency in COPD, Quint [/bib_ref].
## Health-related quality of life
Health-related quality of life data are captured at recruitment and at the 6-and 12-month assessments by questionnaire using the EuroQoL 5D (EQ-5D 3-level version) Index, which has been used widely in COPD. The completed instrument can be translated into quality-oflife utilities suitable for calculation of QALYs through the published UK tariffs [bib_ref] Modelling valuations for health states, Dolan [/bib_ref].
# Disease-related health status
Disease-related health status is ascertained at recruitment and at the 6-and 12-month assessments by questionnaire using the CAT. The CAT is an eight-item unidimensional measure of health status impairment in COPD and is completed by the subject. The CAT has a scoring interval of 0-40; 0-5 is the norm for healthy non-smokers, and more than 30 is indicative of a very high impact of COPD on quality of life. The CAT is reliable and responsive, correlates very closely with the St George Respiratory Questionnaire, and is preferred because it provides a more comprehensive assessment of the symptomatic impact of COPD [bib_ref] The COPD assessment test (CAT): response to pulmonary rehabilitation. A multicentre, prospective..., Dodd [/bib_ref] [bib_ref] Development and first validation of the COPD assessment test, Jones [/bib_ref]. The MRC dyspnoea scale is included in the recruitment and the 6-and 12-month assessments [bib_ref] Standardised questionnaire on respiratory symptoms: a statement prepared and approved by the..., Fletcher [/bib_ref]. The MRC dyspnoea scale has been in use for many years to grade the effect of breathlessness on daily activities. The MRC dyspnoea scale is a single question which assesses breathlessness related to activities. The scoring interval is 1-5; 1 refers to 'Not troubled by breathlessness except on strenuous exercise' , and 5 indicates 'Too breathless to leave the house or breathless when dressing or undressing'. The MRC score has been validated against walking test performance and other metrics of COPD health status (e.g., St George Respiratory Questionnaire [bib_ref] Usefulness of the Medical Research Council (MRC) dyspnoea scale as a measure..., Bestall [/bib_ref].
## Post-bronchodilator lung function
Post-bronchodilator lung function is measured at recruitment and 6 and 12 months by using spirometry performed to American Thoracic Society/European Respiratory Society standards [bib_ref] ATS/ERS task force. Standardisation of spirometry, Miller [/bib_ref].
## Health-care utilisation
In keeping with the NHS perspective adopted for the economic analysis, health-care utilisation is the focus of the costing for the study. This includes the study drug, concomitant medications, general practitioner visits, and any-cause hospitalisations during the previous 6 months and is ascertained at the 6-and 12-month assessments.
## Adverse reactions and serious adverse events
The trial complies with the UK NHS National Research Ethics Service guidelines for reporting adverse events. Adverse reactions and serious adverse events whilst on study medication are ascertained at the 2-week telephone call and the 6-and 12-month assessments. Participants are notified of recognised adverse reactions and encouraged to contact the local study centre if they experience these.
## Mortality
Deaths during the follow-up period are recorded and are reported as serious adverse events.
## Compliance
Compliance with study medication is assessed at the 6and 12-month assessments. Participants will be asked to return empty drug bottles and unused medication; compliance will be calculated by pill counting [bib_ref] Methods for measuring and monitoring medication regimen adherence in clinical trials and..., Farmer [/bib_ref].
## Sample size
The sample size of 1424 was estimated on the basis of the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study reporting the frequency of COPD exacerbation in 2138 patients [bib_ref] Evaluation of COPD longitudinally to identify predictive, surrogate endpoints. Susceptibility to exacerbation..., Hurst [/bib_ref]. For patients identical to our target population (who in a 1-year period have at least two self-reported COPD exacerbations requiring antibiotics or oral corticosteroids), the mean (standard deviation) number of COPD exacerbations within 1 year was 2.22 (1.86) [bib_ref] Evaluation of COPD longitudinally to identify predictive, surrogate endpoints. Susceptibility to exacerbation..., Hurst [/bib_ref]. Given a similar rate in the placebo arm, 669 subjects are needed in each arm of the trial to detect a clinically important reduction in COPD exacerbations of 15 % (i.e., from a mean of 2.22 to 1.89) with 90 % power at the two-sided 5 % significance level. With an estimated 6 % loss to follow-up, 712 participants are required in each study arm (i.e., 1424 in total).
# Statistical methods
All analyses will be governed by a comprehensive statistical analysis plan that is in place and will be in accordance with the intention-to-treat principle with a per-protocol analysis performed as a sensitivity. The per-protocol analysis will exclude participants who were not compliant (at less than 75 %) with their study medication.
## Primary clinical outcome
The number of COPD exacerbations requiring antibiotics or oral corticosteroids in the 52-week treatment period will be compared between randomised groups by using a generalised linear model with log-link function, an appropriate dispersion parameter, and length of time in the study as an offset. Estimates will be adjusted for centre and other baseline covariates that are known to be strongly related to outcome (e.g., age, smoking, and COPD hospitalisations in the year prior to study). An over-dispersion parameter will be used to adjust for between-patient variability.
## Economic evaluation
An NHS perspective will be adopted in keeping with the NICE reference case for health technology assessments. The health economic evaluation will be conducted in two stages. First, the cost-effectiveness of treatment will be calculated for the within-trial period on the basis of observed data. Second, the results of the trial will be extrapolated to patient lifetimes by using costeffectiveness modelling.
The within-trial analysis will make use of the healthcare resource use data (translated to a cost per patient by using unit cost standard reference sources), the exacerbation rate associated with the treatment arms, and the quality-of-life effects estimated from the EQ-5D combined with utility data to calculate QALYs. Nonparametric bootstrapping will be used to capture sampling uncertainty in the observed data, and results will be presented as cost per exacerbation avoided and cost per QALY gained within the trial period with accompanying confidence intervals (or cost-effectiveness acceptability curves if more appropriate). The extrapolation analysis will make use of regression estimates of exacerbation on cost and quality of life from the trial, as well as previously published models of COPD, to guide the extrapolation to patient lifetimes. In addition to sampling uncertainty, extensive sensitivity analysis will be performed to understand the importance of alternative modelling assumptions for the extrapolated results.
# Discussion
COPD is a common disease associated with high morbidity, mortality, and health-care costs despite the widespread use of inhaled corticosteroids. Although inhaled corticosteroids are beneficial in COPD, a relative insensitivity of COPD airway inflammation to the antiinflammatory effects of high-dose inhaled corticosteroids has been demonstrated [bib_ref] Effect of high dose inhaled steroid on cells, cytokines, and proteases in..., Culpitt [/bib_ref] [bib_ref] Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients..., Culpitt [/bib_ref] [bib_ref] The effects of inhaled fluticasone on airway inflammation in chronic obstructive pulmonary..., Hattotuwa [/bib_ref]. TWICS is a randomised double-blind placebo-controlled trial that tests the hypothesis: Does the addition of oral 'low dose' theophylline reduce the rate of exacerbation in patients with COPD established on a treatment regimen including an inhaled corticosteroid?
The primary outcome of COPD exacerbations is clinically important for patients, their carers, and health services; exacerbations of COPD are associated with many adverse outcomes, including mortality, and their management comprises 60 % of NHS expenditure for COPD [bib_ref] The burden of COPD in the U.K.: results from the confronting COPD..., Britton [/bib_ref]. To be eligible in TWICS, participants must have an established diagnosis of COPD on the basis of the spirometric finding of FEV 1 /FVC of less than 0.7 and of at least two exacerbations in the previous year. These criteria reflect the findings of the ECLIPSE study that the single best predictor of exacerbations is a history of exacerbations [bib_ref] Evaluation of COPD longitudinally to identify predictive, surrogate endpoints. Susceptibility to exacerbation..., Hurst [/bib_ref]. Moreover, patients of the frequent-exacerbation phenotype (of at least two exacerbations in a year) are present at all severities of COPD (22 % of GOLD stage 2, 33 % of stage 3, and 47 % of stage 4), and the frequent-exacerbation phenotype is relatively stable over a 3-year period and can be identified on the basis of patient recall.
It is almost certain that a substantial proportion of the participants in TWICS will have severe lung disease and will have limited exercise tolerance. Allowances have been made in the trial design to facilitate participation by this group of patients: at site discretion, participants can be recruited at home, and those unable to attend follow-up assessment visits will be assessed by telephone review and postal collection of quality-of-life questionnaires; the majority (at least 48 of 52 weeks) of study medication will be couriered directly to the homes of participants, thus avoiding travel to study centres to collect supplies.
Oral theophylline has conventionally been used primarily as a bronchodilator in COPD for over 70 years; however, to achieve modest clinical effects, relatively high blood levels (10-20 mg/l) are required. The bronchodilator effect of this 'high dose' theophylline is the consequence of inhibition of phosphodiesterase and consequent relaxation of airway smooth muscle; however, phosphodiesterase inhibition is also associated with the side effects of theophylline, namely nausea, gastrointestinal upset, cardiac arrhythmias, and malaise. The use of high-dose theophylline has declined in recent years, and current COPD guidelines have relegated high-dose theophylline to third-line therapy because of its narrow therapeutic index, modest clinical effect, side effect profile, drug interactions, the need for monitoring and the development of inhaled long-acting β 2 agonists, antimuscarinics, and the widespread use of inhaled corticosteroids. The use of 'low dose' theophylline derives from the demonstration by preclinical studies and two small randomised controlled trials that theophylline at 'low dose' (plasma concentration of 1-5 mg/l) increases the sensitivity of COPD airway inflammation to the anti-inflammatory effects of inhaled corticosteroids [bib_ref] Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation..., Ito [/bib_ref] [bib_ref] Decreased histone deacetylase activity in chronic obstructive pulmonary disease, Ito [/bib_ref] [bib_ref] Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression, Ito [/bib_ref] [bib_ref] Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary..., Barnes [/bib_ref] [bib_ref] Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice, Marwick [/bib_ref] [bib_ref] A molecular mechanism of action of theophylline: Induction of histone deacetylase activity..., Ito [/bib_ref] [bib_ref] Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages, Cosio [/bib_ref] [bib_ref] Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease, To [/bib_ref]. Previous studies have investigated the potential anti-inflammatory effects of 'low dose' theophylline in COPD and asthma (not in conjunction with inhaled corticosteroids). However, they have used a 'one size fits all' dosing approach (e.g., all participants received 100 mg twice daily or 200 mg twice daily) [bib_ref] A molecular mechanism of action of theophylline: Induction of histone deacetylase activity..., Ito [/bib_ref] [bib_ref] Low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of COPD, Cosio [/bib_ref] [bib_ref] Treatment effects of low-dose theophylline combined with an inhaled corticosteroid in COPD, Ford [/bib_ref] [bib_ref] Low-dose theophylline reduces eosinophilic inflammation but not exhaled nitric oxide in mild..., Lim [/bib_ref] [bib_ref] Anti-inflammatory effects of low-dose oral theophylline in atopic asthma, Sullivan [/bib_ref] [bib_ref] A prospective clinical study of theophylline safety in 3810 elderly with asthma..., Ohta [/bib_ref]. In contrast, in TWICS, theophylline dosing is stratified, as determined by IBW and smoking status. Population studies have demonstrated that theophylline pharmacokinetics are influenced by weight, COPD disease status (reduced clearance), and smoking (increased clearance) [bib_ref] Theophylline disposition in acutely ill hospitalized patients. The effect of smoking, heart..., Powell [/bib_ref] [bib_ref] Effect of smoking on theophylline disposition, Hunt [/bib_ref] [bib_ref] The influence of cigarette smoking and sex on theophylline disposition, Powell [/bib_ref] [bib_ref] Role of tobacco smoking in pharmacokinetics, Jusko [/bib_ref] [bib_ref] Enhanced biotransformation of theophylline in marihuana and tobacco smokers, Jusko [/bib_ref] [bib_ref] Disposition of theophylline after a single intravenous infusion of aminophylline, Hendeles [/bib_ref] [bib_ref] Theophylline kinetics in relation to age: the importance of smoking, Cusack [/bib_ref] [bib_ref] Dose response relation to oral theophylline in severe chronic obstructive airways disease, Chrystyn [/bib_ref] [bib_ref] The accuracy and stability of Bayesian theophylline predictions, Chrystyn [/bib_ref] [bib_ref] The accuracy of a pharmacokinetic theophylline predictor using once daily dosing, Chrystyn [/bib_ref] [bib_ref] Submit your next manuscript to BioMed Central and take full advantage of:..., Chrystyn [/bib_ref]. Smoking induces theophylline clearance by approximately 60 %, which gradually returns to normal levels upon smoking cessation, and this has been incorporated into the definition of a non-smoker in TWICS. The use of IBW in preference to actual weight avoids the potential for giving an inappropriately high dose of theophylline to obese participants; furthermore, use of IBW is good clinical practice. In TWICS, theophylline dosing is based on pharmacokinetic modelling incorporating the major determinants of theophylline steadystate concentration, i.e., weight, smoking status, and clearance of theophylline (low, normal, or high), and is designed to achieve a steady-state serum theophylline level of 1-5 mg/l and certainly less than 10 mg/l. Theophylline is metabolised in the liver by the enzyme CYP1A2, which is induced by smoking and inhibited by a number of medications with a consequent increase in serum theophylline levels. For this reason, the exclusion criteria include long-term use of drugs known to increase serum theophylline.
Theophylline in the form of intravenous aminophylline has been used in the treatment of severe acute exacerbations of COPD in hospital settings. However, research does not support this modality of treatment and this is reflected in guideline recommendations, and the use of intravenous aminophylline has rapidly declined. When used, intravenous aminophylline is usually administered as a loading dose followed by a maintenance infusion in patients not established on theophylline, and for patients established on theophylline, only the maintenance infusion is given because of toxicity concerns.
Inevitably, during TWICS, some participants will be admitted to hospital with very severe life-threatening exacerbations of COPD, and the attending physician may wish to use intravenous aminophylline. Pharmacokinetic modelling demonstrates that patients receiving 'low dose' theophylline will not achieve toxic levels of theophylline following the usual loading dose of aminophylline, because their baseline serum theophylline concentrations will vary between 1 and 5 mg/l; and after the loading dose of aminophylline, serum theophylline will remain within the conventional bronchodilating interval of 10-20 mg/l. This is clinically important as the attending physician will not be aware whether a TWICS participant is on theophylline or placebo, and the modelling confirms that an aminophylline infusion can be safely administered if thought by the attending physician to be clinically indicated. Study medication will be suspended whilst the participant receives intravenous aminophylline and restarted when the aminophylline discontinued. In keeping with guideline recommendations, serum theophylline will be measured 24 h after commencing intravenous aminophylline (allocation status will not be discernible from such a level). All participants are given (and advised to carry) a credit cardsized alert card giving brief information about the trial with advice for clinicians, contact details for the local investigator, and the contact details for emergency unblinding. The participant's primary care physician is informed of participation and provided with appropriate clinical advice.
Theophylline has been used for decades, and many clinicians are familiar with its use; moreover, 'low dose' theophylline is considerably less expensive than inhaled therapies and does not incur the costs of monitoring of blood levels. The demonstration that low-dose theophylline increases the efficacy of inhaled corticosteroids in COPD by reducing the incidence of exacerbations will be relevant not only to patients and clinicians but also to health-care providers, both in the UK and globally.
## Trial status
The first subject was recruited on February 6, 2014, and the trial is currently recruiting patients. The anticipated date of last participant assessment is October 2016.
## Additional file
Additional file 1: Title: TWICS study protocol. Description: Full TWICS study protocol. Competing interests PB is a co-founder of RespiVert (now part of Johnson & Johnson, New Brunswick, NJ, USA), which has developed new treatments of asthma and COPD on the basis of the mechanism of action of theophylline. He is on the Scientific Advisory Board of Napp Pharmaceuticals Limited (Cambridge, UK). The other authors declare that they have no competing interests.
Authors' contributions GD and DP helped to conceptualize and design the study and to obtain the funding and are co-chief investigators. SC helped to obtain the funding and is the senior trial manager. PB conceived of low-dose theophylline augmenting inhaled corticosteroids in COPD and helped to conceptualize and design the study and to obtain the funding. AB helped to conceptualize and design the study and to obtain the funding and is responsible for the health economics aspects. GB and SG helped to conceptualize and design the study. RC, LD, ADS, JH, AM, JN, AS, and AW helped to conceptualize and design the study and to obtain the funding. HC helped to conceptualize and design the study and to obtain the funding and conducted the pharmacokinetic modelling. SF shares responsibility for the statistical analysis. AJL helped to conceptualize and design the study and to obtain the funding and shares responsibility for the statistical analysis. KMcC and GMcP helped to obtain the funding. All authors read and approved the final manuscript.
[fig] Figure 1: Flow diagram of study design and schedule. bd, Twice a day (Bis in die); CATest, COPD (Chronic Obstructive Pulmonary Disease) Assessment Test; EQ-5D, EuroQOL five-dimension questionnaire; MRC, Medical Research Council; od, Once a day (Omne in die) [/fig]
[table] Table 1: Schedule of study assessments X boxes represent which aspect of the assessment is conducted. CAT COPD (Chronic Obstructive Pulmonary Disease) Assessment Test, COPD Chronic obstructive pulmonary disease, EQ-5D EuroQOL five-dimension questionnaire, GP General practitioner, MRC Medical Research Council [/table]
|
Antibody Response After SARS-CoV-2 Infection and Implications for Immunity
## Ovid ebm reviews cochrane central register of controlled trials syntax
# Methods
## Protocol and registration 5
Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.
# Methods (page 5)
Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.
## Supplement
Data Extraction and Quality Assessment (Methods, page 7)
Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.
Review protocol11 Section/Topic # Checklist item Reported on page #
## Risk of bias in individual studies
12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.
Data Extraction and Quality Assessment (Methods, page 7)
Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). Point estimates for seroprevalence over time, pages 29-30)
## Synthesis of results 14
Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each meta-analysis.
We did not perform meta-analyses due to limited or differently reported data and study heterogeneity
Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).
Data Synthesis and Analysis (Methods, page 7)
Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified.
## N/a
# Results
## Study selection 17
Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.
Study Characteristics (Results, pages. See also . Literature Flow Chart (page 26)
## Study characteristics 18
For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.
## N/a
# Discussion
## Summary of evidence 24
Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).
# Discussion (pages 15-17)
## Section/topic # checklist item reported on page #
# Limitations 25
Discuss limitations at study and outcome level (e.g., risk of bias), and at reviewlevel (e.g., incomplete retrieval of identified research, reporting bias).
# Discussion (pages 15-17)
Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research.
Future Research (page 18)
# Funding
# Funding 27
Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review.
Role of funding source (Methods, page 8)
## Supplement table 2. inclusion and exclusion criteria*
## Picots inclusion and exclusion criteria
Population Include: KQ 1,4: Adults with RT-PCR-confirmed SARS-CoV-2 infection who underwent serologic testing for antibodies to SARS-CoV-2 (e.g. IgM, IgG, IgA, etc.) using an immunoassay.
## Kq 2-3:
Adults with RT-PCR-confirmed SARS-CoV-2 infection who underwent serologic testing using an immunoassay and had detectable antibodies to SARS-CoV-2 (e.g. IgM, IgG, IgA, etc.).
Exclude: Children less than 18 years of age; studies using serologic assays that have had Emergency Use Authorization revoked by the U.S. FDA.
## Outcomes
Include: KQ 1: Levels of antibodies to SARS-CoV-2 Duration antibodies remain detectable KQ 2: Incidence of reinfection (defined as new clinical or laboratory evidence of COVID-19 after a documented period of recovery, or according to the most recent definition of reinfection per the CDC) (87) KQ 3: Duration of immunity (i.e. length of time between an initial RT-PCR confirmed SARS-CoV-2 infection with clinical recovery to another SARS-CoV-2 infection) KQ 4: Unintended consequences of antibody testing after SARS-CoV-2 infection (e.g., discontinuation of recommended safety practices such as wearing masks or social distancing due to misinterpretation of positive antibody test results as indicative of immunity) We will stratify outcomes by the following factors: Patient characteristics (i.e. age, gender, race/ethnicity, comorbidities) COVID-19 severity (i.e. mild, moderate, severe, and critical as defined in NIH COVID-19 treatment guidelines)(10) Presence of symptoms (asymptomatic or symptomatic) Time from symptom onset or RT-PCR diagnosis to antibody testing Type of test and characteristics of the immunoassay (i.e., name, manufacturer, sensitivity and specificity, test methodology, test kit identifying information, lot number, expiration dates).
## Eligible study designs
Include: Any study design that addresses the key questions and otherwise meets eligibility criteria, including systematic reviews, RCTs, and controlled and uncontrolled observational studies. We will include intervention series (i.e., prospective studies that assess outcomes of interest among a series of patients. Systematic reviews that meet criteria for timeliness, relevance, and quality.
Exclude: Descriptive case series with <25 subjects, case reports, non-peer reviewed articles, commentaries (i.e. editorials, non-systematic reviews), and systematic reviews that do not meet the criteria detailed above. Animal studies, in-vitro studies, and studies of infection with coronavirus other than SARS-CoV-2 (e.g., SARS-CoV-1, MERS-CoV, seasonal coronaviruses). (17), severe (7) Age ( (6) Obesity: 17 (11) Asthma: 11 (7) Robbins, 2020 ( (14) Andrey (15) Chirathaworn (21) Gudbjartsson (29) Jaaskelainen (
## Study settings
[formula] N Y Y Y Y Y Y Y [/formula]
Y Participants were pregnant women (unique population) who may have more frequent care or more access to care than the general population; however, this finding would not be expected to affect seroprevalence results. Universal RT-PCR testing was implemented during the study period. 80% of eligible participants were included in the sample.
## Low
Gudbjartsson,
[formula] 2020(29) Y Y Y Y Y Y Y Y U Population-based sample. [/formula]
The number of individuals eligible for voluntary study participation is not reported; therefore, unable to evaluate adequacy of the response rate.
## Low
Iversen,
[formula] 2020(35) Y Y Y Y Y Y Y Y Y 97% [/formula]
## Sample size justified and satisfactory?
Yes/No/Unclear 3. Non-participants comparable to participants? Yes = Comparability between participants and non-participants is established, and the participation rate is satisfactory. No = The participation rate is unsatisfactory or the comparability between participants and non-participants is unsatisfactory. Unclear = No description of the participation rate or the characteristics of participants and non-participants 4. Ascertainment of the exposure appropriate? Yes = Adequately described, such as by medical record review or participant self-report. Unclear = Ascertainment methods not described.
Comparability 5. Were subjects in different outcome groups comparable, based on the study design or analysis? Yes/No/Unclear Outcome 6. Were outcomes pre-specified and ascertained using appropriate methods? Yes Unclear 7. Appropriate statistical analyses on potential confounders? Yes = Appropriate statistical analyses were conducted. No = No statistical analyses were conducted to adjust for confounders. Unclear = Statistical analysis was conducted but is not well-described and adjustment for confounders cannot be determined.
## Supplement
[formula] Y NA Y Y NA Y Y Y N N U Fair [/formula]
Small sample. Participants included those with a range of disease severity. Methods well-described.
## Low
Hou, 2020 (
[formula] 30) Y U Y Y U Y Y Y N N U Fair [/formula]
Unclear if groups were comparable at baseline. No adjustment for potential confounders.
Unclear
[formula] (31) Y NA Y Y NA Y Y Y Y N U Fair Low Isho, 2020 Y U Y Y U Y Y Y Y Y U Fair [/formula]
Unclear if groups were comparable at baseline.
[formula] Low Iyer, 2020 Y U Y Y N Y Y Y U N U Fair [/formula]
Age and sex distribution differed between RT-PCR+ cases and pre-pandemic controls, but that finding is unlikely to influence antibody prevalence among RT-PCR+ cases.
Unclear Cohort 1: Subjects exposed to SARS-CoV-2 infection Cohort 2: Subjects with active SARS-CoV-2 infection Cohort 3: Subjects recovered from SARS-CoV-2 infection Comparison of disease specific T cell repertoire signatures in patients and controls Identification of the immunodominant antigens that elicit a T cell response to COVID-19 Risk stratification based on an individual's immune "signature" Determine whether an immune signature can be detected in individuals exposed to SARS-CoV-2 earlier than currently available tests Prevalence of anti-SARS-CoV-2 antibodies among patients with chronic pulmonary conditions Active, recruiting, as of September 2020
[formula] (38) Y U Y Y U Y Y Y N N U [/formula]
[fig] High: Abbreviations: N=No; NA=Not Applicable; P=Partial; RT-PCR+ = Reverse Transcription Polymerase Chain Reaction (positive result); U=Unclear; Y=Yes. *Criteria (Adapted from the Newcastle-Ottawa Scale for Cross-Sectional Studies(12)): Selection 1. Sample representative? Yes = Truly representative of the average in the target population (all subjects or random sampling); or somewhat representative of the average in the target population (non-random sampling). No = Selected group of users Unclear = No description of the sampling strategy. [/fig]
[table] Table 5b: Quality Assessment of Cross-sectional Studies* [/table]
[table] Table 5c: Quality Assessment of Cohort Studies* [/table]
|
The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome
While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a highresolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.are available in the supplementary materials(Table S1). Data mining for the specific metabolites rutin, IAA and ACC was performed by searching the detected masses of the features in the XCMS positive datasets for masses corresponding to [M + H] + ions and the negative XCMS datasets for masses corresponding to [M − H] − ions. Features were not observed that corresponded to either IAA or ACC, while the details regarding the assignment of rutin are included inTable S1. transcriptome data analysis. Microarray datasets for root samples generated under identical conditions 11,12 were examined for changes in the expression of genes associated with the synthesis, inactivation, or transport of hormone-responsive metabolites, identified from the KEGG database 78 or the recent literature (e.g., in the case of members of the GH, DAO, and PIN families). Results for genes exhibiting a SLR of ≥ 0.5 or ≤ −0.5 in all three biological replicates for at least one time point are presented inTables 4 and 5, with the data for all examined genes provided in Datafile S5.
The phytohormones, auxin and ethylene, regulate physiological and developmental processes in plants, synergistically controlling some responses, while antagonistically regulating others. The morphological changes effected in roots by these two hormones are well characterized, with both hormones inhibiting primary root elongation and stimulating root hair formation. In contrast, the hormones have opposite effects on lateral root development, with auxin stimulating and ethylene inhibiting this process.
Substantial progress has been made in elucidating the auxin and ethylene signaling and transcriptional response pathways and the processes that control the homeostasis and intracellular movement of these two distinct, but highly interactive and interdependent hormone (reviewed in. Auxin, most commonly found in plants in the form of indole-3-acetic acid (IAA), is an aromatic molecule synthesized predominantly from tryptophan, while ethylene is a simple gaseous hydrocarbon (C2H4) derived from methionine via 1-aminocyclopropane-1 -carboxylic acid (ACC). In Arabidopsis, ethylene and auxin responses are initiated by binding of the hormones to their receptors, with ETR1 and TIR1 being the best characterized members of ethylene and receptor families. Binding to these receptors initiates a cascade of well-defined processes that in both cases involves inhibition of negative regulators, but via very different mechanisms. The signaling cascades ultimately results in transcriptional control of a variety of target genes, with cross talk occurring through activation of promoters containing both auxin and ethylene regulatory elements and formation of transcription factor complexes. Secondary crosstalk occurs through genes that respond to one of the hormones but then regulate the synthesis, transport, or response pathways of the other. Auxin gradients are critical to driving directed development in plants and are established through a combination of homeostasis mechanisms that include de novo synthesis, conjugation to sugars and amino acids, and oxidation 7 , as well as polar auxin transport mediated by PIN-FORMED (PIN)and ABCBAUX1/LIKE-AUX1 10 transporters. Auxin transport is regulated by PIN protein localization as well as the activity of specialized metabolites such as the flavonol, quercetin. Ethylene, which is a gas, is synthesized at or near its site of action, and is able to move between cells by diffusion.
To identify downstream targets of the auxin and ethylene signaling machinery that drive changes in growth and development, transcriptome analyses of the immediate response of Arabidopsis seedling roots to auxin and ethylene application were previously undertaken at high temporal resolution. These time courses comprised eight time points over a 24 h period, which were overlaid on time-matched developmental controls. This large-scale effort identified clusters of genes exhibiting similar kinetic patterns that, together with subsequent genetic analyses, provided new insights into the immediate impact of these hormones on global gene expression. This included identification of cell wall remodeling enzymes that participate in auxin-stimulated lateral root developmentt. Much less is known about metabolic enzyme targets that mediate metabolite synthesis, transport, and function to produce specific outcomes at the cellular and organismal levels.
The current study examined the root metabolome over the same high-resolution time course and with the same global approach used for the transcriptome studies. This made it possible to ask whether changes in the profile of metabolites in these tissues were correlated with changes in expression of genes encoding metabolic enzymes, or whether other mechanisms may contribute to the early response to hormone exposure. Prior studies examining the influence of auxin and ethylene on root metabolism have generally focused on a specific class of metabolites, a single time point, or mutants with altered abilities to respond to or synthesize auxin and/or ethylene (e.g.,. In an effort to match the global approach undertaken in the transcriptome studies, the metabolomics analysis presented here utilized two modes of chromatographic separation, reversed phase (RP) and hydrophilic interaction chromatography (HILIC), both performed in positive and negative ion modes, generating an overview of the chemically-diverse root metabolome at each time point. The resulting data files were processed into four datasets, using untargeted metabolomics methods for unsupervised discovery of features that changed in response to hormone treatment. Fragmentation analysis was performed on features of interest and the resulting spectra were compared to those published in metabolite databases to obtain putative identifications of compounds that exhibited substantial changes relative to time-matched controls across the root metabolome, and included sugars, amino acids, glucosinolates, phenylpropanoids, oligolignols, phospholipids, and indole-containing compounds. These experiments provide evidence of a rapid, transient change in the root metabolome primarily involving a small subset of specialized metabolites. Based on comparison with microarray data compiled for a parallel time course, the observed alterations in the metabolome generally did not appear to result from changes in gene expression, but rather from reconfiguration of existing biochemical and/or transport networks. Moreover, despite extensive crosstalk between auxin and ethylene at the transcriptional level and in mediating developmental and stress responses, the immediate effects of the two hormones on the root metabolome were quite distinct, both with respect to timing and the specific metabolites that were altered.
# Results
experimental design and metabolomic data analysis. The goal of this study was to examine the root metabolome in the first 24 h following exposure to the endogenous auxin, IAA, or the ethylene precursor, ACC. Experiments were carried out under conditions that paralleled earlier transcriptome studies so as to also enable exploration of cause and effect relationships at these two levels of cellular phenotyping. Seedlings were grown for 5d on nylon filters overlaid on MS-sucrose-agar. The filters were then moved to medium containing 1 µM IAA or 1 µM ACC. Root samples were collected at 0 (initiation), 0.5, 1, 2, 4, 8, 12 and 24 h after transfer, with biological replicates obtained from three independent experiments. The resulting samples (24 each for IAA-treated, ACCtreated, and untreated controls) were analyzed using four different LC/MS methods that utilized two modes of chromatographic separation (reversed phase and HILIC), each with two mass spectrometer polarities (positive and negative). These complementary approaches maximized coverage and provided a robust sampling of the metabolome. Each sample was analyzed in technical triplicate, a total of 864 injections. A representative chromatogram from each type of LC-MS analysis is shown in.
The raw chromatograms were processed into a data matrix to allow for characterization of changes in the metabolome across hormone treatments and time. Data from each of the four analyses modes were analyzed separately, each generating between 400 and 1600 features, defined as an exact mass/retention time pair (EMRT) and its corresponding peak area. The full datasets are provided as supplementary material (Datasets S1-S4). Redundant features resulting from naturally-occurring isotopes, adducts, and in-source fragmentation were identified and merged using RamClustR 22 to generate a deconvoluted data set. Features exhibiting robust changes in response to hormone exposure were defined as those that displayed at least a two-fold difference between treated and untreated tissues in all three biological replicates. The results of this processing are summarized in .
Overall less than 15% of the features detected exhibited a two-fold difference.
Overview of hormone effects on the root metabolome. Statistical analysis was used to generate an overview of the metabolome over the time course of exposure to IAA or ACC.shows the scores plots derived from sparse partial least squares-discriminant analysis (sPLS-DA) of the reversed phase negative mode dataset (Dataset S1), which exhibited the largest number of changes for either hormone treatment. This analysis enables the selection of the most discriminative features in the data to help distinguish between individual samples, with the resulting plots providing a powerful visualization, in this case of changes in the root metabolome over time. Panel A provides an overview of the response of the root metabolome after IAA treatment across the 24 h time course utilizing 17.0% and 21.4% of the data in components 1 and 2, respectively. The sPLS-DA shows substantial overlap of the IAA elicited root profiles at the early time points, from 0 to 2 h, but displays a distinct separation of the profiles at 4 h and later. Panel B provides an overview of the metabolome after ethylene elicitation displaying 26.1% and 19.6% of the data in components 1 and 2, respectively. The scores plot shows a rapid and distinct separation at the first sampling point after elicitation, at 30 min, which is transient. The 4 h and later time points separate from the origin but do not display clear distinction among each other. Similar results were observed for the sPLS-DA analysis of datasets generated using the other three analytical methods (Datasets S2-S4).
Time series analysis reveals specific metabolites that change in abundance following hormone exposure. Multivariate statistical methods were used to uncover features displaying distinctive temporal patterns that were driving the profile differences observed in the sPLS-DA score plots. ANOVA Simultaneous Component Analysis (ASCA) uncovered two features (273_352.1028 and 271_278.0659) that exhibited a strong response to IAA treatment over the 24 h time course. These were putatively identified by fragmentation analysis as 2-oxindole-3-acetic acid-hexose (oxIAA hexose) and coumaroyl aspartate . ASCA did not identify any features whose abundance exhibited a distinct trend across the time course in response to exposure to ACC.
The separation of time points in the sPLS-DA indicated that the metabolome was changing over time, even though ASCA only correlated two features with a response to hormone treatment. Because ASCA relies on the presence of sustained trends over time, this suggested that additional features could have exhibited changes that were transient or occurred at nonconsecutive time points. To examine this possibility, the reversed phase negative mode dataset was surveyed for a feature associated with the abundant root flavonoid, quercetin hexose deoxyhexose (rutin). Quercetin has previously been shown to exhibit rapid changes in response to IAA and ACC treatments of roots. The time course profile for rutin did indeed reveal a previously-undetected difference, which was an increase at 1 h in response to IAA relative to the untreated control, present in all three biological replicates; blue versus green data points).
A paired univariate approach was therefore applied to all four datasets to identify features that exhibited two-fold differences within each biological replicate at one or more time points, which uncovered approximately 80 additional features of interest for each hormone treatment . Analysis of fragmentation patterns and assignment of duplicate features from the same molecular species resulted in 26 putative identifications for compounds in the IAA datasets (Tables 2 and S1) and 20 for ACC (Tables 3 and S1). These metabolites belong to a few distinct classes of metabolites, primarily phenylpropanoids, glucosinolates, and several derivatives of indole. Many of the changes within these classes were transient, with elevated levels detected at a single early time point www.nature.com/scientificreports www.nature.com/scientificreports/ that then rapidly returned to the levels of untreated time-matched controls, often by the next sampling event. The responses to treatment with IAA or with ACC were also quite distinct, both in terms of the timing and the types of metabolites that exhibited altered levels, with only five metabolites in common between the two datasets. Thus, despite substantial crosstalk between auxin and ethylene at the transcriptional and developmental levels, the short-term response to exogenous exposure results in substantially different responses with regard to the metabolic profiles of root cells. the response of the root metabolome to exogenous iAA has the hallmarks of an effort to maintain auxin homeostasis. Immediate response to exogenous auxin. A response to auxin exposure was already detectable in the root metabolome at the first time point, 0.5 h after the start of treatment. Particularly striking at this stage was a substantial (5.18 fold) decline in the levels of IAA-hexose relative to the untreated controls, followed by an even larger (13.8-fold) reduction at 2 h, one of the largest differences observed for any metabolite in this experiment. This is consistent with very rapid activation of a mechanism for achieving auxin homeostasis through the reversible sequestration and/or inactivation of free IAA (reviewed inand discussed in, followed by buffering of IAA levels by hydrolysis of hex-IAA as other mechanisms for sequestration, exclusion, or transport come into play. This interpretation is further supported by the further-reduced levels of IAA-hexose at 2 h, which were accompanied by elevated levels of the glycoside of the IAA oxidation pathway product, oxIAA-hexose, also discussed further below. An inverse relationship between the levels of these two metabolites has previously been reported. The very early activation of IAA homeostasis mechanisms is also consistent with the finding that free hormone was not observed in either control or IAA-treated roots at any of the sampling times under the conditions used for these experiments.
Transcriptome data indicate that expression of genes associated with the synthesis or hydrolysis of IAA-hexose, specifically two IAA glucosyl transferases (UGT74D1 and UGT84B1and members of the IRL1 family (ILR1, ILL2, IAR3/ILL4, and ILL5, homologs of which encode glucosyl hydrolase activity in Medicago and rice, is largely unaffected by exposure to auxin under the current conditions (Datafile S5). This suggests that key enzymes remain to be identified or that post-transcriptional processes are responsible for the observed early changes in IAA-glucose levels. On the other hand, the glycosyl hydrolysis reaction is energetically favored and could potentially proceed instantaneously in response elevated levels of auxin, without the need for changes in a specific enzyme activity. Another possibility, which is also consistent with a role for IAA conjugation in maintaining auxin homeostasis, is that the rapid drop in IAA-hexose levels within the first 0.5 h of exposure of roots to auxin reflects activation of transport machinery to rapidly move excess free and/or conjugated hormone out of the root to other sites in the plant or to the rhizosphere. Although transporters able to mobilize IAA-hexose remain to be identified, the transcriptome data show that genes encoding the auxin efflux carrier PIN-FORMED (PIN) proteins, PIN1, PIN3, and PIN7, are rapidly induced . The current findings thus suggest the possibility that activation of transport machinery for various forms of auxin may help mediate the early response to abrupt changes in this hormone.
A similar early and transient decrease in abundance was observed at 0.5 h for two glycosides of 6-hydroxyindole-3-carboxylate, approximately 3-fold in each case. These compounds are downstream components of the indole glucosinolate pathway, the intermediates of which can be used for IAA synthesis in Brassicaceae. The reduced levels may thus reflect a rapid downregulation of indole glucosinolate metabolism to mitigate the de novo production of additional IAA. Indeed, a four-fold reduction was observed slightly later, at 2 h, one of two terminal products of this pathway, 4-methoxy-3-indolylmethyl glucosinolate. However, these changes were again not well correlated with changes in the transcriptome. Although transcripts encoding several enzymes of indole glucosinolate metabolism (CYP79B2/3, CYP81F2 and UGT74B1) exhibit decreases, these are first observed at 2-4 h, well after the reduction in the two downstream carboxylates relative to untreated controls. Moreover, no changes were observed for transcripts encoding several other enzymes of aromatic and and coumaroyl aspartate (B), and one uncovered by targeted analysis, the flavonoid glycoside rutin (C). Note that the x axis is not drawn on a linear scale; this is to allow for better visualization of the data points for early sampling times.
indole glucosinolate biosynthesis (CYP79A2, CYP83B1, SUR1, UGT74B1, and SOT16) or those required for synthesis of the carboxylate derivatives (CYP71B6 and AAO1) (Datafile S5). Conversely, there were no substantial changes in the levels of aliphatic glucosinolates, despite changes in the transcripts for a number of the corresponding enzymes (BCAT4, CYP79F1/2, CYP83A1). Thus, here too, it appears that post-translational mechanisms may be key to the early response at the level of the metabolome, including transport of glucosinolates to other parts of the seedling 32 or secretion into the rhizosphere.
Implementation of a distinct response was apparent at the 1 h and 2 h time points, with a sudden, short-lived increase in the levels of a number of different flavonol glycosides relative to time-matched controls. These included six quercetin glycosides, two glycosides of isorhamnetin (a 3'-methoxylated derivative of quercetin), and four kaempferol glycosides, all of which exhibited a substantial (2-11 fold) and transient (1-2 h) increase. This finding is consistent with previous studies that reported an increase in flavonols and in the quercetin:kaempferol ratio under similar experimental conditions, although it occurred somewhat later in the time course. A glycoside of coumarate, a precursor for the synthesis of flavonoids and monolignols, exhibited the same short-lived increase, as did a glycoside of the monolignol intermediate, caffeic acid, suggestive of altered flux across the phenylpropanoid pathway, the branches of which are highly interconnected (e.g.. Flavonols, and quercetin in particular, are well-established inhibitors of auxin transport. Thus an early strategy for regaining auxin homeostasis could include shutting down the import of exogenous auxin, both from the medium and from the primary site of synthesis in shoots, by raising flavonol levels in the root.
# Metabolite emrt method
Average log two-fold change after transfer to hormonecontaining medium 0 www.nature.com/scientificreports www.nature.com/scientificreports/ As with the other early responses of the root metabolome to auxin, changes in the transcriptome could not explain the observed accumulation of phenylpropanoids at the 1 and 2 h sampling times. The only phenylpropanoid enzyme for which transcript levels increased during this time frame was the lignin pathway enzyme, ferulate 5-hydroxylase (F5H), which exhibited slightly-elevated levels at 1 h ; Datafile S5). Previous qRT-PCR analyses of Arabidopsis roots exposed to 1 µM IAA or ACC detected 3-to 4-fold increases in transcripts for several flavonoid genes, but in this prior study roots were grown on higher levels of sucrose, which has been shown to increase expression of pathway enzymes, and to result in more profound root developmental phenotypes in mutants with defects in flavonol synthesis. However, even in this case, expression peaked at the 2 h time point, lagging behind the changes in the abundance of flavonols observed here, similar to what has also been reported for the light response of flavonoids and associated genes in roots. Once again, it appears that immediate/early mechanisms for re-establishing auxin homeostasis largely involve post-transcriptional mechanisms for coordination and reconfiguration of existing metabolic networks. These may also involve long-distance translocation or secretion processes, both well documented in the case of flavonoids.
On the other hand, it appears that changes in gene expression occur at later time points that could contribute to the observed return of the quercetin, kaempferol, coumarate, and caffeic acid conjugates to pre-exposure levels, with transcript levels for a number of enzymes of general phenylpropanoid and flavonoid metabolism decreasing relative to untreated controls starting at 1-2 h . Interestingly, Kelch domain-containing F-box proteins have recently been implicated in the ubiquitin-mediated degradation of PALand CHSand transcripts encoding two of the PAL-specific KFBs do exhibit elevated levels in response to auxin (at 1, 4, 8, and 12 h for KFB1 and 1 h for KFB20; Datafile S5). This provides evidence from the transcriptome that post-translational modifications may also help mediate the reduction of flux through the phenylpropanoid pathway to return flavonoid metabolites to pre-exposure levels.
# Metabolite emrt method
Average log two-fold change after transfer to hormonecontaining medium 0 www.nature.com/scientificreports www.nature.com/scientificreports/ Second stage response. The immediate responses of the metabolome appeared to be followed by implementation of two well-established auxin inactivation mechanisms at 2-4 h, both of which persisted through the rest of the sampling times, with peak activity apparent at 8-12 h. This was a unique pattern among the features/metabolites identified in this study, making these the only two uncovered by ASCA, as described above. The first of the mechanisms was evidenced by the appearance of strongly elevated (3-to 8.5-fold) levels of oxIAA hexose, the major primary IAA catabolite in Arabidopsis roots. This oxidized, inactive form of IAA is generated through the action of two DIOXYGENASE FOR AUXIN OXIDATION (DAO) enzymes (reviewed in. However, contrary to expectations, over the same time period transcripts encoding DAO1 and DAO2 are reduced 2-to 3-fold [signal log ratio (SLR) of −1.00 to −1.57; ]. This suggests that posttranscriptional processes such as changes in activity of this enzymatic machinery may drive auxin inactivation mechanisms, even after 8 or more hours, while changes in gene expression may contribute to the return to pre-exposure conditions.
A second metabolite, coumaroyl aspartate (hydroxycinnamoyl aspartic acid), exhibited a very similar pattern of induction, with levels that were substantially higher than in untreated controls at 12 h, among largest changes observed for any metabolite. It was also the only phenylpropanoid pathway-derived product that was elevated at 4 h and thereafter, and thus also the only one correlated with elevated transcripts for a cinnamyl-alcohol dehydrogenase (CAD)-like enzyme (at 2-24 h) and two CAD enzymes (at 12-24 h) . This compound has been reported in only a limited number of prior studies, notably in roasted coffee and cocoa beans (e.g., as the major metabolite after elicitor treatment of cell suspension cultures of European beech, and in cell suspension cultures derived from Arabidopsis cotyledons. It is also one of many compounds proposed to accumulate upon perturbation of the lignin pathway, which derives from p-coumarate in phenylpropanoid metabolism. Thus it is possible that accumulation of coumaroyl aspartate is related to the earlier shift of flux into flavonoids and monolignols. Another possibility is that the higher levels of this compound are indicative of mobilization of another primary auxin inactivation mechanism, conjugation to aspartate. Indeed, the associated transcriptome date show a rapid and strong induction of transcript levels for several GRETCHEN HAGEN 3 (GH3) genes, which encode key enzymes for conjugation of aspartate, as well as other amino acids, to IAA. Defining the position of coumaroyl aspartate in the metabolic network should provide new insights into the role of this compound in the response to auxin and other perturbations. www.nature.com/scientificreports www.nature.com/scientificreports/ Transition to a new state of homeostasis. Changes in one additional class of metabolites characterized the response to auxin, including at the final, 24 h, time point, a reduction in several forms of phosphatidylcholine (PC). These metabolites represent the major class of phospholipids in eukaryote membranes, serving key roles as both structural and signaling molecules. Two forms, 34:2 and 34:3, exhibited a robust elevation early in the time course, at 2 h, together with an 8-fold increase in the fatty acid derivative, trihydroxyhexadecanoic acid. The PCs were then detected at reduced levels at 24 h, together with two 36-carbon PCs, 36:4 and 36:5. Changes in membrane lipid composition have been shown to mediate localization and activity of several PIN efflux carriers, and genes involved in this process are emerging from a number of different studies, including in roots. These include the phospholipases, PLP1 and NPC3, that are proposed to have a role in processes linked to auxin signaling, including lateral root development and modification of root architecture in response to phosphate starvation. A number of genes with roles in PC metabolism exhibit fluctuations in expression starting with the 2 h time point , indicating that transcriptional changes may, at least in part, mediate the observed changes in PC levels. These findings also suggest that modifications in lipid membrane composition may contribute to maintenance of auxin homeostasis in the face of continued elevated levels of the hormone.
Ethylene induces a distinct response at the level of the metabolome. Changes in the root metabolome in response to ACC and the resulting elevated levels of ethylene were substantially different from those observed with auxin, despite the considerable crosstalk occurring between these hormones. Even the few metabolites that were common to both responses exhibited distinct temporal patterns. A unique feature of the ethylene response was a substantial reduction in a large number of glucosinolates. This was initially apparent already at 0.5 h, with an approximately 3-fold reduction in three aliphatic forms. The two octyl forms were also substantially reduced again at 24 h, together with five other aliphatic forms and two aromatic glucosinolates, all between 3.5 and 7-fold lower than in untreated tissues. A number of these metabolites were previously reported to be abundant in roots of mature plants. The reduction in the levels of these compounds suggests that the response to ACC includes a change in the rate of flux through all three pathways of glucosinolate biosynthesis. Analysis of the corresponding transcriptome data 12 showed a striking lack of correlated suppression of expression of genes encoding enzymes in these pathways over the 24 h time course, with the only substantial differences with the time-matched controls being increases at various sampling times, rather than the expected immediate decreases, in several enzymes of aliphatic (BCAT4, CYP83A1, SOT18) and aromatic (CYP83B1) metabolism; Datafile S5). There was also an increase in transcripts for an enzyme of indole glucosinolate biosynthesis (CYP81F2) at 12 and 24 h, with no corresponding change in the corresponding metabolites. Once again, it appears that posttranscriptional mechanisms control the early response of a major pathway of specialized metabolism to hormone exposure.
The other major group of compounds characterizing the response to ethylene were products of phenylpropanoid metabolism. These included elevated levels of three flavonol glycosides, all of which were also found to change in response to auxin, but in this case with quite different temporal patterns that spanned the 2-12 h time points; an unknown quercetin-containing metabolite was also elevated in response to ACC. Several genes of central flavonoid metabolism exhibit differential expression at various time points, although not in a coordinated or sustained manner consistent with the observed elevated levels of flavonols observed response to ACC exposure. However, transcripts encoding flavonoid 3'-hydroxylase (F3'H), which controls flux between kaempferol and quercetin, were downregulated at the 8 h time point, consistent with the shift from quercetin to kaempferol-containing metabolites. An overall elevation in flavonol levels at 12 h of treatment with ACC, accompanied with an increase in the kaempferol:quercetin ratio, was also previously reported based on staining with DPBA 14 . The only other flavonoid genes with altered expression are a flavonol glycosyltransferase (UGT71C3) and O-methyltransferase (OMT1) with elevated levels late in the time course (8-24 h), the former potentially contributing to the enhanced synthesis of the two kaempferol glycosides. As mentioned above, although previous studies have detected increases in expression of several flavonoid genes in response to treatment with 1 µM ACC 14 , this is likely attributable to the higher levels of sucrose used in those experiments.
The step-wise changes in glucosinolate and phenylpropanoid products over the 24 h of exposure to ACC points to a gradual shift in flux across the major pathways of specialized metabolism as an initial response to elevated ethylene. The inverse relationship between these two classes of metabolites is consistent with the previous reports of crosstalk between these pathways. However, those cases were the reverse what was observed here, with elevated levels of glucosinolates driving repression of phenylpropanoid metabolism, driven in part by the aldoxime-and MED5-mediated degradation of PAL. Although the changes in the metabolome were again not correlated with altered levels of transcripts encoding pathway enzymes, expression of several PAL KFB's and the CHS KFB were enhanced, particularly at later time points. Overall, it appears that post-transcriptional processes, including modulation of pathway flux through post-translational control of enzyme levels as well as long-distance transport or secretion of metabolites, may be at play.
Another unique aspect of the ethylene response was an approximately 2.5-fold increase in the levels of three oligolignols at 12 h, products of the lignin branch of phenylpropanoid metabolism that are characteristic cell wall components. This latter change is consistent with the alterations in root hair initiation and elongation that occur during the 24 h of ACC exposure, which has also been shown to be accompanied by changes in expression of numerous genes associated with cell wall biogenesis, biosynthesis, and organizationwww.nature.com/scientificreports www.nature.com/scientificreports/ Previous experiments have shown that ethylene positively regulates auxin synthesis. Although free IAA was not detected, IAA-hexose, which exhibited reduced levels at 0.5 h of IAA exposure, was also significantly reduced at 24 h in response to ACC. In this case, the difference was preceded by changes in the expression of two DAO genes ( www.nature.com/scientificreports www.nature.com/scientificreports/ homeostasis via oxidation. However, as with the response to IAA, no substantial changes were observed in transcripts encoding known IAA glycosylase or hydrolase enzymes (Datafile S5). Moreover, changes in the transcripts encoding GH3.3 and 3.6, CYP71A13, and UGT74E2 were not reflected in altered levels of the corresponding IAA metabolites. These observations suggest the possibility of yet-to-be-identified degradation processes or transport/ secretion mechanisms specific for IAA conjugates.
# Discussion
The current study represents the first comprehensive analysis of the early response of the root metabolome to auxin and ethylene, phytohormones with well-established synergistic and antagonistic effects on root development. High-resolution data generated using a global untargeted LC-MS approach showed that, in the presence of exogenous IAA and ACC, seedling roots exhibit a series of reproducible and substantial (2-fold or greater) changes to a limited number of classes of metabolites, with the majority of changes being evident within small windows of time. The analysis provided evidence that three distinct strategies are used in response to elevated auxin, one following the other, as plants seek to regain homeostasis in the face of high external levels of the hormone. The compounds exhibiting substantial changes belonged predominantly to three classes: phenylpropanoids, fatty acids, and derivatives of IAA. A distinct response was observed for elevated levels of ethylene that was characterized primarily by changes in glucosinolate and flavonoid levels with minimal overlap with those observed to change in response to IAA. There is evidence in the transcriptome for effects of both hormones on ACC oxidase (ACO) and ACC synthase (ACS) gene expressionand thus also crosstalk of auxin on ethylene metabolism. However, ACC was not detected in roots under the conditions used in these experiments and these were also not designed to detect volatile compounds such as ethylene. A reduction in IAA-hexose levels at 24 h were the one evidence of crosstalk with IAA signaling pathways.
The finding that altered levels were detected for only a subset of compounds within a particular pathway indicated that certain metabolites are particularly indicative of change in network flux, possibly due to small steady-state pools or short half lives. These compounds may represent additional "sensor" metabolites or biomarkers that can provide insights into the metabolic status of the system as a whole. It is of note that previously-described sensor molecules (e.g., free IAA, L-phenylalanine, naringenin chalcone, eriodictyol, and several glucosinolates including 7-methylthioheptyl and 4-benzyloxybutyl glucosinolates and sinapoyloxy conjugates) did not consistently display differences in the short-term auxin/ethylene response even where there was evidence of altered pathway flux. Thus additional compounds such as those identified in the current study may prove useful, perhaps especially for tracking dynamic responses to biotic or abiotic stress.
Another consistent finding is that changes in the metabolome were not well correlated with previously-published transcriptomics and proteomics datasets for matching tissues. Although the majority of changes at the transcript level were also transient, rapidly returning to the levels present in time-matched controls, there was little evidence that these were responsible for the observed changes in the metabolome except possibly in the case of auxin-mediate changes in phosphatidylcholine metabolism and ethylene-induced changes in lignin biosynthesis. For both hormones, the largest differentially-expressed clusters were comprised of known auxin-and ethylene-responsive genes, genes related to RNA and DNA processes, and cell wall biogenesis and organization, among a few others; none were related to specialized metabolism.
This was also true for proteomic profiles generated for the response of roots to auxin at 0.5 and 2h 57 and at 8, 12, and 24h 58 under similar or identical conditions, respectively. Although the top auxin-responsive proteins were not related to metabolic processes in either of these studies, GO analysis of the early time points did show evidence of changes in the proteome related to organization of transporters and the cytoskeleton. Profiles of metabolites present in the root exudates of 5-6 week-old hydroponically-grown plants are consistent with secretion as one mechanism for rapidly reducing the levels of specific metabolites, including phenylpropanoids and glucosinolates, as well as other indole-containing compounds. A similar outcome has recently been reported in whole seedlings exposed to 1 µM auxin for 3 h, where the majority of differently-expressed proteins were also not correlated with changes in the corresponding transcripts.
This outcome is not particularly surprising in the context of the growing number of studies, including in plants, suggesting that immediate changes in metabolic activity and flux are controlled largely at the post-transcriptional and even post-translational levels (reviewed in 62 ). In fact, it has recently been shown that the response to auxin is essentially instantaneous, with elongation of primary roots inhibited within 30 s of exposure to auxin. Even at later time points in the current experiment, it consistently appeared that the response relied on rewiring or mobilizing existing systems through posttranslational mechanisms, an observation for which there is substantial precedent conclusion Correlating changes in the metabolic status of plant cells with the underlying mechanistic processes will require extending the elucidation of changes in network flux to the pathways of specialized metabolism, including those that occur immediately following stresses that disrupt equilibrium. It will also require accounting for redistribution, not only of metabolites, but likely also microRNAs, transcripts, and proteins, within the plant via long-distance transport, as well as secretion or volatilization of metabolites away from the plant itself. So, too, must there be information at the level of the individual protein components, including reorganization of intracellular interaction networks and localization. Even the role of post-translational modifications, as in the case of the phenylpropanoid and flavonoid pathways, is just beginning to be uncovered. Technologies to address these questions in a comprehensive manner are increasingly within reach and will help write a new understanding of the processes by which cells execute re-equilibration of their metabolic status, both as part of immediate responses and to achieve new long-term steady states.
Peak identification and data processing. Raw data files (*.raw) were converted to the NetCDF format using DataBridge (Waters Corp., Milford, MA). Chromatogram alignment, peak detection, and peak integration were performed in the R statistical programing environment 71 using the XCMS package 72 with the following processing parameters: centWave peak detection, 15 ppm mass deviation, peak width (s), 5 min/40 max, signal/ noise ratio 2, noise 999, and lockMass adjustment applied (Datasets S1-S4). The XCMS data set was deconvoluted using RamClustR, which identified and merged redundant features resulting from naturally-occurring isotopes, adducts, and in-source fragmentation, before averaging all technical replicates.
sPLS-DA and ASCA were performed on the reduced dataset using the time-series module of the web-based program MetaboAnalyst 73 . A univariate statistical approach was used to discover additional features that exhibited at least a two-fold change in all three biological replicates at one or more time points. Comparison with calculations performed using a 1.5, 2.0 and 2.5-fold cutoff showed that the two-fold criterion was sufficiently high to select against noise, while still uncovering a substantial number of changes. Putative metabolite identities were assigned with the aid of MS/MS and MS E fragmentation patterns, commercial standards, and online databases, including ReSpect 74 , Metlin 75 , MassBankand KNApSAcK. The details of each metabolite assignment |
Autophagy and Its Association with Genetic Mutations in Parkinson Disease
Parkinson disease is the second most common neurodegenerative disorder, affecting 0.1-0.2% of the general population. It is a progressive debilitating disorder caused by degeneration of dopaminergic neurons in the substantia nigra pars compacta. It is characterized by motor and non-motor symptoms. Parkinson disease can be caused by mutations in genes that encode proteins involved in the autophagic process, resulting in impaired autophagy. Indeed, autophagy has been implicated in the pathogenesis of Parkinson disease, particularly because its impairment causes the buildup of proteins. Thus, this review aims to provide an overview of Parkinson disease-related genetic mutations and their association with autophagy impairment in Parkinson disease, which can be helpful in improving the understanding of the pathogenesis of Parkinson disease, illustrating the potential therapeutic implications of agents that can enhance autophagy in Parkinson disease. Additionally, we will highlight the essential need for the development of highly sensitive and specific assays for gene-based diagnostic biomarkers. Finally, we will provide an overview on the potential gene-based therapeutic approaches for Parkinson disease, which have been most advanced and are associated with the most common targets being alpha-synuclein (SNCA), leucine-rich repeat kinase-2 (LRRK2), and glucocerebrosidase (GBA).
# Background
Parkinson disease is the second most common neurodegenerative disorder, affecting 0.1-0.2% of the general population [bib_ref] MicroRNA-181a regulates apoptosis and autophagy process in Parkinson's disease by inhibiting p38..., Liu [/bib_ref]. Its prevalence increases with age, affecting 1% and 4% of individuals who are older than 60 years and 80 years, respectively [bib_ref] A meta-analysis of alpha-synuclein multiplication in familial Parkinsonism, Book [/bib_ref].
Parkinson disease is a progressive disorder caused by degeneration of dopaminergic neurons in the substantia nigra pars compacta [bib_ref] Parkinson's disease: Clinical review and update, Cabreira [/bib_ref] [bib_ref] LRRK2 modifies alpha-syn pathology and spread in mouse models and human neurons, Bieri [/bib_ref] [bib_ref] Interleukin-1 beta and tumor necrosis factor alpha upregulation and nuclear factor kappa..., Erekat [/bib_ref] [bib_ref] Association of Parkinson disease induction with cardiac upregulation of apoptotic mediators P53..., Erekat [/bib_ref]. It is characterized by motor and non-motor symptoms. Non-motor symptoms often precede the motor symptoms. Chief motor symptoms characteristic of Parkinson disease are at-rest tremor, bradykinesia, rigidity, and postural instability [bib_ref] Overexpression of microRNA-133a inhibits apoptosis and autophagy in a cell model of..., Lu [/bib_ref]. Thus, Parkinson disease is a debilitating disorder that can have a serious impact on the quality of life.
Parkinson disease can be either familial or sporadic [bib_ref] Apoptosis and its therapeutic implications in neurodegenerative diseases, Erekat [/bib_ref]. Familial Parkinson disease cases are due to autosomal dominant and autosomal recessive mutations [bib_ref] Apoptosis and its therapeutic implications in neurodegenerative diseases, Erekat [/bib_ref]. Parkinson disease is characterized by the presence of Lewy bodies, which are aggregated protein inclusions that contain a-synuclein as their main constituent.
Autophagy is a form of programmed cell death that is required for the turnover of long-lived organelles and elimination of impaired proteins [bib_ref] Programmed cell death in cerebellar Purkinje neurons, Erekat [/bib_ref]. Impaired autophagy has been shown to be involved in the pathogenesis of Parkinson disease, since it can lead to disrupted elimination of a-synuclein, resulting in its build up and subsequent misfolding [bib_ref] Impaired autophagy in microglia aggravates dopaminergic neurodegeneration by regulating NLRP3 inflammasome activation..., Qin [/bib_ref] [bib_ref] Parkinson disease epidemiology, pathology, genetics, and pathophysiology, Simon [/bib_ref]. Consequently, misfolded a-synuclein can participate in the formation of Lewy bodies, which are considered as the pathological hallmark in Parkinson disease [bib_ref] Age-related increase in caveolin-1 expression facilitates cell-to-cell transmission of alpha-synuclein in neurons, Ha [/bib_ref] [bib_ref] Observation of an alpha-synuclein liquid droplet state and its maturation into Lewy..., Hardenberg [/bib_ref].
Thus, this article aims to review the process of autophagy and its role in Parkinson disease, illustrating its association with the various genetic mutations of Parkinson disease, and highlighting its potential therapeutic implications in Parkinson disease. It also aims to highlight the essential need for development of highly sensitive and specific assays for gene-based diagnostic biomarkers, and to provide an overview on the most advanced gene-based potential therapeutic approaches for Parkinson disease, which are associated with the most common targets: alpha-synuclein (SNCA), leucine-rich repeat kinase-2 (LRRK2), and glucocerebrosidase (GBA).
## Autophagy
Autophagy is physiologically involved in many biological processes, such as organelle turnover and intracellular homeostasis [bib_ref] Autophagy in neurons, Stavoe [/bib_ref] [bib_ref] Biological functions of autophagy genes: A disease perspective, Levine [/bib_ref] [bib_ref] Programmed cell death in diabetic nephropathy: A review of apoptosis, autophagy, and..., Erekat [/bib_ref]. Pathologically, autophagic activity is altered and it is consequently involved in the pathogenesis of diseases, such as neurodegenerative diseases, including Parkinson disease [bib_ref] The vicious cycle between alpha-synuclein aggregation and autophagic-lysosomal dysfunction, Bellomo [/bib_ref].
Autophagy is classified into three types, which are: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [bib_ref] Programmed cell death in cerebellar Purkinje neurons, Erekat [/bib_ref]. Macroautophagy is possibly the best-described type of autophagy, and it is referred to as autophagy throughout this review [bib_ref] Ferroptosis is a type of autophagy-dependent cell death, Zhou [/bib_ref] [bib_ref] Chaperone-mediated autophagy in neurodegenerative diseases: Molecular mechanisms and pharmacological opportunities, Wang [/bib_ref]. It starts with the formation of autophagosomes, which are double-membrane vesicles that contain the substrate to be degraded [bib_ref] Autophagy precedes apoptosis among at risk cerebellar Purkinje cells in the shaker..., Erekat [/bib_ref].
Autophagy occurs via four stages, which are induction, vesicle nucleation, autophagosome membrane elongation, and termination/fusion and degradation, and they involve autophagy-related genes (Atg) and proteins [bib_ref] GAN (gigaxonin) E3 ligase and ATG16L1: Master and commander of autophagosome production, Bomont [/bib_ref]. Under normal conditions, autophagy is inhibited due to the interaction of the mammalian target of rapamycin 1 (mTORC1) complex with the unc51-like kinase 1 (ULK1) complex [bib_ref] An mTORC1-to-CDK1 switch maintains autophagy suppression during mitosis, Odle [/bib_ref]. However, this interaction stops the prevention of autophagy under stressful circumstances, such as starvation and energy depletion [bib_ref] Fine-tuning of AMPK-ULK1-mTORC1 regulatory triangle is crucial for autophagy oscillation, Holczer [/bib_ref].
Activated adenosine monophosphate-activated protein kinase (AMPK) prevents mTOR resulting in the induction of autophagy [bib_ref] Induction of autophagy by baicalin through the AMPK-mTOR pathway protects human skin..., Zhang [/bib_ref]. The second stage in the autophagic process, which is vesicle nucleation, requires Beclin-1 complex, which consists of class III phosphoinositide 3-kinase (PI3K), p150, Beclin-1, and Atg14 [bib_ref] Harnessing autophagic network is essential for trophoblast stem cell differentiation, Chakraborty [/bib_ref].
Autophagosome membrane engagement is performed by the Atg12 and light chain 3 (LC3) ubiquitin-like conjugation systems [bib_ref] Autophagy and autophagy-related proteins in cancer, Li [/bib_ref]. Thus, ubiquitin-like Atg12 is conjugated to Atg5, Atg7, and Atg10, in which sequential reactions of E1 enzyme (Atg7) and E2 enzyme (Atg10) conjugate Atg12 to Atg5, resulting in the subsequent formation of Atg5-Atg12/Atg16L multimeric complex that localizes to the convex surface of the isolation membrane [bib_ref] Autophagy and autophagy-related proteins in cancer, Li [/bib_ref]. Atg5-Atg12/Atg16L complex serves as an E3 enzyme for the conjugation reaction of LC3, which is Atg8, promoting the transformation of LC3-I to LC3-II, which is essential for autophagosome formation [bib_ref] Autophagy and autophagy-related proteins in cancer, Li [/bib_ref]. In the last stage of autophagy, the autophagosome fuses with lysosomes, forming autophagolysosomes, so that contents of the autophagosome are degraded by hydrolases within the lysosome [bib_ref] Autophagy and apoptosis in the midgut epithelium of millipedes, Rost-Roszkowska [/bib_ref] [bib_ref] Epithelial haven and autophagy breakout in gonococci infection, Mendes [/bib_ref]. Additionally, selective forms of autophagy eliminate substrates according to the various cargoes [bib_ref] A diversity of selective autophagy receptors determines the specificity of the autophagy..., Kirkin [/bib_ref]. For instance, protein aggregates are cleared by aggrephagy and damaged mitochondria are degraded by mitophagy [bib_ref] A diversity of selective autophagy receptors determines the specificity of the autophagy..., Kirkin [/bib_ref] [bib_ref] MAPK1/3 kinase-dependent ULK1 degradation attenuates mitophagy and promotes breast cancer bone metastasis, Deng [/bib_ref].
In contrast, microautophagy is the direct uptake of part of the cytoplasm by lytic organelles [bib_ref] Microautophagy regulates proteasome homeostasis, Li [/bib_ref] [bib_ref] Microautophagy -distinct molecular mechanisms handle cargoes of many sizes, Schuck [/bib_ref]. Thus, the lysosomal membrane invaginates to engulf the cytoplasmic component so that it can be degraded accordingly [bib_ref] Lysosome biology in autophagy, Yim [/bib_ref]. On the other hand, CMA is a selective pathway in which the substrate to be degraded has a specific pentapeptide motif, which is identified and subsequently bound by the cytosolic heat-shock cognate 70 (HSC70) [bib_ref] Analysis of Chaperone-mediated autophagy, Juste [/bib_ref] [bib_ref] The coming of age of chaperone-mediated autophagy, Kaushik [/bib_ref]. Consequently, it is linked to lysosome-associated membrane protein 2A (LAMP2A), leading to its translocation into the lysosomes [bib_ref] The coming of age of chaperone-mediated autophagy, Kaushik [/bib_ref].
## E938519-2
## Role of autophagy in parkinson disease
Defective autophagy, CMA, and mitophagy have been shown in dopaminergic neurons in the substantia nigra in postmortem tissue of Parkinson disease patients [bib_ref] Parkinson disease epidemiology, pathology, genetics, and pathophysiology, Simon [/bib_ref] [bib_ref] Mitophagy in Parkinson's disease: Pathogenic and therapeutic implications, Gao [/bib_ref] [bib_ref] Age-dependent accumulation of oligomeric SNCA/alpha-synuclein from impaired degradation in mutant LRRK2 knockin..., Ho [/bib_ref]. Impaired mitophagy was illustrated by the accumulation of defective mitochondria within the autophagosomes in the neurons of Parkinson disease patients [bib_ref] Mitophagy in Parkinson's disease: From pathogenesis to treatment, Liu [/bib_ref].
Most Lewy bodies in the substantia nigra of Parkinson disease brains displayed immunoreactivity for the autophagy-associated protein microtubule-associated protein 1 light chain 3 (LC3), which is involved in autophagosome formation [bib_ref] Autophagy in synucleinopathy: The overwhelmed and defective machinery, Arotcarena [/bib_ref]. Indeed, LC3 immunoreactivity was demonstrated, particularly in a-synuclein-positive Lewy bodies [bib_ref] Autophagy in synucleinopathy: The overwhelmed and defective machinery, Arotcarena [/bib_ref]. Additionally, CMA markers, such as HSC70 and LAMP2A, were selectively reduced in association with a-synuclein buildup in the same Parkinson disease samples [bib_ref] Role of Chaperone-mediated autophagy dysfunctions in the pathogenesis of Parkinson's disease, Sala [/bib_ref].
Parkinson disease is characterized by protein aggregates, namely a-synuclein-containing Lewy bodies [bib_ref] alpha-Synuclein aggregation modulation: An emerging approach for the treatment of Parkinson's disease, Singh [/bib_ref]. Deterioration of the ubiquitin proteasome system (UPS) and autophagy has been proposed to participate in a-synuclein buildup [bib_ref] Modulation of histone deacetylase, the ubiquitin proteasome system, and autophagy underlies the..., El-Saiy [/bib_ref].
Promoting autophagy has been assumed to exert a protective effect in neurodegenerative diseases, including Parkinson disease [bib_ref] Relevance of autophagy and mitophagy dynamics and markers in neurodegenerative diseases, Giorgi [/bib_ref]. Indeed, it is believed that the elimination of Lewy bodies via the activation of the autophagic pathway would impede their possible toxicity, thus interfering with the progression of Parkinson disease [bib_ref] Relevance of autophagy and mitophagy dynamics and markers in neurodegenerative diseases, Giorgi [/bib_ref] [bib_ref] Therapeutic potential of autophagy-enhancing agents in Parkinson's disease, Moors [/bib_ref]. Additionally, deletion of autophagy-related genes, which are essential for the induction and execution of macroautophagy, leads to the presence of protein aggregates and associated neurodegeneration [bib_ref] Autophagy in neurodegenerative diseases: A hunter for aggregates, Park [/bib_ref] [bib_ref] Autophagy and human diseases, Jiang [/bib_ref].
Autophagy is necessary for the turnover of defective mitochondria in Parkinson disease [bib_ref] Neuronal autophagy and neurodegenerative diseases, Son [/bib_ref]. In addition to that, inability to trigger effective mitophagy might serve as another pathogenic mechanism of Parkinson disease [bib_ref] Parkinson's disease and mitophagy: An emerging role for LRRK2, Singh [/bib_ref]. Furthermore, inadequate activation of autophagy can prevent the elimination of protein aggregates and impaired mitochondria [bib_ref] Autophagy in Parkinson's disease, Hou [/bib_ref].
## Association between parkinson disease-related gene mutations and autophagy
Parkinson disease is associated with genetic mutations, and a number of these genes are involved in the regulation of autophagy. Thus, recognition of the particular genes that can be mutated in association with Parkinson disease can greatly improve the understanding of the mechanisms involved in the pathogenesis of Parkinson disease. Thus, in this section, we will review the genetic mutations and their correlation with the impairment of autophagy in Parkinson disease.
## Association between snca gene mutation and autophagy
SNCA gene encodes a-synuclein protein, which is present chiefly in the axon terminals of presynaptic neurons [bib_ref] A novel SNCA A30G mutation causes familial Parkinson's disease, Liu [/bib_ref]. Mutations and multiplications in SNCA gene encoding a-synuclein are linked to familial cases of Parkinson disease [bib_ref] A meta-analysis of alpha-synuclein multiplication in familial Parkinsonism, Book [/bib_ref] [bib_ref] The genetics of Parkinson's disease and implications for clinical practice, Day [/bib_ref].
Wild-type a-synuclein is unfolded and can be sequestered via many pathways, such as autophagy and CMA [bib_ref] Autophagy in a-Synucleinopathies -an overstrained system, Fellner [/bib_ref]. On the contrary, proteins that are encoded by mutant genes can misfold and begin a vicious cycle [bib_ref] Locked in a vicious cycle: the connection between genomic instability and a..., Huiting [/bib_ref]. For example, overexpression of wild-type or mutant a-synuclein can compromise the degradation pathways [fig_ref] Figure 1: Association between a-synuclein and autophagy in Parkinson disease [/fig_ref] , causing abnormal buildup of asynuclein that assembles, thus contributing to the formation of Lewy bodies in Parkinson disease brains [bib_ref] A meta-analysis of alpha-synuclein multiplication in familial Parkinsonism, Book [/bib_ref] [bib_ref] Therapeutic potential of autophagy-enhancing agents in Parkinson's disease, Moors [/bib_ref] [bib_ref] A30P mutant alpha-synuclein impairs autophagic flux by inactivating JNK signaling to enhance..., Lei [/bib_ref]. Indeed, overexpression of wild-type a-synuclein impairs autophagy in mammalian cell lines and in transgenic mice by preventing RAB1A [fig_ref] Figure 1: Association between a-synuclein and autophagy in Parkinson disease [/fig_ref] , which is a GTPase implicated in the early secretory pathway, by causing mislocalization of the early autophagy protein ATG9 [bib_ref] alpha-Synuclein impairs macroautophagy: Implications for Parkinson's disease, Winslow [/bib_ref] [bib_ref] Golgi-associated Rab GTPases implicated in autophagy, Lu [/bib_ref]. However, such defects may possibly be saved by RAB1A overexpression [bib_ref] alpha-Synuclein impairs macroautophagy: Implications for Parkinson's disease, Winslow [/bib_ref].
Additionally, overexpression of wild-type or mutant a-synuclein has been shown to block autophagy in PC12 cells [fig_ref] Figure 1: Association between a-synuclein and autophagy in Parkinson disease [/fig_ref] by binding to both cytosolic and nuclear high mobility group box 1 (HMGB1) in rat PC12 cells, compromising the cytosolic translocation of HMGB1, inhibiting HMGB1-Beclin 1 (BECN1) binding, and enhancing BECN1-BCL2 binding [bib_ref] HMGB1 is involved in autophagy inhibition caused by SNCA/alpha-synuclein overexpression: A process..., Song [/bib_ref]. Conversely, autophagy could be repaired by deleting HMBG1 [bib_ref] HMGB1 is involved in autophagy inhibition caused by SNCA/alpha-synuclein overexpression: A process..., Song [/bib_ref].
Moreover, a-synuclein is degraded primarily by CMA due to its being identified by the HSC70 and bound to LAMP2A at the lysosomal membrane [bib_ref] Suppression of autophagy in the brain of transgenic mice with overexpression of..., Pupyshev [/bib_ref]. However, mutant a-synuclein inhibits CMA [fig_ref] Figure 1: Association between a-synuclein and autophagy in Parkinson disease [/fig_ref] by acting as uptake blockers blocking LAMP2A-channel for the translocation of proteins into lysosomes [bib_ref] Suppression of autophagy in the brain of transgenic mice with overexpression of..., Pupyshev [/bib_ref]. Consequently, mutant a-synuclein accumulates, leading to the compensatory activation of macroautophagy in the brain of transgenic mice with overexpression of A53T-mutant a-synuclein [bib_ref] Suppression of autophagy in the brain of transgenic mice with overexpression of..., Pupyshev [/bib_ref].
Dopamine modifies a-synuclein making it similar to missense mutants that inhibit CMA, and thus it may further contribute to the selective susceptibility in Parkinson disease [bib_ref] Dopamine induces soluble alpha-synuclein oligomers and nigrostriatal degeneration, Mor [/bib_ref] [bib_ref] Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy, Martinez-Vicente [/bib_ref]. Furthermore, a-synuclein has been reported to interact with mitochondria and the mitophagy pathways, both directly and indirectly, since a-synuclein buildup has been correlated with the commencement of mitochondrial impairment [bib_ref] Mitochondrial dysfunction and alpha-Synuclein synaptic pathology in Parkinson's disease: Who's on first?, Zaltieri [/bib_ref]. It is possible that either a-synuclein accumulation occurs first and induces mitochondrial impairment, or mitochondrial insufficiencies cause neuronal deficits and a-synuclein build up [bib_ref] Mitochondrial dysfunction and alpha-Synuclein synaptic pathology in Parkinson's disease: Who's on first?, Zaltieri [/bib_ref]. Additionally, mutant a-synuclein has been shown to trigger mitophagy in dopaminergic neurons of a transgenic mouse model overexpressing mutant a-synuclein, where prominent e938519-3 mitochondrial defects and augmented autophagic cytoplasmic inclusions enclosing mitochondrial remnants were illustrated prior to the degeneration of dopaminergic neurons [bib_ref] A53T human alpha-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects..., Chen [/bib_ref]. Moreover, overexpressing human wild-type and mutant a-synuclein in yeast cells augmented autophagy and mitophagy activities [bib_ref] Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy, Choubey [/bib_ref] [bib_ref] SNCA (alpha-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated..., Sampaio-Marques [/bib_ref]. Under pathological circumstances, a-synuclein is translocated to the inner mitochondrial membrane [bib_ref] Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic..., Devi [/bib_ref] [bib_ref] Alpha-Synuclein and mitochondrial dysfunction in Parkinson's disease: The emerging role of VDAC, Risiglione [/bib_ref]. Consequently, wild-type a-synuclein accumulates in the mitochondria of human dopaminergic neurons, suppressing mitochondrial complex I activity and promoting the formation of ROS [bib_ref] Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic..., Devi [/bib_ref] [bib_ref] Alpha-Synuclein and mitochondrial dysfunction in Parkinson's disease: The emerging role of VDAC, Risiglione [/bib_ref]. Indeed, overexpression of mutant a-synuclein in dopaminergic neurons of transgenic mice led to its presence in monomeric and oligomeric forms onto the mitochondrial membranes and concomitant augmentation of mitophagy [bib_ref] Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy, Choubey [/bib_ref] [bib_ref] Impairment of mitochondria dynamics by human A53T alpha-synuclein and rescue by NAP..., Melo [/bib_ref].
Moreover, incomplete autophagic breakdown of a-synuclein resulted in the formation of a truncated phosphorylated asynuclein species called "pa-syn*", which is hugely neurotoxic [bib_ref] Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial damage and mitophagy..., Grassi [/bib_ref]. Indeed, it induces mitochondrial damage and mitophagy, and thus plays a key role in Parkinson disease pathogenesis [bib_ref] Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial damage and mitophagy..., Grassi [/bib_ref]. pa-syn* has been recognized in neuronal cultures, mouse brains, and in postmortem brains from Parkinson disease patients [bib_ref] Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial damage and mitophagy..., Grassi [/bib_ref].
Studies conducted in cell cultures, animal models, and human postmortem studies suggest a strong correlation between the overexpression of wild-type, mutant, or modified a-synuclein species with impaired autophagy, CMA, and mitophagy routes [bib_ref] Therapeutic potential of autophagy-enhancing agents in Parkinson's disease, Moors [/bib_ref] [bib_ref] A30P mutant alpha-synuclein impairs autophagic flux by inactivating JNK signaling to enhance..., Lei [/bib_ref] [bib_ref] Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial damage and mitophagy..., Grassi [/bib_ref].
## Association between lrrk2 gene mutation and autophagy
LRRK2 gene encodes LRRK2 protein, which is principally present in membrane microdomains, multivesicular bodies, and autophagic vesicles [bib_ref] LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a..., Alegre-Abarrategui [/bib_ref]. Thus, LRRK2 protein is implicated in many cellular signaling pathways such as autophagy, and it possesses two different enzymatic domains, which are the kinase domain that catalyzes phosphorylation, and the Ras of complex (Roc)-GTPase domain that hydrolyses GTP-GDP [bib_ref] LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a..., Alegre-Abarrategui [/bib_ref] [bib_ref] LRRK2 autophosphorylation enhances its GTPase activity, Liu [/bib_ref]. Thus, mutations in LRRK2 can change its expression levels and/or kinase activity [bib_ref] Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced..., Henry [/bib_ref] [bib_ref] Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration..., Reinhardt [/bib_ref]. Additionally, genetic mutations in LRRK2 are associated with the majority of autosomal dominant cases of Parkinson disease [bib_ref] LRRK2 phosphorylation, more than an epiphenomenon, Marchand [/bib_ref].
Furthermore, LRRK2 has been associated with maintaining lysosomal homeostasis by its substrate Rab GTPases [bib_ref] LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease..., Ysselstein [/bib_ref]. Thus, lysosomal overload stress causes translocation and subsequent activation of endogenous LRRK2 onto the overloaded lysosomes, where its substrates are phosphorylated and subsequently stabilized [bib_ref] LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease..., Ysselstein [/bib_ref].
Inhibition of LRRK2 kinase reduced a-synuclein intracellular inclusions by enhancing autolysosome formation and function [bib_ref] Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous..., Obergasteiger [/bib_ref]. Autophagic dysregulation has been demonstrated in familial cases of Parkinson disease that resulted from LRRK2 mutation [bib_ref] Autophagy and lysosomal activity, Madureira [/bib_ref]. For instance, LRRK2-positive cytoplasmic puncta have been shown in association with autophagic vacuoles in human brain sections and in cultured human cells [bib_ref] Genes associated with Parkinson's disease: Regulation of autophagy and beyond, Beilina [/bib_ref] [bib_ref] Autophagy and LRRK2 in the aging brain, Albanese [/bib_ref]. Additionally, overexpression of LC3-II has been shown in transgenic mice expressing mutant LRRK2 [bib_ref] ULK1 and JNK are involved in mitophagy incurred by LRRK2 G2019S expression, Zhu [/bib_ref] [bib_ref] Leucine-rich repeat kinase 1 regulates autophagy through turning on TBC1D2-dependent Rab7 inactivation, Toyofuku [/bib_ref]. Moreover, SH-SY5Y cells expressing mutant LRRK2 displayed prominent increases in autophagic vacuoles, indicating the occurrence of autophagy, which was mediated by mitogen-activated protein kinase/ extracellular signal-regulated protein kinase (MAPK/ERK) [bib_ref] Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells, Plowey [/bib_ref].
Wild-type LRRK2, in particular, can be degraded by CMA [bib_ref] Interplay of LRRK2 with chaperonemediated autophagy, Orenstein [/bib_ref]. However, mutant LRRK2 or overexpressed wild-type LRRK2 can compromise CMA [bib_ref] Interplay of LRRK2 with chaperonemediated autophagy, Orenstein [/bib_ref]. Indeed, mutant LRRK2 triggers LAMP2A and HSC70 buildup, obstructing its translocation at the lysosomal membrane and thus inhibiting CMA [bib_ref] Age-dependent accumulation of oligomeric SNCA/alpha-synuclein from impaired degradation in mutant LRRK2 knockin..., Ho [/bib_ref]. Consequently, a-synuclein breakdown was blocked, resulting in the buildup of oligomeric a-synuclein [bib_ref] Age-dependent accumulation of oligomeric SNCA/alpha-synuclein from impaired degradation in mutant LRRK2 knockin..., Ho [/bib_ref]. Additionally, mutant LRRK2 induced mitophagy by direct interaction with ULK1, which is required for regulating autophagy [bib_ref] ULK1 and JNK are involved in mitophagy incurred by LRRK2 G2019S expression, Zhu [/bib_ref] , and expression of mutant LRRK2 augmented mitophagy by impairing calcium homeostasis [bib_ref] Mutant LRRK2 elicits calcium imbalance and depletion of dendritic mitochondria in neurons, Cherra Sj 3 Rd [/bib_ref].
On the other hand, LRRK2 is recruited from the cytosol to the mitochondria, where wild-type LRRK2 forms a complex with the mitochondrial transport factor RHOT1/Miro1, which is an outer mitochondrial membrane protein, enhancing its elimination that precedes the initiation of mitophagy [bib_ref] Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy, Wang [/bib_ref]. However, mutant LRRK2 disturbed this event, reducing RHOT1/Miro1 removal from damaged mitochondria and subsequently delaying mitophagy [bib_ref] Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy, Wang [/bib_ref]. Additionally, LRRK2 mutations stop depolarization-associated mitophagy by preventing mitochondrial buildup of RAB10 [bib_ref] LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10, Wauters [/bib_ref].
## Association between pink1 and prkn gene mutation and autophagy
Phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) and Parkin (PRKN or PARK2) proteins are functionally correlated and they control mitophagy [bib_ref] LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10, Wauters [/bib_ref]. Their homozygous or heterozygous mutations cause the most common cases of autosomal recessive early-onset Parkinson disease [bib_ref] Disruption of mitochondrial homeostasis: The role of PINK1 in Parkinson's disease, Vizziello [/bib_ref].
PINK1 kinase becomes stabilized on the outer membrane of impaired mitochondria, and it activates the E3 ubiquitin ligase PRKN to induce selective autophagy of damaged mitochondria [bib_ref] PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity, Kane [/bib_ref]. Consequently, PRKN is triggered and recruited to the outer membrane of the impaired mitochondria, affording further Ub moieties for PINK1-dependent phosphorylation in a feed forward loop, which intensifies PINK1 and PRKN actions labeling mitochondria for mitophagy [bib_ref] Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL, Koyano [/bib_ref]. Subsequently, the mitochondria are degraded [bib_ref] Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL, Koyano [/bib_ref].
Thus, the entire loss of PINK1 function due to its mutations accompanied by heterozygous Parkinson disease-associated PRKN mutations inhibit PINK1-PRKN dependent mitophagy [bib_ref] Targeting mitophagy in Parkinson's disease, Clark [/bib_ref]. Consistently, reduced mitophagy or even its absence has been demonstrated in postmortem Parkinson disease brains with PINK1 or PRKN mutations [bib_ref] Mitophagy in Parkinson's disease: From pathogenesis to treatment, Liu [/bib_ref]. Additionally, reduced mitophagy has been shown to result in the buildup of impaired mitochondria, which probably leads to neurodegeneration, particularly in dopaminergic neurons that are selectively susceptible to mitochondrial dysfunction [bib_ref] Mitophagy regulates neurodegenerative diseases, Cen [/bib_ref].
PRKN translocation to mitochondrial membranes, resulting in cell death in cell lines and human iPSC-derived neurons [bib_ref] S-nitrosylation of PINK1 attenuates PINK1/ Parkin-dependent mitophagy in hiPSC-based Parkinson's disease models, Oh [/bib_ref]. Indeed, S-nitrosylated PINK1 has been shown in the brains of a-synuclein transgenic mice, suggesting its involvement in the pathogenesis of Parkinson disease [bib_ref] S-nitrosylation of PINK1 attenuates PINK1/ Parkin-dependent mitophagy in hiPSC-based Parkinson's disease models, Oh [/bib_ref].
Furthermore, PRKN function is progressively lost in dopaminergic neurons during aging and Parkinson disease [bib_ref] Age-associated insolubility of parkin in human midbrain is linked to redox balance..., Tokarew [/bib_ref] , probably due to its being covalently changed by dopamine in substantia nigra only in the normal human brain [bib_ref] Age-associated insolubility of parkin in human midbrain is linked to redox balance..., Tokarew [/bib_ref] , which reduces its solubility and deactivates its E3 ubiquitin ligase [bib_ref] Age-associated insolubility of parkin in human midbrain is linked to redox balance..., Tokarew [/bib_ref].
Altogether, the PINK1-PRKN-dependent mitophagy pathway is necessary for the elimination of impaired mitochondria, and its deficiency is possibly involved in the pathogenesis of Parkinson disease [bib_ref] Mitophagy in Parkinson's disease: From pathogenesis to treatment, Liu [/bib_ref] [bib_ref] Disruption of mitochondrial homeostasis: The role of PINK1 in Parkinson's disease, Vizziello [/bib_ref] [bib_ref] Age-associated insolubility of parkin in human midbrain is linked to redox balance..., Tokarew [/bib_ref].
## Association between vps35 gene mutation and autophagy
Vacuolar protein-sorting 35 (VPS35) gene encodes a subunit of the retromer complex, which mediates the retrograde transport of endosomes to the Golgi complex, thus stimulating the recycling of particular membrane proteins [bib_ref] VPS35, the retromer complex and Parkinson's disease, Williams [/bib_ref]. Additionally, retromer is required for endosomal recruitment of the Wiskott-Aldrich syndrome protein and SCAR homolog complex (WASH), which mediates protein sorting [bib_ref] VPS35, the retromer complex and Parkinson's disease, Williams [/bib_ref].
Mutations in VPS35 are associated with autosomal dominant Parkinson disease [bib_ref] The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse..., Mir [/bib_ref]. D620 mutation of VPS35 decreases its communication with the WASH complex, resulting in abnormal transferring of the autophagy protein Atg9A and impaired autophagosome formation, leading to obstruction of autophagy [bib_ref] Mutation in VPS35 associated with Parkinson's disease impairs WASH complex association and..., Zavodszky [/bib_ref]. Furthermore, mutation of VPS35 enhances LRRK2-mediated phosphorylation of Rab protein in Parkinson disease patients [bib_ref] The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse..., Mir [/bib_ref].
Moreover, mutated or depleted VPS35 has been illustrated to impair endosome-to-Golgi retrieval of LAMP2A and increase LAMP2A breakdown, thus impairing CMA, which is essential for the degradation of a-synuclein, which subsequently builds up, leading to the development of Parkinson disease [bib_ref] VPS35 in dopamine neurons is required for endosome-to-golgi retrieval of Lamp2a, a..., Tang [/bib_ref].
## Association between gba gene mutation and autophagy
The GBA gene encodes glucocerebrosidase (GCase), which is a lysosomal hydrolase enzyme that cleaves glucosylceramide into glucose and ceramide [bib_ref] GBA variants and Parkinson disease: Mechanisms and treatments, Smith [/bib_ref]. Homozygous mutations in GBA gene results in Gaucher disease (GD), which is the most common sphingolipidosis lysosomal storage disorder, and it is classified into three subtypes [bib_ref] GBA variants and Parkinson disease: Mechanisms and treatments, Smith [/bib_ref]. Some patients with type 1 display parkinsonism [bib_ref] GBA variants and Parkinson disease: Mechanisms and treatments, Smith [/bib_ref].
Thus, homozygous or heterozygous mutations in GBA prevent GCase protein and lysosomal degradation, leading to asynuclein accumulation [bib_ref] GBA variants and Parkinson disease: Mechanisms and treatments, Smith [/bib_ref] [bib_ref] GBA deficiency promotes SNCA/alpha-synuclein accumulation through autophagic inhibition by inactivated PPP2A, Du [/bib_ref]. Accordingly, the diminished GCase function probably disrupts the CMA pathway, augmenting a-synuclein accumulation and resulting in dopaminergic neuron degeneration [bib_ref] Mutant glucocerebrosidase impairs alpha-synuclein degradation by blockade of chaperone-mediated autophagy, Kuo [/bib_ref].
## Association between atp13a2 gene mutation and autophagy
ATP13A2 is mutated in some types of juvenile Parkinson disease [bib_ref] ATP13A2 novel mutations causing a rare form of juvenile-onset Parkinson disease, Suleiman [/bib_ref]. It encodes a lysosomal ATPase that is suggested to be a regulator of the autophagy-lysosome pathway [bib_ref] The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common..., Bento [/bib_ref]. Consistently, ATP13A2 promotes intracellular a-synuclein accumulation by impairing lysosome exocytosis using iPSC-derived neurons from Parkinson disease patients [bib_ref] Contribution of autophagy-lysosomal pathway in the exosomal secretion of alpha-Synuclein and its..., Sepulveda [/bib_ref]. Furthermore, ATP13A2 mutation or depletion can contribute to forms of Parkinson disease-associated neurodegeneration, since it negatively regulates another Parkinson disease-correlated gene called synaptotagmin11 (SYT11) at both transcriptional and post-translational levels, leading to mTORC1 activation and decreased levels of SYT11, which cause lysosomal dysfunction and impaired degradation of autophagosomes [bib_ref] The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common..., Bento [/bib_ref]. Additionally, ATP13A2 has been reported to facilitate the recruitment of histone deacetylase 6 (HDAC6) to lysosomes to stimulate autophagosome-lysosome fusion and subsequent autophagy [bib_ref] ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome-lysosome fusion, Wang [/bib_ref].
## Potential therapeutic involvement of autophagy in parkinson disease
Based on the current understanding of the involvement of autophagy in association with the genetic mutations in the pathogenesis of Parkinson disease, augmenting autophagy to prevent protein aggregation and/or eliminate impaired organelles might be considered promising therapeutic approaches.
Ganoderma lucidum extract (GLE) has been shown to suppress parkinsonian phenotype by regulating both mitochondrial function and the autophagic response to oxidative stress [bib_ref] Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative..., Ren [/bib_ref]. Its administration improved the motor deficits and the selective loss of dopaminergic neurons in an MPTP model of Parkinson disease [bib_ref] Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative..., Ren [/bib_ref]. In vitro, GLE treatment reduced MPP + -induced mitochondrial dysfunction and repaired the compromised transport of impaired mitochondria, leading to enhanced autophagy in primary cultured mesencephalic neuronal cells [bib_ref] Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative..., Ren [/bib_ref]. GLE has been shown to protect neuronal cells by stimulating the activation of both the AMPK/mTOR/ULK1 and PINK1/Parkin pathways, and thus stimulating mitophagy in Parkinson disease [bib_ref] Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative..., Ren [/bib_ref]. Thus, GLE might be a promising neuroprotective agent for Parkinson disease [bib_ref] Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative..., Ren [/bib_ref].
## E938519-6
Resveratrol has been demonstrated to induce autophagy in dopaminergic neuronal cell lines, including SH-SY5Y and PC12 cells, leading to enhanced elimination of impaired mitochondria and the degradation of mutant a-synuclein [bib_ref] Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease, Wu [/bib_ref]. Furthermore, resveratrol has been illustrated to induce autophagic clearance of mutant a-synuclein, possibly by activating the AMPK/SIRT1 pathway, which might occur via inhibiting the mTOR pathway [bib_ref] Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease, Wu [/bib_ref].
Rosuvastatin has been shown to ameliorate rotenone-induced neurotoxicity in SH-SY5Y cells through autophagy modulation [bib_ref] Autophagic modulation by rosuvastatin prevents rotenone-induced neurotoxicity in an in vitro model..., Kang [/bib_ref]. Indeed, treatment with rosuvastatin enhanced autophagy by restoring the Beclin-1 expression, increasing the expression of AMPK, and decreasing mTOR expression in a rotenone-induced neurotoxicity model in SH-SY5Y cells [bib_ref] Autophagic modulation by rosuvastatin prevents rotenone-induced neurotoxicity in an in vitro model..., Kang [/bib_ref].
Thus, rosuvastatin appears to exert neuroprotective effects on rotenone-induced dopaminergic neurotoxicity via modulating autophagy, serving as a new potential therapeutic strategy for the treatment of Parkinson disease [bib_ref] Autophagic modulation by rosuvastatin prevents rotenone-induced neurotoxicity in an in vitro model..., Kang [/bib_ref].
The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that plays a vital role in many processes, including autophagy regulation [bib_ref] The role of mammalian target of rapamycin (mTOR) in insulin signaling, Yoon [/bib_ref]. Rapamycin interferes with the assembly of mTOR, blocking its kinase activity [bib_ref] New perspectives on mTOR inhibitors (rapamycin, rapalogs and TORKinibs) in transplantation, Waldner [/bib_ref]. Moreover, rapamycin improved motor dysfunction induced by 6-OHDA, probably by stimulating autophagy subsequent to the obstruction of mTORC1 activation [bib_ref] Therapeutic potential of autophagy-enhancing agents in Parkinson's disease, Moors [/bib_ref]. In the wild-type SNCA transgenic mouse model, rapamycin induced autophagy and decreased the accumulation of neuronal cell bodies and synaptic regions of a-synuclein [bib_ref] Parkinson's disease: Acid-glucocerebrosidase activity and alpha-synuclein clearance, Blanz [/bib_ref]. Additionally, rapamycin ameliorated the Parkinson disease phenotype of Parkin or PINK1 gene deletion in Drosophila and improved the toxic impact of paraquat on Drosophila.
Moxibustion has been illustrated to improve the behavioral performance and to suppress mTOR, enhancing autophagy and promoting the clearance of a-synuclein in a rat model of rotenone-induced Parkinson disease [bib_ref] Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats, Wang [/bib_ref].
Metformin has improved motor impairment and enhanced autophagy via AMPK activation in MPTP/p Parkinson disease model mice [bib_ref] Metformin prevents dopaminergic neuron death in MPTP/P-induced mouse model of Parkinson's disease..., Lu [/bib_ref].
Lithium chloride and valproic acid have been shown to enhance autophagy by suppressing inositol-monophosphatase (IMPase), which cleaves inositol-monophosphate (IMP) producing inositol (Ins), which eventually reduces autophagy [bib_ref] Lithium protects dopaminergic cells from rotenone toxicity via autophagy enhancement, Hou [/bib_ref] [bib_ref] Lithium induces autophagy by inhibiting inositol monophosphatase, Sarkar [/bib_ref] [bib_ref] Therapeutic effects of valproate combined with lithium carbonate on MPTP-induced parkinsonism in..., Li [/bib_ref].
In addition, glycogen synthase kinase3b (GSK3b) has been reported to play a chief role in methamphetamine-induced neurotoxicity by stimulating a-synuclein buildup and obstructing autophagy leading to neurodegeneration [bib_ref] Role of GSK3beta/alpha-synuclein axis in methamphetamine-induced neurotoxicity in PC12 cells, Li [/bib_ref]. Thus, GSK3b inhibition might be considered as a possible therapeutic approach in Parkinson disease [bib_ref] Role of GSK3beta/alpha-synuclein axis in methamphetamine-induced neurotoxicity in PC12 cells, Li [/bib_ref].
Trehalose is a natural disaccharide that inhibits protein accumulation or misfolding and promotes autophagy, thus contributing to the clearance of aggregated wild-type and mutant a-synuclein in MPTP-induced Parkinson disease [bib_ref] Trehalose as a promising therapeutic candidate for the treatment of Parkinson's disease, Khalifeh [/bib_ref]. Accordingly, trehalose has been shown to counteract neurotoxicity in Parkinson disease [bib_ref] Trehalose as a promising therapeutic candidate for the treatment of Parkinson's disease, Khalifeh [/bib_ref].
Corynoxine B is a natural autophagy inducer that repairs the deficient cytosolic translocation of HMGB1 and autophagy in cells overexpressing a-synuclein, blocking a-synuclein-HMGB1 interaction [bib_ref] B1 is involved in autophagy inhibition caused by SNCA/a-synuclein overexpression: A process..., Song [/bib_ref]. Additionally, overexpression of BECN1 or HMGB1 in cells overexpressing a-synuclein repairs autophagy and enhances the elimination of a-synuclein [bib_ref] B1 is involved in autophagy inhibition caused by SNCA/a-synuclein overexpression: A process..., Song [/bib_ref].
Geraniol is an acyclic monoterpene alcohol that is present in the essential oils of several aromatic plants [bib_ref] Geraniol protects against the protein and oxidative stress induced by rotenone in..., Rekha [/bib_ref]. Geraniol has been reported to exert a neuroprotective effect in Parkinson disease by reducing a-synuclein expression and improving autophagy [bib_ref] Geraniol protects against the protein and oxidative stress induced by rotenone in..., Rekha [/bib_ref]. Thus, geraniol has been suggested as a novel therapeutic avenue for clinical intervention in Parkinson disease [bib_ref] Geraniol protects against the protein and oxidative stress induced by rotenone in..., Rekha [/bib_ref].
Baicalein has been shown to exert neuroprotective effects in the substantia nigra of 6-hydroxydopamine-induced Parkinson disease model rats by stimulating mitochondrial autophagy via enhancing the phosphorylation level of AMPK and reducing mTOR.
Venlafaxine rescued dopaminergic neurons, restored the dopamine levels in the striatum, promoted autophagy, and attenuated the buildup of a-synuclein, resulting in motor recovery in rotenone-induced Parkinson disease model rats [bib_ref] Modulation of histone deacetylase, the ubiquitin proteasome system, and autophagy underlies the..., El-Saiy [/bib_ref].
Caffeic acid (CA) has been shown to be neuroprotective, preserving dopaminergic neurons and resulting in motor recovery in a mouse model of Parkinson disease by stimulating autophagy and decreasing A53T a-synuclein [bib_ref] Caffeic acid reduces A53T alpha-synuclein by activating JNK/Bcl-2-mediated autophagy in vitro and..., Zhang [/bib_ref].
However, RNA interference knockdown of LC3 or Atg7, which are involved in autophagy, reversed the effects of LRRK2 mutation reducing autophagy in SH-SY5Y cells expressing mutant LRRK2 [bib_ref] Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells, Plowey [/bib_ref]. Additionally, 4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), which is MAPK/ERK kinase (MEK) inhibitor 1, reduced LRRK2-induced neuritic autophagy in SH-SY5Y cells expressing mutant LRRK2 [bib_ref] Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells, Plowey [/bib_ref].
RHOT1 RNAi repairs mitophagy in neurons expressing mutant LRRK2, and protects them from oxidative stress [bib_ref] Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy, Wang [/bib_ref]. Additionally, the neuroprotective impact of reducing RHOT1 protein levels was tested in vivo in a Drosophila model, which overexpressed human mutant LRRK2 and displayed locomotor deficits and dopaminergic neuronal loss [bib_ref] Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy, Wang [/bib_ref]. However, knocking down Miro by RNAi entirely alleviated the locomotor defects and prevented dopaminergic neurodegeneration [bib_ref] Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy, Wang [/bib_ref].
## E938519-7
Mitophagy compromised in LRRK2 mutant patient cells was repaired by deletion of the mutant LRRK2 or inhibition of LRRK2 kinase [bib_ref] LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10, Wauters [/bib_ref].
## Future developments in gene-based diagnosis and treatment of parkinson disease
## Diagnosis of parkinson disease
Unfortunately, urgent medical requirements in the treatment of Parkinson disease are unmet, since no clinical biomarkers are currently available for early diagnosis of Parkinson disease, and there is no effective therapy that can interfere with the pathogenesis or progression of Parkinson disease [bib_ref] The future of targeted gene-based treatment strategies and biomarkers in Parkinson's disease, Polissidis [/bib_ref].
Diagnostic biomarkers of Parkinson disease, including a-synuclein, LRRK2, and b-glucocerebrosidase (GCase), have been illustrated in biofluid studies using cerebrospinal fluid, blood cells, and urine [bib_ref] The future of targeted gene-based treatment strategies and biomarkers in Parkinson's disease, Polissidis [/bib_ref]. Thus, there is a critical need to establish highly sensitive and specific assays of the specific biomarkers that are reliable for differential diagnosis [bib_ref] Recent advances in biomarkers for Parkinson's disease, He [/bib_ref].
Clinical assessment, neuroimaging, and electrophysiology have been helpful in the diagnosis and prognosis of Parkinson disease, since they can help in detecting the abnormalities that can precede the disease onset [bib_ref] Imaging in Parkinson's disease, Pagano [/bib_ref]. For instance, magnetic resonance imaging (MRI), which illustrates structural brain differences, might be useful in differentiating idiopathic Parkinson disease from other parkinsonian phenotypes [bib_ref] Imaging in Parkinson's disease, Pagano [/bib_ref]. Furthermore, considering the functional level, alterations in circulating oxygen levels might be useful in differentiating idiopathic Parkinson disease from a healthy state [bib_ref] Recent advances in biomarkers for Parkinson's disease, He [/bib_ref].
Moreover, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) assesses variations among biomolecules and macromolecules, such as dopamine transporter (DAT), which is regarded as a diagnostic tool [bib_ref] Recent advances in biomarkers for Parkinson's disease, He [/bib_ref] [bib_ref] Brain 18F-FDG PET imaging in the differential diagnosis of parkinsonism, Akdemir [/bib_ref]. Additionally, electrophysiology might be helpful in assessing the neuronal activity and the subsequent monitoring of the disease.
However, because Parkinson disease is heterogeneous and multifactorial in its pathophysiology, the degree of the reliability of these approaches cannot be determined, and a combination of many methods and procedures may be needed [bib_ref] The future of targeted gene-based treatment strategies and biomarkers in Parkinson's disease, Polissidis [/bib_ref].
## Potential therapies targeting a-synuclein
Since a-synuclein plays a key role in the pathogenesis of Parkinson disease, it is considered as a potential therapeutic target in many ongoing studies [fig_ref] Table 1: Future developments in gene-based treatment of Parkinson disease [/fig_ref] that aim at attenuating its burden by decreasing its gene and protein expression, repairing its proteostasis, or restricting its spread to anatomically interconnected brain regions [bib_ref] alpha-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease, Cole [/bib_ref].
A potential therapeutic approach that is expected to reduce asynuclein expression is the use of antisense oligonucleotides [bib_ref] alpha-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease, Cole [/bib_ref] , which are complimentary single-stranded RNA oligomers that can bind particular sequences within an mRNA molecule, resulting in the digestion of mRNA [bib_ref] alpha-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease, Cole [/bib_ref]. Preclinically, antisense oligonucleotides have been successful in reducing a-synuclein expression in animals [bib_ref] alpha-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease, Cole [/bib_ref].
Repairing a-synuclein proteostasis can potentially interfere with the progression of Parkinson disease, and it can be accomplished via preventing protein misfolding and accumulation or promoting its elimination [bib_ref] Nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic..., Hebron [/bib_ref] [bib_ref] The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an..., Price [/bib_ref]. For instance, an inhibitor of a-synuclein called NPT200-11 has been shown to be potentially useful in ameliorating a-synuclein and motor function, Antisense oligonuleotides Suppressing the overall kinase activity of LRRK2 [bib_ref] LRRK2 antisense oligonucleotides ameliorate alpha-Synuclein inclusion formation in a Parkinson's disease mouse..., Zhao [/bib_ref] GCase PR001A Augmenting GCase activity using gene therapy [bib_ref] Gene therapy for Parkinson's disease associated with GBA1 mutations, Abeliovich [/bib_ref] Ambroxol Increasing GCase activity [bib_ref] Ambroxol for the treatment of patients with Parkinson disease with and without..., Mullin [/bib_ref] LRRK2 -leucine-rich repeat kinase 2; GCaseb-glucocerebrosidase.
## E938519-8
and completed phase I clinical trials [bib_ref] The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an..., Price [/bib_ref]. Furthermore, nilotinib, which is an anti-cancer drug that inhibits c-Able tyrosine kinase augmenting the autophagic elimination of a-synuclein, is currently in phase 2 trials [bib_ref] Nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic..., Hebron [/bib_ref] [bib_ref] Nilotinib effects in Parkinson's disease and dementia with Lewy bodies, Pagan [/bib_ref].
a-synuclein can spread among neurons, since it can be secreted extracellularly and subsequently taken up by adjacent neurons [bib_ref] Therapeutic approaches to target alpha-Synuclein pathology, Brundin [/bib_ref]. Thus, such extracellular a-synuclein has been targeted therapeutically by passive or active immunotherapy, in which a-synuclein-specific antibodies are used or synthesis of endogenous antibodies is induced consequent to the administration of modified a-synuclein [bib_ref] Safety and tolerability of multiple ascending doses of PRX002/RG7935, an anti-alpha-Synuclein monoclonal..., Jankovic [/bib_ref] [bib_ref] Safety and tolerability of active immunotherapy targeting alpha-Synuclein with PD03A in patients..., Poewe [/bib_ref]. Passive and active immunotherapies might be useful in attenuating extracellular a-synuclein and the burden of its toxic accumulation and consequently reducing its dissemination to anatomically interconnected brain areas [bib_ref] Safety and tolerability of multiple ascending doses of PRX002/RG7935, an anti-alpha-Synuclein monoclonal..., Jankovic [/bib_ref] [bib_ref] Safety and tolerability of active immunotherapy targeting alpha-Synuclein with PD03A in patients..., Poewe [/bib_ref].
Passive immunotherapy method is being attempted in clinical trials using many drugs, such as prasinuzemab, which is currently being assessed in phase II [bib_ref] Safety and tolerability of multiple ascending doses of PRX002/RG7935, an anti-alpha-Synuclein monoclonal..., Jankovic [/bib_ref]. Similarly, an active immunotherapy approach has been attempted in clinical trials using drugs, including PD03A, which has completed phase 1 trials [bib_ref] Safety and tolerability of active immunotherapy targeting alpha-Synuclein with PD03A in patients..., Poewe [/bib_ref].
## Potential therapies targeting lrrk2
Neuronal death caused by LRRK2 mutation in the PARK8 locus on chromosome 12 was shown to be kinase-dependent [bib_ref] Kinase activity of mutant LRRK2 mediates neuronal toxicity, Smith [/bib_ref]. Thus, selective inhibitors of LRRK2 kinase activity were regarded as potential therapeutic candidates [bib_ref] Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease, Jennings [/bib_ref]. For instance, the selective LRRK2 kinase inhibitor, DNL201, completed a phase I clinical trial, suggesting LRRK2 kinase activity as a potential therapeutic target [fig_ref] Table 1: Future developments in gene-based treatment of Parkinson disease [/fig_ref] in Parkinson disease [bib_ref] Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease, Jennings [/bib_ref].
Alternatively, antisense oligonucleotides can potentially be used in suppressing the overall kinase activity of LRRK2 by obstructing LRRK2 function [bib_ref] LRRK2 antisense oligonucleotides ameliorate alpha-Synuclein inclusion formation in a Parkinson's disease mouse..., Zhao [/bib_ref]. Indeed, LRRK2 antisense oligonucleotides have been reported to decrease Ps-129-a-synuclein inclusions by about 50% compared to controls [bib_ref] LRRK2 antisense oligonucleotides ameliorate alpha-Synuclein inclusion formation in a Parkinson's disease mouse..., Zhao [/bib_ref]. Additionally, LRRK2 antisense oligonucleotides have been demonstrated to be useful in inhibiting Parkinson disease-associated pathology and phenotypes [fig_ref] Table 1: Future developments in gene-based treatment of Parkinson disease [/fig_ref] [bib_ref] LRRK2 antisense oligonucleotides ameliorate alpha-Synuclein inclusion formation in a Parkinson's disease mouse..., Zhao [/bib_ref].
## Potential therapies targeting gcase
GCase is reduced in the brains of patients with Parkinson disease, particularly those with GBA1 mutations [bib_ref] Ambroxol for the treatment of patients with Parkinson disease with and without..., Mullin [/bib_ref]. GCase insufficiency may result in the buildup of a-synuclein by maintaining oligomers, which subsequently lead to more reduction in GCase activity, resulting in a positive forward feedback loop [bib_ref] Gaucher disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loop in synucleinopathies, Mazzulli [/bib_ref] [bib_ref] Glucocerebrosidase mutations in subjects with parkinsonism, Lwin [/bib_ref]. Additionally, GCase inadequacy causes lysosomal impairment, leading to proteinopathy in synucleinopathies [bib_ref] Gaucher-related synucleinopathies: The examination of sporadic neurodegeneration from a rare (disease) angle, Sardi [/bib_ref].
Based on their effectiveness in GD treatment, therapies like enzyme replacement therapy and glucosylceramide synthase inhibitors have been considered as potential therapeutic approaches [fig_ref] Table 1: Future developments in gene-based treatment of Parkinson disease [/fig_ref] in GBA-associated Parkinson disease [bib_ref] Augmenting CNS glucocerebrosidase activity as a therapeutic strategy for parkinsonism and other..., Sardi [/bib_ref]. For instance, PR001A, which augments GCase activity using gene therapy, is being assessed in a phase I safety trial [bib_ref] Gene therapy for Parkinson's disease associated with GBA1 mutations, Abeliovich [/bib_ref]. Another example is ambroxol, which is an inhibitory small-molecule chaperone that increases GCase activity and decreases a-synuclein levels [bib_ref] Ambroxol for the treatment of patients with Parkinson disease with and without..., Mullin [/bib_ref]. It is currently in phase 2 clinical trials in Parkinson disease [bib_ref] Ambroxol for the treatment of patients with Parkinson disease with and without..., Mullin [/bib_ref].
# Conclusions
There is no effective treatment that is capable of preventing neurodegeneration and disease progression in Parkinson disease. Instead of that, the current treatment for Parkinson disease is merely for symptomatic relief. The pathogenesis of Parkinson disease involves autophagy impairment. Various agents, which can stimulate the clearance of toxic a-synuclein by enhancing autophagy, could be regarded as potential therapeutic agents to halt or delay the progression of Parkinson disease. Genetic mutations associated with Parkinson disease have been shown to impact autophagy, leading to its impairment. Additionally, there is a critical need to establish highly sensitive and specific assays of the particular gene-based diagnostic biomarkers of Parkinson disease, including a-synuclein, LRRK2, and GCase, which are reliable for differential diagnosis. Furthermore, potential therapeutic targets that are gene-based in Parkinson disease are being currently developed.
## Declaration of figures' authenticity
All figures submitted have been created by the authors, who confirm that the images are original with no duplication and have not been previously published.
e938519-9
[fig] Figure 1: Association between a-synuclein and autophagy in Parkinson disease. (A) Overexpression of wild-type a-synuclein impairs autophagy by preventing Ras-associated binding 1A (RAB1A) protein, leading to the mislocalization of the early autophagy protein ATG9. (B) Overexpression of wild-type or mutant a-synuclein was shown to block autophagy by binding to both cytosolic and nuclear high mobility group box 1 (HMGB1), compromising the cytosolic translocation of HMGB1, inhibiting HMGB1-Beclin 1 (BECN1) binding, and enhancing BECN1-B-cell lymphoma 2 (BCL2) binding. (C) Mutant a-synuclein inhibits chaperone-mediated autophagy (CMA) by acting as uptake blockers blocking lysosome-associated membrane protein 2A (LAMP2A)channel for the translocation of proteins into lysosomes. Subsequently, mutant a-synuclein accumulates, leading to the compensatory activation of macroautophagy. This figure was generated using Microsoft PowerPoint Software, version 10, Microsoft Corp., USA. [/fig]
[table] Table 1: Future developments in gene-based treatment of Parkinson disease. [/table]
[bib_ref] Historical perspective: Models of Parkinson's disease, Chia [/bib_ref] [bib_ref] Historical perspective: Models of Parkinson's disease, Chia [/bib_ref] [bib_ref] Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice, Kitada [/bib_ref] [bib_ref] Loss of Pink1 modulates synaptic mitochondrial bioenergetics in the rat striatum prior..., Stauch [/bib_ref] [bib_ref] Mitophagy in Parkinson's disease: From pathogenesis to treatment, Liu [/bib_ref] [bib_ref] Parkin and PINK1 patient iPSCderived midbrain dopamine neurons exhibit mitochondrial dysfunction and..., Chung [/bib_ref] [bib_ref] S-nitrosylation of PINK1 attenuates PINK1/ Parkin-dependent mitophagy in hiPSC-based Parkinson's disease models, Oh [/bib_ref] [bib_ref] Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced..., Seibler [/bib_ref] [bib_ref] S-nitrosylation of PINK1 attenuates PINK1/ Parkin-dependent mitophagy in hiPSC-based Parkinson's disease models, Oh [/bib_ref] |
Case Report of Two Cases of Patella Subacute Osteomyelitis in Diabetic Women A Rare Entity
Journal of Orthopaedic Case Reports 2016 April -June: 6(2):Page 13-15 Case Report Introduction: Patella osteomyelitis is a rare entity in adults. Most often it is seen in children of five to twelve years of age because of its unique ossification and vascularity. Immuno compromised states like HIV, tuberculosis, intravenous drug abuse and trauma have been predisposing factors for adult patellar osteomyelitis. We report two cases of patellar osteomyelitis in adult diabetic women with uncontrolled glycemic levels and having no previous history of any trauma or systemic infection. Case Report: A 43-year-old diabetic woman presented with complaints of left knee pain and swelling with no history of trauma. On examination, pointed tenderness was present over anterior aspect of patella with patellar grind test positive. Radiography and MRI revealed solitary well circumscribed patellar cyst. Lateral chondral blisters were noted while doing arthroscopy and secretions oozed out on puncturing. Curettage was carried out for the same. Culture and sensitivity revealed no growth and the patient was prescribed antibiotics for 6 weeks. Second case was a 46-year-old diabetic lady with similar presentation. MRI additionally showed abscess in intermuscular plains around knee joint. An aspirated fluid was negative for growth of organisms. Knee arthrotomy and curettage of patellar sinus tracts was done with evacuation of intramuscular abscess. Antibiotics were given for 6 weeks. Both patients had complete relief of symptoms. Conclusion: Patella osteomyelitis in adults is very rare. In patients with uncontrolled diabetes, vague anterior knee pain, elevated ESR and CRP, one should keep patellar subacute osteomyelitis as a differential diagnosis which can be further confirmed by X-ray, MRI or bone scan. An appropriate early treatment with antibiotics and surgical intervention can give a satisfactory result.
# Introduction
Patella is the largest sesamoid bone in our body. It appears as a cartilaginous structure at birth and starts to ossify at the age of five years. Patella osteomyelitis is a rare condition, with highest incidence found in children between ages of five to twelve years. In 1829, Thirion from Belgium was first to report a case of patella osteomyelitis. Primary hematogenous osteomyelitis of patella in adults is a rare entity often seen in immunocompromised patients with predisposing factors like HIV, intravenous drug abuse, disseminated tuberculosis and trauma. With recent surge in incidence of diabetes mellitus which also produces an immunocompromised state, may predispose to a similar condition. Here, we report two cases of patellar osteomyelitis in adult diabetic women.
## Case 1:
A 43-year-old lady presented to us with complaints of severe left anterior knee pain and swelling with no history of any trauma. Patient has been taking treatment with her family physician for past six weeks with no relief of symptoms but aggravation of symptoms for past few days. On examination patient was having low grade fever and rest of vitals were normal. No history of any other joint involvement, activities of daily living were affected and because of the knee pain, unable to squat and walk pain free. Patient is a known case of diabetes on medications but with uncontrolled glycemic levels. Local examination revealed local rise of temperature, minimal effusion, no discoloration of skin or any signs of trauma, point tenderness over the anterior aspect of patella, patella grind test positive and knee range of movements painful and restricted, no instability present. Investigations revealed uncontrolled sugars (fasting & postprandial and glycosylatedhemoglobin), elevated ESR 82mm at 1sthour and CRP2.9mg/L. X-ray showed solitary localized, well circumscribed patellar cyst and MRI confirmed it. Knee aspiration was sterile. Patient was subjected to knee arthroscopy where chondral blister was present on lateral facet of patella . On probing secretion oozed out, which was sent for culture and sensitivity. Curettage and thorough lavage was given. Culture reports had no growth but few pus cells were noted. Intravenous clindamycin and amikacin were given for seven days followed by oral clindamycin for next five weeks. Patient was immobilized in long knee brace with isometric and intermittent ROM exercises. Patient has excellent relief of symptoms and on further physiotherapy regained complete pain free range of movements in next four weeks.
## Case 2:
A 46-year-old diabetic lady with knee pain on and off for past few months, presented with severe anterior knee pain, swelling over knee and distal thigh, inability to walk, squat and complete restriction of knee movements. Patient was having high grade fever with chills and rigor. On examination, local warmth, pre patellar swelling with extension in to distal thigh and minimal joint effusion present. Tenderness over patella, patella grind test positive, ROM was painful and restricted terminally (50 -1000) and no instability was noted. Blood test revealed uncontrolled sugars, elevated ESR (82mm at the end of 1 hour), CRP (12.8mg/dl). Radiograph had a lytic lesion in patella which was identified retrospectively. MRI showed an abscess involving intermuscular plains of quadriceps encircling the distal femur and posterior compartmental muscles of leg with osteomyelitis of patella . No evidence of osteomyelitis in femur and tibia. Knee aspiration was sterile. Venous Doppler study was done to rule out DVT. Patient was started on intravenous antibiotics, but had no relief of pain, so subjected to surgical intervention, knee arthrotomy with incision and drainage of abscess in quadriceps muscle was done. On patellar eversion chondral blister was seen, curettage and debridement was done. Tissue sent for histopathological examination revealed necrotic and granulation tissue consistent with osteomyelitis, tuberculosis was ruled out but no specific organism was identified. Patient had significant relief of symptoms with intravenous antibiotics for two weeks and oral antibiotics for four weeks. Patient was immobilized in long knee brace with isometric and intermittent ROM exercises. An adequate glycemic control was achieved with appropriate insulin and antidiabetic drugs.
# Discussion
The literature search revealed patellar osteomyelitis is a rare condition and is predominantly seen in the age group of 5 to 12
year. Patella is a cartilaginous structure with limited blood supply till the age of 5 year. Ossification begins from the age of 5 year with rich vascularisation till the age of 12 and is completed by the age of 15, beyond which the vascularity again diminishes and hence explains higher incidence of osteomyelitis between 5 to 12 year of age and decreased incidence in adults. The cartilage layer which is resistant to infection is thick in children and hence infection remains confined to patella without extending into the knee joint. Whereas in adults cartilage gets thinned out and may lead to septic arthritis of knee. Also lack of periosteum over patella explains absence of periosteal reaction which is typical of osteomyelitis at any other site. The sympathetic reaction would produce a sterile knee effusion.series most of the patients were between five and fifteen years, only one of his patients was infant and none adult.suggested that the rarity of the disease under the age of five was explained by the fact that before that age the patella is cartilaginous.showed by injection experiments that until the age of about four years the patella is largely a cartilaginous mass, the very little blood supply. Vascularisation then proceeds with ossification, small vessel passing into the patella from a plexus on its anterior surface. Vascularisation reaches the maximum at twelve. Very few cases of adult patella osteomyelitis have been notified in the literature. History of trauma can predispose to it, can also be as a sequalae to infected prepatellar bursitis [bib_ref] Osteomyelitis of the patella, Evans [/bib_ref]. Staphylococcusaureus is found to be most common organism in hematogenous osteomyelitis [6] followed by tubercular etiology. Immunocompromised states like HIV, intravenous drug abuse, patient on chemotherapy for malignancies are more prone for patellar infections [bib_ref] Pseudomonas Osteomyelitis of the patella, Mund [/bib_ref] [bib_ref] Hematogenous Osteomyelitis of the patella associated with human immunodeficiency virus, Vail [/bib_ref]. Pseudomonas osteomyelitis was reported in intravenous drug abuse by Munddj et al in 1981 [bib_ref] Hematogenous Osteomyelitis of the patella associated with human immunodeficiency virus, Vail [/bib_ref]. In our cases study even though no organisms were identified, tuberculosiswas ruled out and so non-specific organism should have been an etiology and in our patients gradual onset and progressive symptoms were again suggestive of subacute nature of the infection which is more common in diabetic patient. But till now no cases of patellar osteomyelitis in diabetes have been reported in literature. The author believes that immune compromised states which exist in patient with uncontrolled diabetes mellitus are also prone to develop such infections. Few studies have been conducted on immunological function in relation to increased susceptibility to infections in diabetes mellitus [bib_ref] Immunodeficiencies in diabetes and mycobacterial infections, Ce Prasad [/bib_ref]. Bybee and Rogers additionally had shown that phagocyte functional defect was not confined to ketoacidosisbut also occurred in poorly controlled diabetics [bib_ref] The phagocytic activity of polymorphonuclear leucocytes obtained from patients with diabetes mellitus, Bybee [/bib_ref]. Good control of diabetic state reversed the phagocyte defect. Another interesting observation, which suggests the possibility of genetic determination Journal of Orthopaedic Case Reports Volume 6 Issue 2 April -June 2016 Page 13-15 | | | | of the phagocyte defect, by Molenaar [bib_ref] Leucocyte chemotaxis in diabetic patients and their nondiabetic first degree relatives, Molenaar [/bib_ref]. Serum factors played an important role in the phagocyte function of the diabetics; certain serum factors may competitively bind neutrophil receptors thereby preventing complement mediated phagocytosis. Elimination of circulating immune complexes is also poor amongst diabetics. Chemotaxis of neurophils was found to be less in diabetics in a well controlled study of Mowat and Baum [bib_ref] Chemotaxis of polymorphonuclear leucocytes from patients with diabetes mellitus, Mowatt [/bib_ref]. Cell mediated immunity (CMI) is a very important arc of host immunity. Assessment of lymphocyte transformation in response to phytohaemagglutinin showed a decrease in diabetics with poor control. So a high index of suspicion is necessary. Supportive blood investigations, X-rays, MRI and bone scan confirm the diagnosis. When diagnosed early an arthroscopic evaluation can be diagnostic and therapeutic [16] but incases where there is a well set infection with abscess extending into quadriceps muscle an open procedure in indicated. Needle aspiration and lavage technique has been reported in a paper with satisfactory outcome. Most of the time cultures remain sterile, particularly in diabetic patients. So empirical broad spectrum antibiotics will give satisfactory results.
Immobilization with knee brace and isometricand intermittent ROM exercises will avoid knee stiffness.
# Conclusion
Patella osteomyelitis is a rare condition in adults, commonly seen in children. Adults with risk factors like intravenous drug abuse, HIV infection, and trauma have been common cause mentioned in the literature. Recent surge of diabetes mellitus which also produce an immuno compromised state and more prone for such infections which may have an atypical presentation, so one should have an high index of suspicion to reach the diagnosis and intervene early to have a satisfactory outcome.
Atypical Anterior knee pain with poor glycemic control in a diabetic patient, a high index of suspicion toward subacute patellar osteomyelitis will enable early diagnosis and treatment and avoid complications.
## Clinical message
www.jocr.co.in |
Wheezing of unexpected etiology: A case report of pea aspiration mimicking bronchus obstruction caused by a tumor
Introduction: Geriatric patients with foreign body aspiration (FBA) lack a detailed medical history. Meanwhile, FBA can mimic other diseases and present with wheezing. Here, we report on the difficulty of making a diagnosis of FBA in an elderly man with wheezing. Case presentation: An 84-year-old man presented with wet cough. He had progressive lung cancer, for which only supportive care was provided. His physical examination revealed wheezing. We presumptively diagnosed acute exacerbation of chronic obstructive pulmonary disease (AE-COPD) and administered inhaled procaterol and parenteral methylprednisolone, which did not mitigate his symptoms. Computed tomography revealed a round nodule, 1 cm in diameter, in his right intermediate bronchus. Central airway obstruction (CAO) caused by the tumor was initially suspected. However, bronchoscopy revealed a pea (Pisum sativum) lodged in his right bronchus, which was removed using forceps. Discussion: The typical clinical presentations of FBA are sudden-onset cough and dyspnea. However, some geriatric patients do not have cough or lack the cognitive capacity to inform of an episode of FBA. FBA can mimic other entities including AE-COPD and CAO, as seen in our case. Clinicians should not terminate the diagnostic process until all available information explaining the patient's signs and symptoms is collected. Conclusion: Despite the lack of apparent aspiration, FBA is an important differential diagnosis of new wheezing in the elderly. Physicians need to carefully evaluate "trivial" information or collect additional information when encountering airway symptoms in elderly patients to avoid missing an FBA diagnosis.
# Introduction
Foreign body aspiration (FBA) can mimic other diseases and present with wheezing. Here, we report a case of a patient originally diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AE-COPD), then suspected to have central airway obstruction (CAO) caused by a tumor, and finally diagnosed with FBA, highlighting the importance of including FBA in the differential diagnosis when elderly patients present with wheezing. This case was reported according to the SCARE statement [bib_ref] The SCARE 2020 guideline: updating consensus surgical CAse REport (SCARE) guidelines, Agha [/bib_ref].
## Case presentation
An 84-year-old man presented with wet cough. He reported that his cough had started 8 hours prior to this visit and gradually exacerbated, prompting his visit to the emergency department. He had no other respiratory symptoms such as sore throat, nasal discharge, or chest pain.
His medical history included lung cancer and chronic obstructive pulmonary disease (COPD). He denied history of FBA or aspiration pneumonia. His lung cancer had infiltrated the left vagal nerve without metastases, for which he only received supportive care. He also required 2.5 L/min oxygen as home oxygen therapy for COPD. He had been using vilanterol, fluticasone, glycopyrronium, and L-carbocisteine regularly for COPD, and had no known allergies to drugs or foods. His vital signs on arrival were as follows: body temperature, 36.2 - C; heart rate, 93 beats/min; blood pressure, 171/65 mmHg; respiratory rate, 30/min, and oxygen saturation on 3 L/min nasal cannula oxygen, 96%. Wheezing was noted on physical examination. The white blood cell count was 9,100 cells/mcL (neutrophils, 72.9%; and lymphocytes, 20.2%). A chest radiograph revealed the preexisting left lung tumor; no new findings were noted.
We presumptively diagnosed AE-COPD possibly due to a viral infection and administered inhaled procaterol and parenteral methylprednisolone (40 mg) first. However, because the response to these drugs was limited, we considered the possibility of bronchial diseases such as CAO given his history of untreated lung cancer and the lack of new findings on the chest radiograph. Non-contrast computed tomography of the lung showed a round nodule, 1 cm in diameter, in the right intermediate bronchus, which partially obstructed the bronchial lumen [fig_ref] Figure 1: A round nodule in the patient's right intermediate bronchus on non-contrast computed... [/fig_ref]. Considering that the nodule had not been present 2 months prior to this visit, and only supportive care was being provided to the patient for his lung cancer, we suspected progression of the tumor. A pulmonary physician with 15 years' experience performed a bronchoscopy. Bronchoscopy revealed a pea (Pisum sativum) lodged in the patient's right bronchus [fig_ref] Figure 2: A pea [/fig_ref] , which established the diagnosis of pea aspiration. The pea was subsequently removed using forceps, and the wheezing resolved. There were no complications during the bronchoscopy. Repeat history taking from the patient's family suggested that the cough started while the patient ate peas, which also supported the diagnosis. He was discharged with no sequelae on hospitalization day 5. On discharge, we advised the patient and his family to seek medical advice if he experienced an acute exacerbation of his respiratory symptoms while eating because it might indicate aspiration of food. They understood this advice and the patient did not experience recurrent food aspiration during 5 months of follow-up.
# Discussion
Foreign body aspiration (FBA) frequently occurs in children aged < 3 years [bib_ref] A history of recurrent wheezing can delay the diagnosis of foreign body..., Colavita [/bib_ref]. However, FBA can also occur in adults, including geriatric patients, particularly those with neurological impairment and drug or alcohol intoxication. Geriatric patients with FBA often aspirate teeth, pins, and whistles [bib_ref] Foreign body inhalation in the adult population: experience of 25,998 bronchoscopies and..., Sehgal [/bib_ref].
The typical clinical presentations of FBA are sudden-onset cough and dyspnea. However, some geriatric patients do not have cough or lack the cognitive capacity to inform of an episode of FBA. Lin et al. reported that only 29% of geriatric patients can declare an event of aspiration [bib_ref] Cayenne aspiration: an unusual type of lower airway foreign-body aspiration, Lin [/bib_ref]. Furthermore, the asymptomatic period from the timing of aspiration can be longer than the following symptomatic period [bib_ref] Diagnose of occult bronchial foreign body: a rare case report of undetected..., Wang [/bib_ref]. These factors complicate diagnosing geriatric patients with FBA. Furthermore, chest radiographs do not detect approximately 80% of foreign bodies [bib_ref] Ugarte Fornell, Rhonchus and valve-like sensation as initial manifestations of longstanding foreign..., Cherrez-Ojeda [/bib_ref]. Therefore, to directly detect and remove foreign bodies, bronchoscopy should be considered.
FBA can mimic entities such as asthma, anaphylaxis, angioedema, CAO, AE-COPD, congestive heart failure, drug reaction, and vocal cord dysfunction [bib_ref] Clinical mimics: an emergency medicine-focused review of asthma mimics, Kann [/bib_ref]. CAO caused by malignancy is mostly observed in patients with lung cancer [bib_ref] Central airway obstruction: benign strictures, tracheobronchomalacia, and malignancy-related obstruction, Murgu [/bib_ref]. Metastases, mostly from thyroid, breast, or colon cancer, melanoma, or renal carcinoma, can also obstruct the central airways [bib_ref] Central airway obstruction: benign strictures, tracheobronchomalacia, and malignancy-related obstruction, Murgu [/bib_ref]. As both FBA and CAO can be associated with obstructive pneumonia, atelectasis, lung abscess, or pleural effusion, early diagnosis and restoration of the obstructed airway are important.
The symptoms of FBA in geriatric patients can be atypical, and detailed history taking is not always possible. Thus, we should seek other clues for the diagnosis of FBA. For example, there may be some differences in the respiratory sounds between the bilateral lungs. Bronchial foreign bodies can present as unilateral wheezing [bib_ref] Calcium pill aspiration: a case report, Brewer [/bib_ref]. A prone or a head-down position can relieve symptoms of FBA [bib_ref] Effect of body position on relieve of foreign body from the airway, Luczak [/bib_ref]. The respiratory sounds also diminish or change in these positions. Although chest radiographs cannot always detect foreign bodies, hyperinflation of the affected lung can be an indirect sign of FBA. Clinicians should seek subtle physical signs and abnormalities on imaging for a prompt and accurate FBA diagnosis. In our patient, we failed to promptly diagnose FBA for the following reasons. First, we should have obtained the medical history earlier from the patient's accompanying family. Only on repeat history taking, the patient's family informed us that the cough suddenly started while the patient was eating peas. Thus, we should seek more information from any accompanying persons when encountering respiratory symptoms that cannot be easily explained based on the medical history and underlying conditions. Second, we did not screen for laterality of, or changes in, auscultation findings in different body positions. Third, we initially detected no changes in the patient's chest radiograph. However, upon retrospectively reviewing the radiograph , we noted hyperinflation of the affected lung in the newer one , which could indirectly suggest the existence of a check-valve mechanism in the lung. Finally, there might have been availability and anchoring biases involved. Availability bias refers to the tendency to overestimate the likelihood of events when they are easily recalled [bib_ref] Cognitive errors detected in anaesthesiology: a literature review and pilot study, Stiegler [/bib_ref]. Anchoring bias is defined as the tendency to be attached to particular initial traits of a presentation very early in the diagnostic process, making it difficult to correct errors afterward [bib_ref] Cognitive errors detected in anaesthesiology: a literature review and pilot study, Stiegler [/bib_ref]. Our initial diagnosis of AE-COPD was influenced by availability bias because respiratory symptoms in patients with COPD are often related to AE-COPD. The later diagnosis of CAO was susceptible to anchoring bias because this patient had not experienced AE-COPD previously but had an untreated lung cancer, which could potentially manifest as metastasis to the bronchus, thereby developing CAO. Our case emphatically suggests that clinicians should not terminate the diagnostic process until all available information explaining the patient's signs and symptoms is collected.
## List of abbreviations
# Conclusion
Geriatric patients with FBA can present with atypical or scant histories and physical findings. FBA is an important differential diagnosis of new wheezing in the elderly. Physicians need to carefully evaluate "trivial" information or collect additional information when encountering airway symptoms in elderly patients to avoid missing an FBA diagnosis.
# Ethical approval
Not required for case reports. We obtained written informed consent from the patient's family.
# Sources of funding
There are no sources of funding.
# Author contribution
HM and AK looked after the patient, wrote and revised the draft, and approved the submission of the current article.
## Research registration
Not applicable.
## Guarantor
AK serves as the guarantor for this article.
## Provenance and peer review
Not commissioned, externally peer-reviewed.
## Informed consent
Written informed consent was obtained from the patient's family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.
[fig] Figure 1: A round nodule in the patient's right intermediate bronchus on non-contrast computed tomography (arrow) on the lung window setting (A) and mediastinal window setting (B). [/fig]
[fig] Figure 2: A pea (Pisum sativum) lodged in the patient's right bronchus was removed using forceps. [/fig]
|
Combined High-Dose LATTICE Radiation Therapy and Immune Checkpoint Blockade for Advanced Bulky Tumors: The Concept and a Case Report
Although the combination of immune checkpoint blockades with high dose of radiation has indicated the potential of co-stimulatory effects, consistent clinical outcome has been yet to be demonstrated. Bulky tumors present challenges for radiation treatment to achieve high rate of tumor control due to large tumor sizes and normal tissue toxicities. As an alternative, spatially fractionated radiotherapy (SFRT) technique has been applied, in the forms of GRID or LATTICE radiation therapy (LRT), to safely treat bulky tumors. When used alone in a single or a few fractions, GRID or LRT can be best classified as palliative or tumor de-bulking treatments. Since only a small fraction of the tumor volume receive high dose in a SFRT treatment, even with the anticipated bystander effects, total tumor eradications are rare. Backed by the evidence of immune activation of high dose radiation, it is logical to postulate that the combination of High-Dose LATTICE radiation therapy (HDLRT) with immune checkpoint blockade would be effective and could subsequently lead to improved local tumor control without added toxicities, through augmenting the effects of radiation in-situ vaccine and T-cell priming. We herein present a case of non-small cell lung cancer (NSCLC) with multiple metastases. The patient received various types of palliative radiation treatments with combined chemotherapies and immunotherapies to multiple lesions. One of the metastatic lesions measuring 63.2 cc was treated with HDLRT combined with anti-PD1 immunotherapy. The metastatic mass regressed 77.84% over one month after the treatment, and had a complete local response (CR) five months after the treatment. No treatment-related side effects were Frontiers in Oncology | www.frontiersin.org
# Introduction
Lung cancer is the most commonly diagnosed cancer (11.6% of the total cases) and the leading cause of cancer death (18.4% of the total cancer deaths) based on the latest global cancer statistics. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, with approximately 40% of newly diagnosed NSCLC patients presented with stage IV disease. Radiotherapy plays an important role in the definitive, preoperative and postoperative management of early stage and in the palliative treatment of advanced staged NSCLC. The use of radiation as a local ablative therapy is now recommended in the European Society for Medical Oncology (ESMO) guidelines for patients with stage IV disease who subsequently develop oligometastatic progression.
High ablative doses of local radiation therapy (RT), often referred to as stereotactic radiosurgery (SRS) or stereotactic body radiotherapy (SBRT) is usually applied to tumors with limited size. The application of SRS or SBRT in bulky tumors is often considered challenging due to the difficulties in controlling toxicities of the surrounding normal/critical organs. Spatially fractionated radiotherapy (SFRT) with GRID, proposed in 1909 and further developed since early 1950s, has been safely utilized for the treatment of bulky and advanced tumors with impressively accumulated clinical data. In recent years, the 2D GRID technique was extended to a 3D configuration, known as LATTICE radiotherapy (LRT). The safety and clinical efficacy of LRT has been reported in various voluminous tumors.
Based on the results of radiobiological studies, the possible mechanisms behind the effectiveness of SFRT have been attributed to certain bystander effects and abscopal effects, such as the radiation-mediated anti-tumor immunityor perfusion modulation. The key characteristics of SFRT in either GRID or LRT configurations is the Peak-Valley dose distribution, where high dose of radiation is delivered to the peaks or vertices, leaving relatively lower dose in the valleys (between the peaks or vertices). In the context of immune modulation, as long as the peak dose is sufficiently high, although only partial volume of the tumor receives that high dose, the induced anti-tumor immunity can be expected and would subsequently contribute to the enhanced tumor control. As radiation-mediated immune activation follows the pathway of T-cell priming through antigen presentation, for immunogenic tumors, combining immune checkpoint blockades with high dose of radiation is a logical strategy and has been extensively studied with encouraging results. The recent studies also showed that combined ablative dose with low dose of radiation could lead to the reprograming of the immunosuppressive tumor microenvironment (TME) to become more immunogenic and synergistically augment the anti-tumor response. This is an important insight as SFRT intrinsically combines high and low dose in its Peak-Valley dose distribution. All these have suggested that HDLRT, when combined with checkpoint blockade immunotherapy could result in improved tumor control.
In the following case report, a patient with multiple metastatic lesions from a primary NSCLC received various regimens of palliative treatments, including conformal radiotherapy (CRT), intensity-modulated radiotherapy (IMRT), SBRT, LRT, chemotherapy, and immunotherapy. Remarkably, only one lesion treated with high-dose LRT (HDLRT) and anti-PD1 therapy achieved complete local response (CR).
## Case presentation
A 33-year-old female patient initially presented with a lung mass in the right lower lobe, accompanied by cough for one month. She underwent video-assisted thoracoscopic right lower lobectomy and systemic mediastinal lymph node dissection as curative intent resection on May 10, 2017. The surgical histopathological report demonstrated diagnosis of invasive adenocarcinoma in the lower lobe of the right lung. The lung mass measured 7, 4.5, and 3.2 cm in the greatest dimensions. Post-surgical staging was T3N2M0. Postoperatively, she received two cycles of adjuvant chemotherapy of PP regimen (pemetrexed disodium 0.8 g dl + cisplatin 0.4g dl) from June to August 2017.
In September 2017, the patient developed metastatic disease in L2-3 spine diagnosed by magnetic resonance imaging scan. Computed Tomography (CT) scan showed multiple metastases of different sizes in both lungs, a metastatic nodule in thyroid, and a mass in the posterior chest wall measuring 2.0 cc with maximum dimensions 1.8x1.7x1.2 cm. Single-Photon Emission Computed Tomography (SPECT) scan also showed multiple metastases in right parietal bone, cervical vertebra, L2-3 spine, left ilium, and right sacroiliac joint.
The patient was found to have EGFR exon 20 insertion mutation, ALK and ROS-1 negative and over 70% expression of PD-L1. The patient started the first cycle of checkpoint inhibitor therapy using Pembrolizumab, an anti-PD-1 monoclonal antibody (100mg ivgtt d1 q3w) on September 30th, 2017. While the treatment was on going, the metastatic mass in the posterior chest wall grew rapidly from 2.0 cc to 63.2 cc with maximum dimensions 5.0 × 5.4 × 5.3 cm on October 10, 2017, in less than a month,. The lesion was ulcerated with slight local bleeding. New metastases in brain and right sternoclavicular joint were subsequently observed on MRI and CT images.
## High-dose lattice radiation therapy (hdlrt)
The decision was to treat the fast-growing posterior chest wall mass with HDLRT to combine with the on-going anti-PD-1 treatment.
Treatment planning was performed on the MULTIPLAN (Accuray, Incorporated, Sunnyvale, CA). A CyberKnife VSI Robotic Radiosurgery System (Accuray, Incorporated, Sunnyvale, CA) was used for delivering a single fraction of LRT with 20 Gy prescribed to six high-dose vertices.
The HDLRT was configured with six spherical high dose vertices with diameter of 1.0 cm distributed within the GTV and with 2.0 cm of separation (center to center). The optimized plan resulted in the doses covering 98%, 95%, 50%, and 5% of the vertices volume (D98, D95, D50, and D5%) being 20.95, 21.40, 24.88, and 27.85 Gy, respectively; the maximal dose of the spinal cord and rib being 2.67 and 7.45 Gy, respectively. The dose distribution is shown in. The dose-volume histograms (DVHs) of the high-dose vertices, GTV, ribs, and spinal cord were shown in. The Peak-to-Valley dose profileshowed the valley dose between vertices to be about 25% of the peak dose. Note that only 6.5% of the GTV received the prescribed vertex dose of 20 Gy and higher, and that the DVH of the GTV is closely similar to that of the published data with GRID.
The HDLRT was given on October 18, 2017, 18 days after the initiation of the first cycle of Pembrolizumab.
## Other treatments
Subsequent to the HDLRT, from October 30, 2017, to , patient continued to receive another six cycles of Pembrolizumab (100 mg ivgtt d1 q3w, for each cycle).
Additionally, the patient received SBRT with 10 Gy in two fractions, 20 Gy in 1 fraction, and 15 Gy in 1 fraction for a metastatic tumor at the L2-3 spine, a right lung metastasis near anterior chest wall, and a tumor at the T10 spine, respectively; CRT with 8 Gy in two fractions and 20 Gy in five fractions for a spinal metastatic tumor at the C3-5 levels and the whole brain, respectively; IMRT with 30 Gy in 10 fractions for a thyroid and a posterior sternal metastasis, and 8 Gy in four fractions for the metastatic abdominal lymph nodes, respectively; LRT with 12 and 10 Gy in 1 fraction for the spinal metastatic tumors at the L3 spine and psoas, and the metastatic abdominal lymph nodes, respectively. From February 2018 to May 2018, the patient also received four cycles of VEGF targeted therapies with bevacizumab and three cycles of chemotherapy with gemcitabine. The timeline and therapeutic interventions were listed in .
## Clinical outcome
While all metastatic lesions responded to various palliative treatments, only the posterior chest wall metastatic tumor achieved complete response. Under the background of anti- PD1 treatment, the tumor regressed 77.84% over one month after the HDLRT and then continued to shrink. Two months after the HDLRT, in addition to further shrinkage, all symptoms were relieved with the bleeding/discharging totally under control. This posterior chest wall tumor achieved complete local response (based on visual and radiographic exams) five months after the HDLRT without side effects. CT scans of the chest and abdomen on May 10, 2018 showed progression of multiple metastases in both lungs, mediastinum, retroperitoneum, right lower pleura, left upper middle abdominal cavity, double ilium, uterus, and T10 spine. Metastases in the right thyroid, pancreatic neck, bilateral adrenal glands, both kidneys, L3 spine and psoas, and C3-C5 spine were stable. Metastases in the right sternoclavicular joint and right lung near anterior chest wall shrank moderately. Due to the subsequent progression of metastases in multiple sites with cancerous fever and abdominal infection, the patient finally succumbed to the disease, seven months after the HDLRT. The treatment site of the posterior chest wall remained disease-free until patient's death.
# Discussion
Radiation therapy when used for palliative management of advanced cancers employs either conventional fractionation or SBRT regimens with dose lower than that of definitive, curative treatments, and would expectedly result in partial tumor response. LRT when used as palliative treatment would also lead to partial response in general. LRT as a safe boost to conventionally fractionated radiotherapy had shown clinical success in a variety of bulky tumors such as advanced gynaecological tumors and voluminous lung tumors. The first patient with locally advanced lung cancer treated with LRT followed by conventionally fractionated radiotherapy, combined with chemotherapy demonstrated excellent clinical response after 6 years follow-up. Since 2010, over 150 patients have been treated with LRT and more reports of clincal outcomes are anticipated. However, to this day, LRT alone as induction of anti-tumor T-cell immunity, to combine with immune checkpoint blockade treatment has not been reported.
In the reported case, with the anti-PD1 treatment in parallel, except for the posterior chest wall lesion that received HDLRT of 20 Gy, all other lesions achieved only partial response, including the ones treated with SBRT of 20 Gy in a single fraction (full tumor coverage), and LRTs with 10 Gy and 12 Gy of vertex doses. This implies that not only a high dose (20 Gy or higher) is essential; the spatial fractionation with Peak-Valley or High-Low dose alternation within the tumor volume might also be critical to mediate effective anti-tumor immune response. This is consistent with a number of research works favoring high dose for effective anti-tumor T-cell priming, and that when combined with low-dose treatment, radiation-induced immune modulation might be augmented. Additionally, it has been postulated that the low dose regions (valleys) might preserve the perfusion needed for circulating the factors essential for anti-tumor immunity.
It is worth noting that, with only 6.5% of the GTV receiving the dose of 20 Gy and higher, the effective uniform dose (EUD) of the GTV was calculated to be 1.2 Gy, using Niemierko's phenomenological modelwith a=−10 (typically suggested for tumors). Based on the traditionally understood mechanism of radiobiology, the probability of achieving complete local control with such a dose for a tumor of 63 cc would be nearly zero. Given the fact that this tumor was not responsive to the initial anti-PD1 treatment and none of the other tumors showed significant reduction throughout the curse of the treatments, the synergetic effect of combining HDLRT with anti-PD1 becomes a plausible speculation. To summarize the postulated mechanism, in HDLRT the dose in the vertices are sufficiently high (>20 Gy) to induce neo-antigen release and initiate the cascade of APC (antigen presenting cell)-based T-cell priming; the dose in between the vertices is low enough to preserve internal tumor circulation/perfusion to potentially facilitate the infiltration of APCs and the primed cytotoxic T-cells; the highly heterogeneous dose configuration could reprogram the immunosuppressive TME to become more immunogenic; and when synergistically treated by checkpoint inhibitors, the primed T cells could attack tumor cells without being exhausted. Mohiuddin et al. treated a pembrolizumab-refractory patient with locally advanced melanoma who was dramatically resensitised to the same drug by the administration of parallel opposed, spatially fractionated GRID radiation therapy. Their result suggested the similar synergistic effect of high-dose GRID radiation therapy as a primer for immunological response. Our finding echoes with their result. However, abscopal response of other tumors with the appreciable magnitude was not observed in this case study.
# Conclusion
SFRT, with its long history of evolution is currently gaining new momentum and much of the new potentials are awaited for further exploration. LRT can safely deliver potentially immunogenic high dose to partial volume of bulky tumors. When combined with immune checkpoint blockades, therapeutic effects greater than traditional palliation/debulking, and even complete local tumor eradication are possible. The reported case showed the dramatic difference in tumor response between HDLRT and an array of palliative radiation therapy regimens when combined with anti-PD1 immunotherapy in a same individual, suggesting such strategy of combining HDLRT and immune checkpoint blockades might present a universally applicable treatment option if the clinical efficacy and safety can be systemically tested and proven.
# Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.
# Ethics statement
The studies involving human participants were reviewed and approved by Ethical Committee of Fujian Medical University Union Hospital. The requirement for informed patient consent was waived by Ethical Committee of Fujian Medical University Union Hospital due to the retrospective nature of this study.
# Author contributions
BX and XW are responsible for study design. BX, XW, LJ, and XL, contributed to the writing of the manuscript. LJ, XL, JZ, and WL are responsible for developing treatment plan, data analysis, and contributed to the physics portion of the manuscript. FD, CC, QL, CZ, and FZ are responsible for clinical data collection and contributed to the clinical portion of the manuscript. WY and YZ contributed to data analysis discussion and manuscript editing. All authors contributed to the article and approved the submitted version. |
Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards
Recreational water monitoring can be challenging due to the highly variable nature of pathogens and indicator concentrations, the myriad of potential biological hazards to measure for, and numerous access points, both official and unofficial, that are used for recreation. The aim of this study was to develop, deploy, and assess the effectiveness of a quantitative polymerase chain reaction (qPCR) community-based monitoring (CBM) program for the assessment of bacterial and parasitic hazards in recreational water. This study developed methodologies for performing qPCR 'in the field,' then engaged with water management and monitoring groups and tested the method in a real-world implementation study to evaluate the accuracy of CBM using qPCR both quantitatively and qualitatively. This study found high reproducibility between qPCR results performed by non-expert field users and expert laboratory results, suggesting that qPCR as a methodology could be amenable to a CBM program.OPEN ACCESSCitation: Rudko SP, Reimink RL, Peter B, White J, Hanington PC (2020) Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards. PLoS ONE 15(5): e0229701. https://doi.
# Introduction
Community-based monitoring is now routinely used for conservation and environmental monitoring. Citizen science describes both a methodology of conducting large-scale research by recruiting volunteers and refers to the process by which citizens are involved in scientific investigation as researchers. Citizen science can include community-based monitoring (CBM) as a process of collaboration between government, industry, academia, and local community groups to monitor, track, and respond to issues.
The earliest incarnations of citizen science and CBM relied on volunteers as data collectors, but the discipline of CBM has grown and evolved. Recent arguments in favour of CBM suggest the field move away from a paradigm of "using citizens to do science" to an equal power relationship that views citizens as scientists, embracing some of the ideals of participatory action research. CBM is poised to improve environmental decision-making. Its' use has been on the rise due to budgetary constraints in both government and academia and because CBM can be a powerful methodology for generating large spatial or temporal datasets for monitoring/surveillance purposes. CBM improves scientific literacy, builds social capital, improves participation in local issues and benefits the environment [bib_ref] Thinking scientifically during participation in a citizen-science project, Trumbull [/bib_ref] [bib_ref] Establishing the Canadian community monitoring network, Whitelaw [/bib_ref]. Traditional CBM programs have typically relied on volunteers to conduct biodiversity surveys, conduct simple tests (i.e. Secchi disk tests for assessing water clarity), or specimen collection and shipment to central facilities for analysis. However, modern monitoring methods conducted in academia, industry, and government have evolved considerably to include large-scale spatial assessment methods, for example, algal/cyanobacteria bloom-tracking satellites, next-generation sequencing analysis, and eDNA monitoring. CBM programs also must evolve and advance as new technologies become available. In water monitoring, especially, quantitative polymerase chain reaction (qPCR) has emerged as a common method to conduct regulatory testing for sewage impacted recreational water (i.e. USEPA Method 1611), and a common screening method for fecal indicator organisms.
Quantitative PCR methods for the detection of surrogates and hazards in water have existed for decades and can be used to detect minute quantities of an organisms' DNA in a complex matrix such as water, soil, or blood. qPCR is highly sensitive and is very specific for particular regions of DNA. In the last decade, agencies responsible for monitoring the environment and health have begun to capitalize on the potential of qPCR. Some of the greatest strides have been made in health, especially after the USEPA EMPACT study, which found that levels of enterococcus as measured by qPCR correlate with the risk of human gastrointestinal illness, and in correlating the amount of human-associated Bacteroides with human health targets [bib_ref] Human-Associated Fecal Quantitative Polymerase Chain Reaction Measurements and Simulated Risk of Gastrointestinal..., Boehm [/bib_ref]. Screening for toxigenic cyanobacteria species is also moving towards molecular detection methods. For example, in Poland, initial screening for toxin genes in recreational waters is conducted using qPCR, followed by immunochemical analysis to quantify the toxins. In related fields like environmental monitoring, some locales have moved to molecular methods for monitoring for the veliger stage of invasive zebra (Dreissena polymorpha) and quagga (Dreissena rostriformis bugensis) mussels [bib_ref] Tracing the quagga mussel invasion along the Rhine river system using eDNA..., Ventura [/bib_ref] [bib_ref] Specific amplification of the 18S rRNA gene as a method to detect..., Frischer [/bib_ref].
As the effectiveness of qPCR diagnostic tests continues to be realized, it is apparent qPCR is an excellent choice for CBM. qPCR is a platform; once the infrastructure is in place, monitoring for additional targets, or changing targets if new issues arise is as simple as validating a new test. For this reason, qPCR and related molecular techniques have been touted as grand solutions for point of care diagnostics in infectious disease monitoring, yet this future has not yet been realized [bib_ref] Democratizing molecular diagnostics for the developing world, Abou Tayoun [/bib_ref] [bib_ref] Taking connected mobile-health diagnostics of infectious diseases to the field, Wood [/bib_ref]. The idea of portable diagnostic technologies that can be used to detect multiple targets feeding information into a surveillance system is attractive for a number of reasons; however, the development to implementation gap is often wider than one would expect.
It is often presumed that highly skilled personnel are required to execute molecular biology methods such as qPCR. Additionally, technologies to conduct testing portably have only just begun to emerge onto the market and have not been fully vetted. This study is, to our knowledge, the first of its kind to test the rigour of qPCR for detection/quantification of biological hazards and their surrogates in water through a CBM-implementation study. Here, we test the feasibility, reproducibility and reliability of implementing portable qPCR water monitoring amongst a variety of groups (government, NGO, and private enterprise). This was assessed both quantitatively, by conducting our own measurements on CBM partner samples, and qualitatively, through surveying our user groups to capture their perceptions of the technology and its fit within their individual contexts and organizations. salary for JW. The specific roles of these authors are articulated in the 'author contributions' section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
## Competing interests:
The authors have read the journal's policy, and the authors of this manuscript have the following competing interests: JW is owner of Aquality Environmental Consulting LTD and RLR is owner of Freshwater Solutions, LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.
# Materials and methods
# Ethics statement
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This research was approved by the University of Alberta Human Research Ethics Board: Approval # Pro00048511. Oral consent was obtained from all survey participants.
## Implementation study design
We first connected with relevant stakeholders of recreational water in Alberta, and worked with them to determine their monitoring goals. Using a participatory research (PAR) approach, we then developed qPCR tests and testing methodologies that would fill these needs [bib_ref] Participatory action research, Baum [/bib_ref]. Under this PAR approach, CBM partners selected study sites they felt would be appropriate, and we advised and assisted in this selection where it seemed appropriate. In total, CBM partners conducted 985 qPCR tests over the two years of this program. Since the goal of this study was to measure the effectiveness of a CBM monitoring program in a real-world context, participants in the study were instructed to collect a duplicate sample or cut (using disposable, individually packaged sterile scalpel blades the filter membrane in half after filtration and send this to the university laboratory. CBM partners stored their membranes frozen, and samples were transported to the laboratory on ice. Samples were picked up weekly or monthly, depending on sampling frequency. Partners in Michigan shipped samples overnight to Alberta. Samples in our laboratory would be processed in an identical fashion to the field user to compare novice versus expert methodologies [fig_ref] Fig 1: Implementation process of the CBM qPCR program [/fig_ref] as soon as they were received; this was done to monitor method agreement. Additionally, CBM partners sent their extracted DNA to our laboratory, which enabled us to also perform qPCR on their DNA extracts and to perform inhibition reactions. CBM partners were encouraged to extract DNA from their water samples as soon as possible but stored filter membranes in the freezer if they weren't going to perform extraction right away. Extracted DNA was also stored in the freezer until use, which was within one week of sample collection.
## Training of cbm partners
CBM partners were provided with a training video and a written protocol detailing all stages of the method, from sampling DNA extraction and qPCR. Training included using all equipment that was distributed, including thermocyclers and pipettes. Additionally, they were provided with two in-person training sessions. Typically, we would demonstrate the method in our laboratory, and the second training session would on-site at their location, where the CBM partner would run their first samples. As partners processed their samples, the laboratory would also perform DNA extractions and qPCR on the other half of the sample. These results were then compared. If the partner results disagreed with laboratory results, retraining occurred. CBM partners were also instructed on maintaining a clean workspace. This included the use of bleach to disinfect benchtops where DNA extraction occurred, using filter-tipped pipettes, and how to properly dispose of all materials.
## Sample collection
Specific water collection methods are detailed below for each target of interest; regardless of the volume collected, all samples were then filtered through a 0.4 μm polycarbonate filter (Pall FMFNL1050) using an electric vacuum pump (Vaccubrand ME1C). CBM partners had the option of either collecting and filtering a duplicate water sample for analysis, or cutting their filter membranes in half to be analyzed at the university laboratory.
Avian schistosome monitoring. Sample collection was conducted as described in [bib_ref] Use of qPCR-based cercariometry to assess swimmer's itch in recreational lakes, Rudko [/bib_ref]. Briefly, 25L water samples, collected one litre at a time across a shoreline up to ~1m deep were passed through a 20μm plankton tow. Debris from inside was washed down using well water (this is not a contamination risk when monitoring for avian schistosomes as these parasites are shed from snail hosts, and only when those snail hosts also co-occur with locations where the bird definitive host's feces are also present [bib_ref] The control of swimmer's itch in Michigan: Past, present and future, Blankespoor [/bib_ref] followed by a 95% ethanol wash and collection in sterile 50-mL conical tubes.
Toxin-producing cyanobacteria monitoring. Sample collection was conducted from watercraft operated by CBM partners on various lakes. Samples were collected through a oneway foot valve attached to weighted 3/4" Nalgene tubing. Samples were only collected from the euphotic zone as determined by a Secchi disk measurement at each lake's deepest point. Ten sampling locations were selected for each lake, with water being composited from each sampling location into a central container. Water from this container was then poured into 50-mL conical tubes. Equipment was decontaminated between lakes using quaternary ammonium compound (QUAC) to prevent contamination between lakes (1500ppm QUAC for 10 minutes). QUAC disinfectants are membrane-active compounds that interact with the cytoplasmic membranes of eukaryotes and bacteria. Additionally, they bind DNA, making them useful disinfectants for samples that will be used for qPCR [bib_ref] Quaternary ammonium biocides: efficacy in application, Gerba [/bib_ref].
HF183 monitoring. All samples were collected by scooping two 50ml samples in sterile, conical, collection tubes from the surface water 15m from shore every 150m along the entire perimeter of each participating lake.
## Dna extraction
Onsite DNA extraction. DNA extraction was conducted using the MI Sample Prep Kit (Biomeme, USA) according to the manufacturers' instructions. The MI sample prep kit is designed to function in the field. Lysis is accomplished by placing a filter in the lysis buffer and shaking for one minute. Next, the solution is passed through a syringe unit fitted with a DNA binding column. The column undergoes two washes to remove proteins and salts and then is dried using an acetone buffer before elution. Sample blanks were conducted by partners every batch of 24 samples processed. In 2018, the avian schistosomes monitoring group was interested in transitioning to a DNA extraction method that would allow for batch processing of samples. We, therefore, opted to transition their program to the DNeasy DNA extraction kit [bib_ref] Use of qPCR-based cercariometry to assess swimmer's itch in recreational lakes, Rudko [/bib_ref]. To set up this remote laboratory in a cost-effective manner, equipment (centrifuge, heating block, and vortex) were sourced from Dot Scientific (USA) (S2 , and pipettes were from the company VWR (USA). qPCR methods Maintaining workflows. All master mix components were mixed in a cleanroom located at the University of Alberta and aliquoted into 0.2 ml thin-wall PCR tubes (Axygen, USA). All plasmid dilutions and preparation of positive controls occurred in a dead box, a PCR workspace designed to limit airflow and prevent cross-contamination between wells during reaction set up. Standards and reaction tubes were prepared independently to prevent cross-contamination. IDT DNA (USA) Prime Time Gene Expression Master mix was utilized for the field and in-laboratory qPCR because it is both light and temperature stable. That being said, all premixed reactions were stored frozen and transported on ice. Master mix was prepared and delivered to partners monthly.
In laboratory qPCR method. Samples were quantitated relative to a plasmid standard curve, which contained 50,000, 5000, 500, 50, 5 and 0.5 gene copies. Each of the gene targets below was synthesized (IDT DNA) into a puc19 plasmid vector (Genscript. USA). Thermocycling was performed on the ABI 7500 Fast or the QuantStudio 3 using a standard, 40 cycle, two-step reaction. The thermocycling parameters were a 30-second hold at 95˚C, followed by a 30-second denaturation cycle at 95˚C, and a 60˚C annealing cycle. Each qPCR reaction had a final volume of 20μL, and we added 5μL of DNA to each reaction.
Avian schistosomes. The 18S avian schistosomes-targeting qPCR assay was performed as described in [bib_ref] Use of qPCR-based cercariometry to assess swimmer's itch in recreational lakes, Rudko [/bib_ref] [bib_ref] Use of qPCR-based cercariometry to assess swimmer's itch in recreational lakes, Rudko [/bib_ref] (S1 . The LOD 95 of this technique is 3.4 gene copies/ rxn [bib_ref] Use of qPCR-based cercariometry to assess swimmer's itch in recreational lakes, Rudko [/bib_ref]. qPCR master mix (IDT DNA) containing 1x master mix, and 200nm forward reverse primer and a fluorescein-labelled probe were used.
Toxigenic (mcyE gene) cyanobacteria monitoring. The mcyE gene targeting qPCR assay was performed as described in [bib_ref] Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR, Qiu [/bib_ref] [bib_ref] Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR, Qiu [/bib_ref] and [bib_ref] Development of a chip assay and quantitative PCR for detecting microcystin synthetase..., Sipari [/bib_ref] [bib_ref] Development of a chip assay and quantitative PCR for detecting microcystin synthetase..., Sipari [/bib_ref] (T S1 . The LOD 95 of this technique is 6.25 copies/5μL. qPCR master mix (IDT DNA), containing 1x master mix, and 200nm forward reverse primer and 125nm fluorescein-labelled probe was used.
HF183 Bacteroides monitoring. This 16S gene-targeting assay was performed as described in Haugland et al. . The LOD 95 of this technique is 7.2 gene copies/rxn. qPCR master mix (IDT DNA), containing 1x master mix, and 100nm forward reverse primer and 80nm fluorescein-labelled probe was used (S1 .
In-field qPCR method. Mastermix components and concentrations were unchanged between the laboratory method and the field method, nor were the thermocycling parameters. CBM partners received four control tubes, which consisted of a negative control, and three standards (5000, 500, and 50 copies). They were instructed not to open these tubes to prevent contamination. CBM partners also received 12 tubes to add their own samples DNA to [fig_ref] Fig 1: Implementation process of the CBM qPCR program [/fig_ref].
Inhibition controls. Inhibition controls were performed utilizing the inhibition control assay described in [bib_ref] Enterobius vermicularis as a novel surrogate for the presence of helminth ova..., Rudko [/bib_ref] [bib_ref] Enterobius vermicularis as a novel surrogate for the presence of helminth ova..., Rudko [/bib_ref]. Internal control plasmid DNA was spiked in excess into qPCR reactions containing 5μL of water sample DNA, and inhibition was defined as a 3-ct (i.e. 1 log) shift in amplification.
Creation of the field kits. Field kits given to CBM partners contained: the M1 DNA extraction kit (Biomeme), 1.5 ml snap-cap tubes, sample collection vials (Corning), a 20-micron plankton tow (Aquatic Research Instruments, USA), 0.45 μM polycarbonate filter funnels (Pall, FMFNL 1050), a 20 μL pipette, a box of pipette tips, PCR tubes, a laptop (Acer [Taiwan,] and Chromebooks [USA] were distributed) an Open qPCR (USA) thermocycler, all the necessary cables, and reaction strips [fig_ref] Fig 1: Implementation process of the CBM qPCR program [/fig_ref].
## Capturing cbm partners perceptions of the method
CBM partners (six in total) were administered a survey with open-ended questions regarding the implementation of the method (S4 . All six CBM partners submitted a completed survey. Surveys were blinded from the researchers to encourage honesty from participants; a research associate received the surveys via email and edited them to remove any personal identifiers before sending them to the analyst. Data were analyzed using deductive thematic analysis [bib_ref] Demonstrating rigor using thematic analysis: A hybrid approach of inductive and deductive..., Fereday [/bib_ref]. Open coding was used, and codes were developed and modified as the analysis took place. Analyzing the codes enabled the identification of initial themes; these preliminary themes were refined to demonstrate interesting patterns in the data that were important to the successes or failures of the implementation. Themes were realized semantically (i.e. the explicit or surface meaning of the data) and latently, to identify and examine underlying ideas and assumptions that inform the semantic content of the data [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref].
## Bland-altman plots
Bland-Altman plots were created in GraphPad Prism 8 on the log-transformed copy number per 5μL data. Log transformation was performed to normalize data (Shapiro-Wilk test, test statistic 0.22, p < 0.00). [bib_ref] Stastical Methods for Assessing Agreement Between Two Methods of Clinical Measurement, Bland [/bib_ref] [bib_ref] repeatability and reproducibility: analysis of measurement errors in continuous variables, Bartlett [/bib_ref].
## Statistics
Statistical analyses were conducted in IBM SPSS (version 25, USA). Graphs were made in Prism 8 (GraphPad, USA). Limit of Detections were calculated using the POD/LOD calculator [bib_ref] Estimation of the POD function and the LOD of a qualitative microbiological..., Wilrich [/bib_ref]. Maximum log difference was calculated as the upper 95% confidence interval of the average of the log difference between all sets of paired samples. Interclass correlation analysis was performed in SPSS on the log-transformed data using a two-way random-effects model with average measures, and a type c model with a consistency definition. A two-way random-effects model was selected because it models both an effect of the operator and the sample and assumes that both are drawn randomly from larger populations.
# Results
## Thermocycler comparison
Detection limits of the open qPCR thermocyclers. The limit of detection 95 (LOD 95 ) of the Open qPCR thermocyclers is 63.4 gene copies (GC)/5μL (lower limit 43.7 GC/5μL, upper limit 89.2 GC/5μL, n = 40, based on all qPCR tests). This is approximately 1-log higher than the same assays (Avian schistosomes LOD 95: 3.4 GC/5μL; Toxic cyanobacteria LOD 95 : 6.25 GC/5μL; HF183 LOD 95 : 7.2 GC/5μL) performed using our laboratory ABI 7500/QuantStudio 3 thermocycler. All of these assays have been validated in previous papers, the names, sequences, and the references for the primers and probes are found in S1 [fig_ref] Table: Field method materials used in this study [/fig_ref] Standard curves performed well compared to the optimal qPCR standard curve (i.e. slope: -3.3, amplification factor: 1.9-2.0, R 2 : 0.99) using the Open qPCR thermocyclers [bib_ref] The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Bustin [/bib_ref] (S1 .
Comparison between machines. Interclass correlation coefficients (ICC) were calculated to compare CBM partner DNA extracts run on the Chaibio Open qPCR machine, and our laboratory ABI 7500/QuantStudio 3. In 2017, the ICC of the avian schistosomes assay was 0.88 (95% CI: 0.85 lower, 0.90 upper), and in 2018, it was 0.76 (95% CI: 0.56 lower, 0.866 upper), in 2018 this group used 2 Open qPCR machines, and this ICC is a pooled result of both of these machines. In 2018, the ICC of the toxic cyanobacteria assay was 0.57 (95% CI: 0.1 lower, 0.86 upper) . Maximum log differences were also calculated and ranged from 1-1.5 depending on the test and year . CBM partners also prepared sample blanks and performed DNA extraction and qPCR on these. All sample blanks were negative when performed both in the field and on the more sensitive core laboratory equipment.
## Cbm partner comparison
Semi-quantitative analysis using Bland-Altman plots. Reproducibility was assessed using the semi-quantitative Bland-Altman plot. Bland-Altman plots graph the average of two measurements on the X-axis and the difference between these measurements on the Y-axis. The Bland-Altman plot for avian schistosomes monitoring for 2017 and 2018 show a linear pattern at lower copy numbers, but at higher copy numbers show uniform variability [fig_ref] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the... [/fig_ref]. Bland-Altman analysis of the toxic cyanobacteria test shows uniform variability within the limits of agreement (1.96 times the standard deviation). A paired t-test using the log-transformed data was used to compare the within-subject standard deviations of the partner data compared to the laboratory-generated data. They were significantly different based on an F-test and Welch's t-test (p < 0.0001, F = 6288, mean difference ± SEM: 20326 ± 9843).
Interclass correlation analysis. ICC analysis was performed to compare user and laboratory samples. In 2017, the Biomeme MI extraction kit was used for swimmer's itch monitoring. The ICC between user and laboratory extraction samples was 0.539 (95% CI: 0.320 lower, 0.680 upper). The ICC 2018 for avian schistosomes monitoring was 0.593 (95% CI: 0.344 lower, 0.747 upper). The ICC mcyE was 0.640 (95% CI: -0.250 lower, 0.896 upper) . Maximum log differences ranged from 1.3-1.4 .
Inhibition controls. PCR Inhibition was tested on partners' DNA extractions and on DNA extractions performed in house. Five percent (49 samples) of samples were inhibited (as defined by a 3-ct shift in the inhibition control) in both partner and in house extractions. Inhibited samples were excluded from the analyses in this paper because inhibitors will alter the estimates of copy number when present.
# Qualitative analysis
User perceptions. User perceptions of the program were captured through a written survey that was administered to participants. The questions are available in S4 [fig_ref] Table: Field method materials used in this study [/fig_ref] Thirty-three percent (33%) of respondents stated that they had some prior knowledge of molecular biology, PCR (polymerase chain reaction), eDNA, or DNA based detection in general prior to the use of the qPCR field method. Fifty percent (50%) reported having low prior knowledge, and one participant had no prior knowledge. The same 33% of respondents who reported some knowledge with molecular biology and methods also reported having performed some form of PCR in the past. The rest of the respondents reported not having performed PCR (50%), and one participant did not remember. However, prior knowledge did not impact the training all users were provided.
Thematic analysis. User surveys underwent deductive thematic analysis whereby surveys were coded, and then codes were organized into themes [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref]. The codes identified and relevant excerpts from the surveys are presented in S5 [fig_ref] Table: Field method materials used in this study [/fig_ref] The first theme identified is "rapidly responding to hazards." This theme captured the CBM partners' perceptions of the speed of the qPCR method and their perceived ability to respond to issues quickly. The second theme identified was the question of who controls the CBM monitoring system. This theme emerged from CBM partners expressing a desire for independence and control over the interpretation of results. The third theme identified was that the triangulation of training was valuable in that most CBM partners suggested that the written and video protocols (complemented with a few in-person training sessions) were important to them and enhanced their learning. A subtheme that emerged from this theme was "learning and communication."
# Discussion
## Implementation of the cbm qpcr program
We assessed the accuracy of the portable qPCR machines relative to a "core" machine, and the ability for CBM partners to execute the method. Our analyses have demonstrated that a CBM . Interclass correlation coefficients and maximum log difference. Comparing the reproducibility of samples run on the Chaibio Open qPCR thermocycler and the ABI 7500 thermocycler/QuantStudio 3. qPCR monitoring program can yield accurate results for different targets (i.e., eukaryotic versus prokaryotic) when deployment of the method is controlled. Our intention was to implement a CBM qPCR system in a real-world context. [fig_ref] Fig 1: Implementation process of the CBM qPCR program [/fig_ref] we began the development of this project by consulting with local stakeholder groups and assessing their interest in the project and what types of biological hazards and surrogates they might be interested in monitoring for. Our goal was to have partners run a sufficient number of tests so as to test the reproducibility of the method. We did not want to prescribe a particular test for CBM partners to run, as if a particular test was not useful to a partner, it would be unlikely that they would be motivated to continue testing. Therefore, we adapted to the needs of our CBM partners and adapted a variety of existing qPCR tests to the field equipment and testing protocol. Additionally, some of the groups we worked with had their own scientific questions they wanted to answer, and we facilitated this.
## Comparison of partner-extracted dna samples performed on the open qpcr versus the quantstudio 3/abi
Our laboratory distributed all materials required to complete testing to users. Additionally, we prepared all qPCR master mix components (enzyme mix, primers and probes) and aliquoted these into individual reaction tubes for users. The purpose of this was two-fold, to prevent contamination of CBM partners' qPCR reactions and for simplicity for partners. Our laboratory facilities are equipped with a PCR clean room, as well as separate pre and postamplification rooms. By preparing reaction tubes and controls, we could prevent CBM partners from handling high copy number controls (a likely source of contamination). Additionally, CBM partners were instructed not to open tubes that had undergone qPCR. The Biomeme DNA extraction does not utilize pipettes, but all users were supplied with filtertipped 20μL pipettes to add their purified DNA into their reaction tubes. Pre-preparing reaction tubes made running qPCR as simple as adding the DNA and pressing "Start" on the Open qPCR machine. Our laboratory also performed an analysis of the qPCR data. CBM partners would download their spreadsheets from the Open qPCR and send them either via email or Google Drive to our laboratory, where we would analyze control data, and calculate copy numbers and, where possible, organismal numbers for partners. Again, this was done in an attempt to preserve the simplicity of the method, and because analysis of qPCR data is complex and requires an expert eye.
The CBM partners participating in this study ran 985 total samples over the two years of this program. Deductive thematic analysis was performed to analyze CBM partner surveys, which is a method of analysis by which codes and theme development were directed by our existing research questions. Three primary themes emerged from this analysis.
The first theme identified was "Rapidly responding to hazards." Our CBM partners liked that the "time requirement from the qPCR testing method was less than the traditional operational time frame. . ." Our CBM partners seemed to equate the rapidness of the method to a rapid policy response to hazards. This was not the case, as our province has only begun to adapt policy frameworks to qPCR methods.
The second theme was "Independence and verification of a CBM monitoring system." CBM partners expressed a desire for more independence and more control over the interpretation of results. Our study was designed to remove data interpretation from the participant's hands. We thought this would be beneficial because the interpretation of qPCR data is not trivial, and to prevent panic if CBM partners saw positive samples that, while meaningful, might not constitute a real concern. CBM partners said they wished the data was published online, "If the data was available or if there was a way to input the data online into a database. Then we could use the results more easily," they said. Our CBM partners also expressed a desire to validate their results and have access to quality control data. One user suggested, ". . .a visual that compared our results to yours, so we have some idea of if we were capturing the results accurately." Another specifically suggested that ". . .third party verification can be one method to enhance the validity of the results," suggesting a desire for some oversight to ensure data quality, but also a desire for CBM partners to know that they are contributing meaningful and accurate results.
One of the biggest challenges for CBM programs is data validation, storage, and visualization. However, tools are emerging to address this challenge, including the Lake Observer mobile app through the Global Lake Ecological Observatory Network, the DataStream through the Gordon Foundation, or the ABMI's NatureLynx. Allowing community partners to upload and visualize their results may help partners feel that they are part of something bigger. It may allow them to contextualize their results relative to other water bodies, log additional environmental observations, or upload photographs of recreational waters. These apps can also be helpful to track long-term results or to have the data incorporated into reporting by other agencies.
The third theme we identified was that the triangulation of training was valuable. CBM partners appreciated the three forms of training. Most CBM partners found "the training videos were really useful." CBM partners found the written protocol useful as a reference but suggested that after "around 2-3 runs of the machine, this resource was no longer needed." Most CBM partners stressed the importance of the in-person training, and one user stated that "the in-person training went a long way in creating and (sic) increased comfort and confidence in the machine." Other studies that have looked at how to effectively train participants for CBM projects have found that multiple training sessions can improve data accuracy [bib_ref] Data reliability in citizen science: learning curve and the effects of training..., Ratnieks [/bib_ref]. A recent study that focussed specifically on training in molecular biology especially emphasized that multiple training sessions and especially hands-on training was key to participants being able to successfully complete the method [bib_ref] Lessons Learned from Implementing a Wet Laboratory Molecular Training Workshop for Beach..., Verhougstraete [/bib_ref].
## Agreement between cbm partners and scientists
The LOD 95 is the lowest concentration of DNA that can be reliably detected in 95% of samples; it is a measure of sensitivity. The Open qPCR thermocycler has a higher limit of detection when using a Taqman fluorescein probe than the ABI core thermocyclers (63.4 DNA copies/ 5μL versus >10 DNA copies/5μL across all methods, respectively). The field thermocyclers are less sensitive than the core laboratory machine. Understanding this change in the detection limit is important in determining if the CBM qPCR system would be effective for a particular test. For instance, if the concentration of the target that might constitute a risk is below the LOD 95 for the Open qPCR thermocyclers, "risky" samples will appear negative as this thermocycler is not capable of detecting them. When we deployed the human-associated Bacteroides HF183 CBM testing for recreational shoreline source tracking in Michigan, USA, our CBM partners reported only a single positive sample. However, when these DNA extracts were analyzed, 22.7% (54/237), were found to be positive for between 15-35 copies DNA/5ul. Seven of these samples approached the LOD 95 of the Open qPCR thermocycler, and CBM partners detected one of these samples. This is a common issue in PCR based fecal source tracking studies, not just in CBM studies, and highlights the importance of ensuring a particular method fits the research question, especially in a CBM study [bib_ref] Large-scale implementation of standardized quantitative real-time PCR fecal source identification procedures in..., Li [/bib_ref]. Nonetheless, a recent study found that an HF183 gene copy number of 4200 HF183/100ml exceeds the USEPA benchmark risk of GI illness [bib_ref] Human-Associated Fecal Quantitative Polymerase Chain Reaction Measurements and Simulated Risk of Gastrointestinal..., Boehm [/bib_ref]. This level is equivalent to a gene copy number of 210 HF183 GC/5ul-well above the detection limit of the Open qPCR thermocycler. Thus, outbreak scenarios would be clearly discernable. However, this also illustrates an example of how the monitoring project must be clearly rooted in a management outcome. If the intention of the monitoring program is to detect potential outbreak scenarios and initiate action, the increased detection limit is acceptable. However, if the management context is the detection of leaking septic areas, or source tracking fecal markers on a beach, this detection limit would be inappropriate. It is important to work closely with CBM partners to understand their specific monitoring questions, and critically appraise if CBM qPCR is capable and appropriate to answer these questions.
ICC analysis for the avian trematode assays showed a very high level of agreement between the Open qPCR thermocycler and the core thermocyclers . The toxic cyanobacteria test showed much lower levels of agreement between the field thermocycler and the laboratory thermocycler. We discovered through analyzing the control standards that the heated lid on the field thermocycler was loose and therefore was failing to engage properly with the tops of the reaction tubes (i.e. machine failure). However, from a quality control perspective, we were able to detect a probable machine failure with a sample size comparison of 11. This is extremely promising for future larger-scale CBM qPCR systems. It suggests that it would be possible with a relatively low number of samples being confirmed by a core facility or quality control partner to detect user or machine error once a baseline level of agreement for a single test had been established.
The comparison between CBM partners performing DNA extraction and experts performing the DNA extraction was first assessed semi-quantitatively using the Bland-Altman plot [fig_ref] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the... [/fig_ref]. The results of this analysis for the almost all targets show a linear and negative linear pattern at lower gene copy numbers. This can be due to bias between methods, but can also be caused by a difference in the within-subject standard deviation [bib_ref] repeatability and reproducibility: analysis of measurement errors in continuous variables, Bartlett [/bib_ref]. This seems plausible as users with potentially very different skill levels are performing the two methods. A paired t-test using the log-transformed data was used to compare the within-subject standard deviations. They were significantly different, which suggests that the linear pattern observed is due to increased variability in CBM partner data.
Partner extracted samples are typically lower in copy numbers than expert-extracted samples [fig_ref] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the... [/fig_ref]. This is likely due to differences in DNA extraction efficiency between the CBM partners and experts. However, it seems more experienced users become better at DNA extraction over time, as both the avian schistosomes monitoring group and the toxic cyanobacteria monitoring groups seem to improve over time. [fig_ref] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the... [/fig_ref]. Its unsurprised that the ICCs and maximum log differences are higher when comparing partner and expert extracted DNA samples due to the highly variable nature of DNA extraction, and because the duplicate samples run in the central laboratory could never be expected to contain exactly the same amount of organism. The ICCs of the DNA extraction comparison ranged from 0.54 to 0.67, with maximum log differences ranging from 1.3 to 1.4 . It is important to note that for the avian schistosomes monitoring program, a change was made in 2018 to establish a fully functional remote laboratory, and move these partners onto using the Qiagen DNeasy DNA extraction kit. This change was made at the request of the CBM partners, who would typically collect and analyze hundreds of samples each field season. Details about the equipment in this satellite laboratory can be found in S2 [fig_ref] Table: Field method materials used in this study [/fig_ref] Ebentier et al. (2013) conducted a reproducibility analysis of five core laboratories on a panel of microbial source tracking qPCR markers. They calculated reproducibility as the maximum expected log difference (within 95% confidence) between the different laboratories. Their analysis demonstrated reproducibility coefficients for different qPCR assays were highly variable, between 0.09-0.66 log. The methods that were likely to produce higher copy numbers, like Enterococcus qPCR testing via USEPA Method 1611, showed higher reproducibility coefficients than methods that were likely to produce lower copy numbers, like human-associated Bacteroides marker HF183. They also analyzed the contribution to variability of a variety of factors (the sample itself, equipment, procedures) to the measurement. Their paper concluded that when protocols and reagents were not standardized, the agreement between methods decreased. They highlighted the need for standardization of protocols and consumables before the implementation of studies involving multi-laboratory experiments [bib_ref] Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial..., Ebentier [/bib_ref] [bib_ref] Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator..., Shanks [/bib_ref].
The maximum log difference of the CBM qPCR monitoring program higher than the values reported in the Ebentier paper. Reproducibility between the same extract performed by our expert team and the CBM partners ranged from 0.44 to 1.5 log, and reproducibility coefficients of between partner and expert extracted split samples ranged from 1.3 to 1.4 log . It should be noted that the majority of the qPCR methods deployed routinely detected copy numbers in excess of 1 log. Thus, we might expect higher variability between replicates at these larger copy numbers [fig_ref] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the... [/fig_ref]. CBM qPCR monitoring programs will likely generate data that does have higher variability. It's important to weigh the pros of a CBM qPCR approach, notably that a CBM qPCR approach may result in increased numbers of samples from across a larger geographic area and builds relationships and partnerships across sectors.
## Future directions for the cbm qpcr program
Rapid monitoring approaches, including CBM qPCR, should be deployed within the context of a policy framework and management response plan that can support acting upon the results generated. The response plan for samples that might constitute a hazard should be clear to CBM partners. If response plans lack transparency, a CBM partner who encounters a sample that contains a high level of an indicator organism, but upon subsequent tests shows low or no risk, might be dismayed by a lack of response by government. A CBM qPCR monitoring system in recreational water would need to prioritize communication and understanding between regulators and CBM partners, and would likely function best when addressing specific objectives [bib_ref] Advice and Frequently Asked Questions (FAQs) for Citizen-Science Environmental Health Assessments, Barzyk [/bib_ref].
Whether the rapid CBM qPCR monitoring system enables a more rapid response to hazards is yet to be seen; however, CBM qPCR monitoring certainly has the advantage of being able to generate data over a large geographic area and for numerous hazards. It could be adapted to measure organisms not typically considered in monitoring programs; as we have demonstrated in our study, the approach works equally well for eukaryotic hazards like parasitic organisms as it does for the more traditional prokaryotic targets like enteric bacteria. The flexibility inherent in CBM qPCR makes this an attractive and adaptive platform for governments and communities to answer management related questions for their watersheds.
Our vision for the CBM qPCR monitoring system was that data analysis would not occur in the hands of CBM partners [fig_ref] Fig 1: Implementation process of the CBM qPCR program [/fig_ref]. Analysis of qPCR data, while not extremely complex, does require a more comprehensive understanding of qPCR data; additionally, data interpretation is typically the most erroneous component of CBM programs [bib_ref] Citizen Science: Can Volunteers Do Real Research?, Cohn [/bib_ref]. Our study supports a CBM monitoring program that is supported by a central agency. Some central program (i.e., academic, governmental, or not-for-profit) should distribute materials and provide QC support. Participants in our study expressed a desire to know how well they were performing the method. This highlights an important component of a large-scale CBM monitoring program: a compliance testing system that would test and train potential participants to ensure the method is being conducted appropriately. This must include third-party verification of a certain percentage of all samples tested. While verification is important to ensure CBM partners are generating reliable results, it is essential that communication is prioritized. This includes responding quickly to results reported by CBM partners when a potential hazard is detected. It also includes being honest with partners about their performance, and willingness by both the CBM and regulatory partners to collect and assess additional samples when clarification or confirmation is required. Any CBM program should support and empower communities to answer monitoring and research questions they are interested in.
# Conclusion
To our knowledge, this is the first study to comprehensively test the accuracy of a CBM qPCR water monitoring approach in a real-world context. Our results show that when implemented in a controlled manner, such that a central body controls materials and protocols, results can be highly reproducible. Our study also suggests that CBM partners, whose buy-in would be required for ensuring program longevity, value the method and the results it provides.
CBM qPCR could process a large number of samples from a wide geographical area that could aid beach management for health and invasive species. CBM qPCR could act as a valuable component of an environmental monitoring surveillance system. qPCR is a platform, and therefore a myriad of diagnostic tests could be deployed as needed in remote locations. While CBM qPCR programs may be more variable than traditional monitoring programs, they could serve as a comprehensive screening system for traditional monitoring programs. In many contexts, CBM qPCR programs could be as accurate as traditional testing and have the potential to replace traditional testing.
Supporting information S1 [fig_ref] Table: Field method materials used in this study [/fig_ref] Primers and probes used in this study.
[fig] Fig 1: Implementation process of the CBM qPCR program. Cells with blue backgrounds are processes done in collaboration between the central laboratory and the CBM partners, yellow backgrounds indicate processes completed by the central laboratory, and red backgrounds indicate processes completed by the CBM partners. [/fig]
[fig] Fig 2: Bland-Altman graphs of the difference between the CBM partners' data and the central laboratories' data. Limits of agreement (1.96 times the standard deviation) are bounded by the dotted lines. Top: Agreement of the 2017 Avian schistosome monitoring program. Middle: Agreement of the 2018 avian schistosomes monitoring program. Bottom: Agreement of the microcystin gene monitoring program. https://doi.org/10.1371/journal.pone.0229701.g002 [/fig]
[table] PLOS ONE: | https://doi.org/10.1371/journal.pone. [/table]
[table] Table: Field method materials used in this study. (DOCX) S4 Table. Questions administered to users in survey. (DOCX) S5 Table. (DOCX) [/table]
|
Socioeconomic status and neural processing of a go/no-go task in preschoolers: An assessment of the P3b
While it is well established that lower socioeconomic status (SES) is associated with poorer executive functioning (EF), how SES relates to the neural processing of EF in childhood remains largely unexplored. We examined how household income and parent education related to amplitudes of the P3b, an event-related potential component, during one EF task. We assessed the P3b, indexing inhibition and attention allocation processes, given the importance of these skills for academic success. Children aged 4.5-5.5 years completed a go/no-task, which assesses inhibitory control and attention, while recording EEG. The P3b was assessed for both go trials (indexing sustained attention) and no-go trials (indexing inhibition processes). Higher household income was related to larger P3b amplitudes on both go and no-go trials. This was a highly educated sample, thus results indicate that P3b amplitudes are sensitive to household income even within the context of high parental education. Findings build on the behavioral literature and demonstrate that SES also has implications for the neural mechanisms underlying inhibition and attention processing in early childhood.
# Introduction
Socioeconomic status (SES) has broad implications for development, with high SES parents able to invest time and money in their children's development and children from lower SES families at risk for adverse outcomes including poorer health, psychological well-being, and academic achievement (see for review; [bib_ref] Socioeconomic status and child development, Bradley [/bib_ref] [bib_ref] Socioeconomic status and the brain: mechanistic insights from human and animal research, Hackman [/bib_ref] [bib_ref] The widening academic achievement gap between the rich and the poor: new..., Reardon [/bib_ref]. In particular, the SES gap in executive functioning (EF), encompassing higher-order cognitive thinking, is evident by kindergarten [bib_ref] Childhood poverty: specific associations with neurocognitive development, Farah [/bib_ref] [bib_ref] Poverty as a predictor of 4-year-olds' executive function: new perspectives on models..., Raver [/bib_ref]. Despite implications of EF for long-term academic and socioemotional outcomes [bib_ref] Developmental links between children's working memory and their social relations with teachers..., De Wilde [/bib_ref] [bib_ref] Kindergarten children's executive functions predict their second-grade academic achievement and behavior, Morgan [/bib_ref] [bib_ref] Executive function and the promotion of social-emotional competence, Riggs [/bib_ref] , the mechanisms underlying SES-EF associations are not fully understood.
One promising approach is to assess how SES relates to neural mechanisms of EF at school entry, when EF is rapidly developing [bib_ref] Developmentally sensitive measures of executive function in preschool children, Carlson [/bib_ref] [bib_ref] The neuroscience of socioeconomic status: correlates, causes, and consequences, Farah [/bib_ref]. Given that many processes underlie behavioral performance, neural measures help tease apart specific aspects of processing while children perform EF tasks. Indeed, neural processing has been proposed to be a factor underlying socioeconomic gaps in cognitive development [bib_ref] Brain imaging and electrophysiology biomarkers: is there a role in poverty and..., Pavlakis [/bib_ref]. Thus, the brain could be the intermediary in explaining how SES shapes life outcomes [bib_ref] The neuroscience of socioeconomic status: correlates, causes, and consequences, Farah [/bib_ref] and could serve as an underlying mechanism in understanding how SES shapes EF. Additionally, the practical implication of assessing neural processes is their potential to serve as biomarkers with predictive power for later outcomes [bib_ref] The neuroscience of socioeconomic status: correlates, causes, and consequences, Farah [/bib_ref] [bib_ref] Prediction as a humanitarian and pragmatic contribution from human cognitive neuroscience, Gabrieli [/bib_ref] [bib_ref] Effects of socioeconomic status on brain development, and how cognitive neuroscience may..., Raizada [/bib_ref]. EF demands increase throughout school and EF becomes increasingly essential for academic success as children get older [bib_ref] Self-regulation in early childhood: improving conceptual clarity and developing ecologically valid measures, Mcclelland [/bib_ref]. Therefore, neural measures could aid in predicting EF and academic performance [bib_ref] Promoting resilience in children and youth, Greenberg [/bib_ref] [bib_ref] Stability of executive function and predictions to adaptive behavior from middle childhood..., Harms [/bib_ref] [bib_ref] Effects of socioeconomic status on brain development, and how cognitive neuroscience may..., Raizada [/bib_ref]. kindergarten study, P3b amplitudes were reported for the go condition only during a go/no-go where children won and lost points; and larger P3b amplitudes predicted better academic performance in first grade [bib_ref] Relevance of a neurophysiological marker of attention allocation for children's learning-related behaviors..., Willner [/bib_ref]. This demonstrates the practical role of ERPs in predicting outcomes. Further, given that inhibition and attention vary by SES (e.g., [bib_ref] A meta-analysis of the relationship between socioeconomic status and executive function performance..., Lawson [/bib_ref] and these processes are critical for learning and academic success [bib_ref] Relations between inhibitory control and the development of academic skills in preschool..., Allan [/bib_ref] [bib_ref] Executive functions, Diamond [/bib_ref] , an ERP component that indexes these processes is a probable candidate to vary by SES. Moreover, research suggests the P3b increases in amplitude through adolescence [bib_ref] Event-related potential measures of executive functioning from preschool to adolescence, Downes [/bib_ref]. Taken together, it is possible that higher SES would relate to larger P3b amplitudes, perhaps indicating more developed neural processing.
## Income, parent education, and neural processing
When assessing socioeconomic context, it is important to consider how to conceptualize and operationalize SES. SES is a complex construct that reflects financial resources and capital [bib_ref] Socioeconomic status and the developing brain, Hackman [/bib_ref]. The most common indicators are household income, parental education, and parental occupation (see for review, [bib_ref] Neurocognitive development in socioeconomic context: multiple mechanisms and implications for measuring socioeconomic..., Ursache [/bib_ref]. There is debate whether to combine these measures into a composite or assess them separately [bib_ref] Neurocognitive development in socioeconomic context: multiple mechanisms and implications for measuring socioeconomic..., Ursache [/bib_ref]. While using composite SES measures is common, others argue that SES constructs (e.g., income and parent education) have different implications for development and are conceptually distinct . For instance, household income has been more related to academic success while parent education has been associated with both academic and behavioral outcomes [bib_ref] Off with hollingshead: socioeconomic resources, parenting, and child development, Duncan [/bib_ref].
Additionally, these aspects of SES differentially relate to structural brain development as income was associated with cortical thickness while parent education related to hippocampal volume . Moreover, other studies have assessed how only one aspect of SES (such as parent education) relates to the brain in childhood [bib_ref] Differences in the neural mechanisms of selective attention in children from different..., Stevens [/bib_ref]. It is possible that income and parent education could relate to brain development and adaptive outcomes via different mechanisms. Parent education may be more related to parenting style while income could enable access to learning materials and higher quality child-care. Therefore, assessing how income and parent education separately relate to neural EF processes would provide a more comprehensive and specific assessment of the role of the socioeconomic context.
## Current study
Given that low SES children are at risk for poor EF by kindergarten entry, it is critical to understand the neural mechanisms that contribute to this disparity. The goal of this study was to take a first step and examine how indices of SES relate to the P3b in a go/no-go task in 4.5-5.5 year olds. This task taps two aspects of EF including inhibition and attention processes. Based on previous literature, we expected the P3b to be larger on no-go trials compared to go trials [bib_ref] Neural correlates of response inhibition in early childhood: evidence from a Go/No-go..., Rahman [/bib_ref] [bib_ref] The X-trials: neural correlates of an inhibitory control task in children and..., Davis [/bib_ref]. We explored the separate contributions of household income and parent education to provide specificity in how socioeconomic context may relate to neural processing. We assessed the P3b on go and no-go trials to examine whether income and parent education mattered for the P3b on sustained attention (go) or inhibition (no-go) trials, or whether there were global effects. We expected higher income and higher levels of parent education would be associated with larger P3b amplitudes.
# Methods
## Participants
Participants were 69 children (40 females) aged 4.5-5.5 years and their primary caregivers. Children spoke and understood English.
Children were full-term singletons with no known hearing, visual, neurological, or developmental disorders. The primary goal of this study was to assess how income and parent education related to the P3b on go and no-go trials. Therefore to be included in analyses, (1) children had to have useable ERP data for both trial types and (2) children needed to understand the task. This ensured that ERPs included in analyses were only from children engaging in the task. Therefore while 125 children participated in the study, 56 were excluded leaving a final sample of 69. Children were excluded for the following reasons: did not understand the task (N = 3), which was defined as go accuracy was less than 70% and no-go accuracy was higher than their go accuracy; declined to complete the task (N = 6); did not have usable ERPs for both trial types (go and no-go; N = 29); had EEG technical difficulties (N = 5); had braided hair, which prevented electrodes from sufficiently contacting the scalp (N = 4); and declined to wear the EEG cap (N = 9). There were no differences in child age, child gender, income, or parent education, between the group of children included in the final sample and those excluded.
Of the final sample, 26 children attended preschool, 15 children were in kindergarten, and 28 children did not attend preschool or kindergarten. See [fig_ref] Table 1: Demographics. [/fig_ref] for demographic information of the final sample.
Effort was made to recruit a sample across the SES spectrum. Thus 38.20% of the sample was at or below an income-to-needs (ITN) ratio of 3.0, meaning they had an income less than three times the federal poverty line, given their household size. Given the high cost of living in the city where this study was conducted, an income below the threshold of 3 times the federal poverty line is considered financially strained (Ames, Lowe, Dowd, Liberman, & Youngblood, 2013). See [fig_ref] Table 2: ITN and Parent Education Information [/fig_ref] for a breakdown of the income and parent education distribution in the sample.
## General procedure
Participants were recruited from a department-maintained database of families interested in research; from publicly available state birth records; from online advertising; and through face-to-face-recruitment events at Head Starts, diaper banks, community play groups, and a community health center. This study was approved by the university Institutional Review Board. Parent-child dyads visited the laboratory for one session lasting 1.5-2.0 h. Following informed consent, the experimenter presented the child with a sticker card with the child's name on it and explained that by working hard at the game, they could earn stickers. Next the experimenter placed the EEG cap on the child's head. While EEG was recording, the child completed a computerized go/nogo task in an electrically shielded booth to prevent interference with the EEG signal. The task was administered via E-Prime 2.0 software (Psychology Software Tools, Pittsburgh, PA, USA). Next the child completed assessments of receptive language and nonverbal IQ. The parent completed a demographics questionnaire.
## Measures
## Go/no-go
Children were told that all of the animals had escaped from their cages at the zoo and the zookeeper needed their help to catch them [bib_ref] Anger and approach motivation in infancy: relations to early childhood inhibitory control..., He [/bib_ref] [bib_ref] Cognitive control moderates early childhood temperament in predicting social behavior in seven..., Lamm [/bib_ref] , but that the friendly orangutans are helping them catch the animals. Therefore children were told to press a button to catch the animals each time they saw an animal (go trial) but not to press the button when they saw an orangutan (no-go trial). Thus children had to inhibit their dominant responses on no-go trials. Children completed 18 practice trials and the rules were repeated halfway through the task. Each trial consisted of an animal stimulus presented for 750 ms and then a blank screen presented for 500 ms. Each trial was followed by a blank screen with a randomized inter-trial interval between 200-300 ms. Children could respond during the presentation of the stimulus or on the blank screen. Therefore, the trial ended either at the end of the 500 ms blank screen or when the child pushed the button, whichever came first [fig_ref] Figure 1: Visual depiction of the go/no-go task [/fig_ref].
Children completed a total of 280 trials of which 75% were go trials and 25% were no-trials. The trials were broken up into four blocks. Children were shown a map of the zoo at the beginning of the task and after each block so they could track their progress and they received two stickers at the end of each block. Accuracy was computed for each trial type (go and no-go) and block. The go trials index sustained or selective attention while the no-go trials index actual inhibition processes [bib_ref] Evidence of substantial development of inhibitory control and sustained attention between 6..., Lewis [/bib_ref] [bib_ref] Relevance of a neurophysiological marker of attention allocation for children's learning-related behaviors..., Willner [/bib_ref]. Reaction time was calculated as the mean reaction time on correct go trials only. Trials with reaction times < 150 ms were excluded prior to computing the mean reaction time. The task took around 12 min to complete. See [fig_ref] Table 3: Task Descriptive Statistics [/fig_ref] for task descriptive statistics.
## Household income
Parents reported on household income and household composition. An ITN ratio based on the federal poverty level was computed with income and household composition.
## Parent education
The parent reported highest maternal and paternal educational level. They were coded from 1 (some middle school or high school) to 5 (graduate degree). Scores were averaged to yield a parent education composite.
## Tested covariates 2.4.2.1. language.
Children completed the picture vocabulary test (normed for ages 3-85) from the National Institutes of Health Toolbox Cognition Battery [bib_ref] Language measures of the NIH toolbox cognition battery, Gershon [/bib_ref]. This measures receptive vocabulary and uses a computerized adaptive format based on performance. The child hears a word and sees four photographs on the screen and is asked to select the picture that most closely matches the meaning of the word. Age-adjusted scores were used. Two children did not have scores due to technical difficulties.
## Nonverbal iq. the matrices sub-scale of the kaufman brief
Intelligence Test, Second Edition, was used. The assessment is multiple choice and involves the child pointing to pictures that reflect an understanding of both meaningful and abstract relationships. The task takes 5-10 min to complete. Age-adjusted scores were used. This assessment was not administered to two children.
## Electrophysiological recording and analysis
EEG was recorded to a vertex reference using NetStation acquisition software and a Net Amp 300 amplifier (Electrical Geodesics, Inc.: Eugene, OR) connected to a Geodesic Sensor Net with 128 electrodes spaced ∼1 cm apart over the scalp. Prior to use, the 128 lead highdensity net was soaked for 10 min in an electrolyte solution (6cc potassium chloride/liter distilled water) to facilitate electrical contact between the scalp and electrodes. Prior to recording, impedances were lowered by administering small amounts of the electrolyte solution to electrodes with poor contact. Data were sampled from all channels at 500 Hz. Offline data was processed using NetStation. A bandpass filter of .3-30 Hz was applied. Continuous EEG data was then was segmented time-locked to trial onset from -100 prior to the trial to 1000 ms after the trial. As with the behavioral data, only trials in which the child responded correctly were included and additionally for go trials, when reaction time was > 150 ms. Each segment was baseline corrected, using the mean voltage in the 100 ms prior to stimulus onset. Next, an automatic artifact rejection paradigm identified channels with excessive artifact (> 150 μV). In addition, segments with eye blinks (> 140 μV differential average) or eye movements (> 100 μV differential average) were excluded. Next bad channels were replaced via interpolation and segments for each child were averaged within each trial type (go and no-go) and re-referenced to the average reference. Each child had to have at least 10 useable trials for each trial type to be included in analyses. For the final sample, this resulted in an average 63.45 (SD = 32.45) go trials and 24.59 (SD = 12.11) no-go trials.
Mean P3b amplitude was computed in the time window 400-700 ms post-stimulus in the parietal region for go trials and no-go trials. This time window was selected to be generally consistent with past research [bib_ref] Early adversity and neural correlates of executive function: implications for academic adjustment, Mcdermott [/bib_ref] [bib_ref] Relevance of a neurophysiological marker of attention allocation for children's learning-related behaviors..., Willner [/bib_ref]. Visual inspection of the grand-averaged waveforms confirmed that the P3b occurred in the window from 400 to 700 ms post-stimulus. Further, individual waveforms were inspected and time windows were adjusted if needed to ensure that the P3b was represented. This was the case for four children. For one child, the revised time window was 300-600 ms and for three other children, the time windows were adjusted to 350-650 ms. Visual inspection indicated that the P3b was maximal in the parietal region, consistent with past research [bib_ref] The X-trials: neural correlates of an inhibitory control task in children and..., Davis [/bib_ref] [bib_ref] Early adversity and neural correlates of executive function: implications for academic adjustment, Mcdermott [/bib_ref] [bib_ref] Relevance of a neurophysiological marker of attention allocation for children's learning-related behaviors..., Willner [/bib_ref]. The following electrodes with their corresponding 10-20 system sites were averaged into one parietal region: 61 (P1), 62 (Pz), 67 (PO3), 72 (POZ), 77 (PO4), 78 (P2; see [fig_ref] Figure 2: The parietal electrodes used in the current study [/fig_ref]. The mean amplitude of the go P3b was not correlated with the number of useable go segments, r (67) = .008, p = .95, and the mean amplitude of the no-go P3b was not correlated with the number of useable no-go segments, r (67) = −.06, p = .65.
# Analysis plan
A preliminary model was first run to check that the paradigm elicited the expected differences in P3 amplitude. We expected larger P3b amplitudes on no-go trials compared to go trials. A repeated measures analysis of variance (RM-ANOVA) was used with trial type as a withinsubjects factor. Next, Pearson correlations were used to assess relations of behavioral performance to the P3b. In addition, Pearson correlations were used to assess relations of ITN and parent education to behavioral performance.
To examine possible associations of child gender, age, language, and nonverbal IQ to the P3b, each covariate was included in the repeated measures model. Gender was included as a between-subjects factor and continuous variables were included as covariates. Whenever there was an effect of the covariate in the model, the covariate was included in subsequent models. In addition, the relations between child age with income and parent education were tested using Pearson correlations.
To assess relations of ITN and parent education to the P3b, we used repeated measures analyses of covariance (RM-ANCOVAs) with the P3b as the dependent measure. Trial type (go or no-go) was included as a within-subjects factor. ITN and parent education were included as predictors in separate models. This allowed us to test for main effects of ITN and parent education as well as interactions. Statistically all continuous variables were entered as covariates. We use the term "predictor" for ITN and parent education to distinguish from variables that we treated as potential covariates (e.g., age).
For all ANOVAs, post hoc analyses followed significant main effects, using Bonferroni corrections for multiple testing. In ANOVAs where the assumption of sphericity was violated, we used Greenhouse-Geisser corrections.
# Results
## Preliminary analyses
We first used a RM-ANOVA to test for an effect of trial type. As would be expected, there was a main effect of trial type, F (1, 68) = 18.10, p < .001, ηp 2 = .21, such that P3b amplitudes were larger on no-go trials (M = 19.83 μV, SD = 11.86) compared to go trials (M = 16.68 μV, SD = 9.47). See .
Accuracy on go trials was significantly correlated with the go P3b, r (67) = .27, p = .03, such that higher accuracy related to larger go P3b amplitudes. No other correlations associating behavioral performance and P3b amplitudes were significant. See [fig_ref] Table 4: Correlations of Study Variables [/fig_ref] for correlations of study variables. In regards to behavioral performance and SES indices, ITN and parent education did not relate to accuracy or reaction time.
Age, language, and nonverbal IQ did not relate to P3b amplitude when included in the RM-ANCOVA, with trial type (go and no-go) as a within-subjects factor. When gender was included, there as a trial type x gender interaction, F (1, 67) = 6.31, p = .014, ηp 2 = .09. While the parameter estimates did not show a significant gender difference on either trial type, the pattern suggested that females had larger P3b amplitudes on go trials (M = 17.91 μV, SD = 9.38) compared to males (M = 14.99 μV, SD = 9.51). There were no differences in no-go P3b amplitude in females (M = 19.53 μV, SD = 12.02) compared to males (M = 20.23 μV, SD = 11.84). Gender was thus included in subsequent models. Finally, child age was not significantly correlated with ITN or parent education (see [fig_ref] Table 4: Correlations of Study Variables [/fig_ref].
## Relations of itn and parent education to p3b amplitudes
We ran two RM-ANCOVAs to test the roles of ITN and parent education. In the first model, trial type (go or no-go) was a within-subjects factor, gender was a between-subjects factor, and ITN was a predictor. There was a main effect of ITN, F (1, 65) = 4.21, p = .04, ηp 2 = .06. Assessment of the parameter estimates indicated that higher ITN was associated with larger P3b amplitudes (see . There was no trial type x ITN interaction.
In the next model to test for effects of parent education, we used a RM-ANCOVA with trial type (go or no-go) as a within-subjects factor, gender as a between-subjects factor, and parent education as a predictor. There was no main effect of parent education or interactions with parent education.
# Discussion
We examined how indices of SES related to electrophysiological processing of a go/no-go task in early childhood. We focused on the P3b, an index of inhibition and attention allocation processes, given the relevance of this ERP component as a predictor of later academic outcomes. Children aged 4.5-5.5 years completed a go/no-go task and P3b amplitudes were calculated for go and no-go trials. Thus we focused on two aspects of EF that were assessed in the task: inhibitory control and attention processes. Even though the sample was highly educated, results showed that higher household income was associated with larger P3b amplitudes of both go and no-go trials. Given the SES disparities in EF and ultimately academic achievement, results highlight the potential relevance of neural processing as a mechanism to understand these behavioral differences.
To our knowledge, this is the first study to demonstrate that the P3b in early childhood varies by socioeconomic context, specifically household income. Studies have begun to assess the neural bases of EF in early childhood (e.g., [bib_ref] Event-related potential measures of executive functioning from preschool to adolescence, Downes [/bib_ref] and the association between SES and behavioral measures of EF is well-established (e.g., [bib_ref] A meta-analysis of the relationship between socioeconomic status and executive function performance..., Lawson [/bib_ref]. Yet to our knowledge this is one the first studies to examine how SES also matters for neural processing of some aspects of EF during a go/no-go task. We focused on the P3b as it indexes complex attention processes and inhibition [bib_ref] Updating P300: an integrative theory of P3a and P3b, Polich [/bib_ref] , all relevant aspects of EF that are critical for academic success [bib_ref] Relations between inhibitory control and the development of academic skills in preschool..., Allan [/bib_ref]. Results indicate the P3b is sensitive to the environment, generally consistent with one study finding that P3b amplitudes were larger on no-go trials for never institutionalized children compared to a foster care group [bib_ref] Early adversity and neural correlates of executive function: implications for academic adjustment, Mcdermott [/bib_ref]. However, P3b amplitudes did not differ on go trials and the nature of this sample should be acknowledged, such that the foster care group was comprised of children who were originally raised in institutional care and thus experienced extreme early psychosocial deprivation. In addition, this result builds on the small body of literature that has found SES differences on ERPs of auditory selective attention [bib_ref] Children's event-related potentials of auditory selective attention vary with their socioeconomic status, D'angiulli [/bib_ref] [bib_ref] Differences in the neural mechanisms of selective attention in children from different..., Stevens [/bib_ref] and visual target detection [bib_ref] Socioeconomic disparities affect prefrontal function in children, Kishiyama [/bib_ref]. Our finding demonstrates that SES also is important for neural attention and inhibition processes that are required to perform go/no-go task.
Past research speaks to the predictive power of the P3b such that higher P3b amplitudes in kindergarten predicted better adaptive learning behaviors in first grade [bib_ref] Relevance of a neurophysiological marker of attention allocation for children's learning-related behaviors..., Willner [/bib_ref]. Concurrent relations have also been found between the P3b and academic achievement [bib_ref] Impulsiveness, aggression, reading, and the P300 of the event-related potential, Harmon-Jones [/bib_ref] [bib_ref] From ERPs to academics, Hillman [/bib_ref] , suggesting the importance of neural inhibition and attention processing for school success. We extend this literature by assessing how the socioeconomic context may play a role in the development of the P3b, as we show SES linked differences in the P3b by school entry. Future research is needed to longitudinally assess these constructs throughout early childhood to more comprehensively understand how the P3b may mediate relations between SES and later EF and academic outcomes.
An important question is why we see differences in P3b amplitude by household income. This is notable, given that this sample was highly educated. Thus, even within this highly educated sample, income mattered. SES co-occurs with other risks and income could thus reflect different aspects of the child's environment. Families with lower incomes may only be able to afford to live in areas with more environmental risks and toxin exposure (see for review, [bib_ref] The environment of childhood poverty, Evans [/bib_ref] [bib_ref] Socioeconomic status and the developing brain, Hackman [/bib_ref] which could negatively affect brain development. Chronic stress also is a probable mechanism [bib_ref] Socioeconomic status and the developing brain, Hackman [/bib_ref]. Living on a low income could be stressful for many reasons including worry about affording rent or enough food. Indeed, lower income families have more food insecurity, which affects cognitive development [bib_ref] Associations between household food insecurity in early childhood and children's kindergarten skills, Johnson [/bib_ref]. Lower-income parents may work multiple jobs and have less time for quality interactions with their children. Finally, parents may not be able to financially invest in cognitively stimulating learning materials and trips [bib_ref] Socioeconomic status and child development, Bradley [/bib_ref]. It may be that these aspects of SES (e.g., chronic stress) are more affected by income and less closely associated with parent education, and could in part help explain our result linking household income and child neural processing. For instance, even if parents are less educated, if they have a higher income, they likely would not experience the stressors and risks discussed above.
While a portion of our sample was economically strained, given the high cost of living where this study was conducted, the majority of parents were college educated. Thus, we may not have had the variability necessary to detect effects of parent education. Future research including a more diversely educated sample is critical to assessing the role of parent education for the P3b. It is possible with more education variability we would see differences in the P3b. Indeed, income and parent education were linked to different aspects of brain structure . Parent education has also related to ERPs of auditory selective attention [bib_ref] Differences in the neural mechanisms of selective attention in children from different..., Stevens [/bib_ref]. Future research including a sample that spans income and parent education continuums, as well as assessing ERPs of different aspects of EF, is needed to better understand how these SES indices matter for EF neural processing. A strength of our study is that we assessed SES continuously. We demonstrate that differences in neural processing are not only present . a. Grand-averaged waveform of go and no-go trials for the entire sample in the parietal region. The P3b was calculated as the mean amplitude from 400 to 700 ms (seen with the dashed lines). Four children had adjusted time windows to ensure the P3b was represented. For one child, their time window was 300-600 ms and three children had time windows of 350-650 ms. Time 0 ms indicates stimulus onset. b. Grand-averaged ERP waveforms for low and high ITN groups for go trials. The P3b was calculated as the mean amplitude from 400 to 700 ms (seen with the dashed lines). Time 0 ms indicates stimulus onset. Four children had adjusted time windows to ensure the P3b was represented. For one child, their time window was 300-600 ms and three children had time windows of 350-650 ms. Note. A median split was used to visually depict the relation between ITN and the P3b on go trials. ITN was analyzed continuously. c. Grand-averaged ERP waveforms for low and high ITN groups for no-go trials. The P3b was calculated as the mean amplitude from 400 to 700 ms (seen with the dashed lines). Time 0 ms indicates stimulus onset. Four children had adjusted time windows to ensure the P3b was represented. For one child, their time window was 300-600 ms and three children had time windows of 350-650 ms. Note. A median split was used to visually depict the relation between ITN and the P3b on no-go trials. ITN was analyzed continuously.
when comparing extreme SES groups, but are evident across the income spectrum. This is consistent with a study showing variation in brain structure on an SES continuum and also with a meta-analysis, which showed child behavioral EF varied across the SES spectrum [bib_ref] A meta-analysis of the relationship between socioeconomic status and executive function performance..., Lawson [/bib_ref]. In addition, we build on past studies which have used dichotomous groups to demonstrate SES differences in neural processing [bib_ref] Children's event-related potentials of auditory selective attention vary with their socioeconomic status, D'angiulli [/bib_ref] [bib_ref] Socioeconomic disparities affect prefrontal function in children, Kishiyama [/bib_ref] [bib_ref] Differences in the neural mechanisms of selective attention in children from different..., Stevens [/bib_ref]. However, a limitation is that we did not preregister our hypotheses, specifically that we expected higher levels of income and education to be associated with higher P3b amplitudes.
We also assessed how SES related to behavioral performance on the go/no-go task.
ITN and parent education did not relate to any behavioral measure. Thus, within our subsample that had useable ERP data, SES differences in P3b amplitudes were more pronounced than behavioral differences. However, past research has found SES differences on behavioral performance of the go/no-go task [bib_ref] Socioeconomic gradients predict individual differences in neurocognitive abilities, Noble [/bib_ref] [bib_ref] Neurocognitive correlates of socioeconomic status in kindergarten children, Noble [/bib_ref]. Future research including a larger sample in a longitudinal study is critical to more thoroughly examine the role of SES for both the P3b and behavioral performance.
Our data suggested that the go/no-go task did elicit the expected P3b amplitudes. As we anticipated and consistent with the literature (Abdul [bib_ref] Neural correlates of response inhibition in early childhood: evidence from a Go/No-go..., Rahman [/bib_ref] [bib_ref] The X-trials: neural correlates of an inhibitory control task in children and..., Davis [/bib_ref] [bib_ref] ERP components in Go/Nogo tasks and their relation to inhibition, Falkenstein [/bib_ref] , P3b amplitudes were larger on no-go trials compared to go trials. In addition, there was a link between behavioral performance and the P3b such that children who had higher accuracy on go trials had larger P3b amplitudes on go trials. This suggests that the P3b does indeed index neural processing of sustaining or maintaining attention.
While to our knowledge, no studies have assessed how indices of SES relate to P3b amplitudes on a go/no-go task, we did not expect an SES by trial type interaction as behavioral differences by SES are typically seen for both inhibitory control and attention [bib_ref] Do maternal stress and home environment mediate the relation between early income-to-need..., Dilworth-Bart [/bib_ref] [bib_ref] Alerting, orienting, and executive attention: developmental properties and sociodemographic correlates in an..., Mezzacappa [/bib_ref] [bib_ref] Neurocognitive correlates of socioeconomic status in kindergarten children, Noble [/bib_ref]. In our study, SES did not differentially relate to the neural processing of sustained attention (i.e., go trials) and to inhibition (i.e., no-go trials). Instead, we saw a global effect such that children from families with higher incomes had larger P3b amplitudes on both of these trial types. This is consistent with SES-linked behavioral differences in both inhibitory control and attention. It is possible that the variation in the P3b by income level indicates the extent to which children recruit neural systems. Research suggests that P3b amplitudes increase with age through adolescence (see for review, [bib_ref] Event-related potential measures of executive functioning from preschool to adolescence, Downes [/bib_ref]. Thus it is possible that children from higher income families are showing more mature neural processing and therefore show increased P3b amplitudes on both go and no-go trials. A longitudinal study assessed ERPs in low and higher SES children at ages 4 and 5 using an auditory selective attention task [bib_ref] Development of selective attention in preschool-age children from lower socioeconomic status backgrounds, Wray [/bib_ref]. Results showed that low SES children at age 5 showed similar ERP patterns to the high SES children at age 4, suggesting that low SES children were delayed relative to their high SES peers. Future research exploring how income level relates to the P3b over time is needed to further tease apart this possibility.
Assessing the neural correlates of inhibition and attention is important for its potential to help explain SES differences in behavioral EF and academic success. An important future direction is to assess longitudinal relations between SES, ERPs, and later outcomes to move towards understanding how neural processing may be a mechanism for understanding how SES impacts later outcomes. Further, the current study only included one task that tapped certain aspects of EF. Future research including tasks that index additional EF skills, such as working memory and cognitive flexibility, are critical for understanding the role of SES for children's neural processing. Additionally, while this study cannot speak to causal relations between SES and neural processing, it contributes to characterizing SES differences in cognitive functioning. This can help inform the development of experimental designs to test for causal relations, with eventual implications of designing target interventions [bib_ref] Socioeconomic status and the developing brain, Hackman [/bib_ref]. Moreover, our findings expand upon the broader literature on effects of SES for the developing brain by demonstrating that SES relates to the neural processing of inhibition and attention processes. Together, this body of research underscores how early in life the brain is sensitive to socioeconomic context. This has serious implications for policy efforts to address the socioeconomic gap early before SES differences are entrenched.
Given that ERPs may be a tool to identify children at risk, information on how specific indices of SES are shaping ERPs can better inform policy and intervention efforts. We demonstrate that by preschool age, children are already showing differences in neural processing, with implications for later inhibition and attention processes. It is noteworthy that income was implicated for neural processing, despite the high level of education in our sample. This fits in with a recent movement for boosting family incomes with the hopes of improving child outcomes [bib_ref] Boosting family income to promote child development, Duncan [/bib_ref].
The nature of the cognitive processes indexed by the P3b is complex and researchers offer different interpretations of what the P3b practically means. In addition to being interpreted as an index of attention, inhibition and controlled processing [bib_ref] Updating P300: an integrative theory of P3a and P3b, Polich [/bib_ref] , the P3b has also been proposed to index context updating and working memory processes [bib_ref] Surprise!… surprise, Donchin [/bib_ref]. In a way, working memory is inherently involved in all tasks, given that to perform a task correctly, one must hold Note. ITN = income-to-needs ratio; EDU = parent education; amp = amplitude; ACC = accuracy; RT = reaction time. † p < 0.10.
* p < 0.05. ** p < 0.01. *** p < 0.001. the instructions/rules in working memory. Indeed, larger P3b amplitudes have been related to better performance on the backward digit span, an assessment of working memory [bib_ref] Predictive validity of the N2 and P3 ERP components to executive functioning..., Brydges [/bib_ref]. Regardless of the exact interpretation of the P3b, our study is a first step in demonstrating that neural processing of inhibition and attention processes varies by socioeconomic context. Future research is needed to more precisely characterize the nature of this relation.
# Conclusion
The relation of lower SES and poorer behavioral EF is well established. We demonstrate that by kindergarten entry, there are already SES-linked differences in the neural processing of inhibitory control and sustained attention. Specifically, higher income related to larger P3b amplitudes in 4.5-5.5 year olds, despite the highly educated nature of the sample. This study adds to the growing literature on effects of SES on the developing brain and demonstrates that household income is important for neural processes of attention and inhibition.
## Declaration of competing interest
The authors have no conflicts of interest to report.
[fig] Figure 1: Visual depiction of the go/no-go task. Children could respond during the stimulus presentation or during the 500 ms blank screen. [/fig]
[fig] Figure 2: The parietal electrodes used in the current study.A.M. St. John, et al. Developmental Cognitive Neuroscience 38 (2019) 100677 [/fig]
[fig] Funding: This research was supported by the American Psychological Association of Graduate Students Basic Psychological Research Grant and the Boston University Clara Mayo Memorial Fellowship to Ashley St. John. [/fig]
[table] Table 1: Demographics. [/table]
[table] Table 2: ITN and Parent Education Information.Note: ITN refers to the level of a household's income relative to the federal poverty line. Thus an ITN of 1.00 means that a family has an income at the federal poverty line. An ITN of 2.00 means that family has an income 2x that of the federal poverty line, etc. [/table]
[table] Table 3: Task Descriptive Statistics. Reaction time in accurate go trials (ms) 658.87 (66.60) 504.18 816.84 69 [/table]
[table] Table 4: Correlations of Study Variables. [/table]
|
Left atrial appendage flow velocity predicts recurrence of atrial fibrillation after catheter ablation: A systematic review and meta-analysis
2022) Left atrial appendage flow velocity predicts recurrence of atrial fibrillation after catheter ablation: A systematic review and meta-analysis. Front. Cardiovasc. Med. 9:971848.Purpose: There is increasing evidence that left atrial appendage flow velocity (LAAFV) is linked to the recurrence of atrial fibrillation (AF) after catheter ablation (CA), suggesting the potential predictable significance of LAAFV in this setting. We performed a systematic review and meta-analysis to assess whether LAAFV is association with AF recurrence after CA.Methods: Up to May 1, 2022, six databases (PubMed, EMBASE, Web of Science, Cochrane Library, Scopus, and CINAHL) were searched for literature reporting the association between LAAFV and AF recurrence after CA. All statistical analyses were carried out using STATA version 16 software. Heterogeneity was determined by the Cochrane's Q test and I 2 statistics. The Newcastle-Ottawa Scale (NOS) was used to assess the methodological quality of each included study, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method was adopted to evaluate the quality of evidence.Result: Sixteen studies with 5,006 AF patients after CA (1,479 patients with AF recurrence, 3,527 without AF recurrence) were included in the metaanalysis. The meta-analysis of 15 studies (16 data sets) showed that patients with recurrence exhibited lower LAAFV values than those without recurrence [standardized mean difference (SMD): −0.65, 95% CI: −0.88 to −0.42, P < 0.01]. Moreover, we evaluated the association of LAAFV and the risk of AF recurrence after CA. Nine studies (11 data sets) defined LAAFV as continuous variables, and the pooled analysis suggested that for every 1 cm/s rise in LAAFV values, the risk of AF recurrence after CA decreased by 3% [Odds Ratio (OR): 0.97, 95% CI: 0.95 to 0.99, P < 0.01]. Seven studies defined LAAFV as categorical variables, and the pooled analysis showed that lower LAAFV were associated with an increased risk of AF recurrence after CA [OR: 2.28, 95% CI: 1.46 to 3.57, P < 0.01]. The subgroup analyses showed that the association Frontiers in Cardiovascular Medicine 01 frontiersin.org Chen et al. 10.3389/fcvm.2022.971848 between LAAFV and AF recurrence after CA was not significantly affected by the AF type and ablation procedure. The NOS indicated that included studies were moderate to high quality, while the GRADE assessment suggested a low certainty of the evidence.Conclusion: Lower LAAFV may be associated with an increased risk of AF recurrence after CA. Further studies with well designed and randomized studies for LAAFV should be conducted. Systematic review registration: [https://www.crd.york.ac.uk/PROSPERO/], identifier [CRD42022333627]. KEYWORDS left atrial appendage flow velocity, atrial fibrillation, catheter ablation, recurrence, systematic review, meta-analysis
# Introduction
Atrial fibrillation (AF), affecting more than 46.3 million individuals worldwide, is the most common sustained arrhythmia [bib_ref] American heart association council on epidemiology and prevention statistics committee and stroke..., Benjamin [/bib_ref]. As one of the main risk factors for heart failure, myocardial infarction, and thromboembolic strokes, AF is associated with high mortality and hospitalization rates and ultimately imposes a considerable burden on individuals and society [bib_ref] Global epidemiology of atrial fibrillation: an increasing epidemic and public health challenge, Lippi [/bib_ref]. Catheter ablation (CA), an effective therapeutic option for drug-refractory symptomatic AF (3), was recommended as first-line therapy for patients with paroxysmal AF in a recent meta-analysis [bib_ref] Assessment of catheter ablation or antiarrhythmic drugs for first-line therapy of atrial..., Turagam [/bib_ref]. However, AF recurrence remains a challenging issue with a recurrence rate of up to 30%, which make it more essential to use screening factors to predict patients at high risk of AF recurrence and post-operative complications. The risk factors for AF recurrence include age, type of AF, duration of AF, left atrial (LA) enlargement, left ventricular ejection fraction, structure and function of left atrial appendage (LAA), atrial natriuretic peptide level, sleep apnea, obesity, and hypertension [bib_ref] Long-term outcomes after ablation for paroxysmal atrial fibrillation using the second-generation cryoballoon:..., Knight [/bib_ref]. The investigations of the association between LAAFV and AF recurrence following CA have increased exponentially in recent years. Nevertheless, these studies were small and contradictory. Therefore, we performed a systematic review and meta-analysis to evaluate whether LAAFV is association with AF recurrence after CA.
# Methods
This systematic review and meta-analysis was reported followed the criteria outlined in the Meta-Analysis of Observational Studies in Epidemiology (MOOSE) and the PRISMA 2020. The systematic evaluation program for this study was registered in PROSPERO (number: CRD42022333627).
## Search strategy
A systematic literature search was conducted independently by two investigators (Peng-fei Chen and Yu-jiao Shi) in six databases (PubMed, EMBASE, Web of Science, Cochrane Library, Scopus, and CINAHL). We searched for Englishlanguage literature published up to May 1, 2022. The following search MESH terms and keywords were used: "left atrial appendage flow velocity, " "atrial fibrillation, " "catheter ablation, " "radiofrequency ablation, " "cryoablation, " "recurrence." Detailed search strategies were shown in the Supplementary material. The disagreements were resolved by consulting a third investigator (Jian-peng Du).
## Eligible studies
Two investigators (Peng-fei Chen and Yu-jiao Shi) independently screened titles, abstracts, and full-text material to select eligible studies. The criteria for inclusion in this meta-analysis were as follows: [bib_ref] American heart association council on epidemiology and prevention statistics committee and stroke..., Benjamin [/bib_ref] observational studies with at least 6 months and completeness of follow-up period; (2) the target population was patients with AF (including paroxysmal AF and persistent AF) after CA (including radiofrequency and cryoballoon ablation); (3) comparing means and standard deviations (SDs) of LAAFV values in individuals with AF recurrence after CA to those without recurrence; and (4) reporting odds ratios (ORs) or hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) for LAAFV as a predictor of AF recurrence after CA. The abstracts, editorial, animal experiment, or review were excluded.
## Data extraction
Pre-specified data variables were extracted independently by two investigators (Peng-fei Chen and Yu-jiao Shi) using a
## Quality evaluation
The quality of the included research was assessed according to the Newcastle-Ottawa Scale (NOS), an assessment tool focused on three aspects: participant selection, comparability, and exposure. The NOS score ranged from 0 to 9, with more than or equal to 8 stars defined as high quality, 6-8 stars as moderate quality, and less than 6 stars as low quality [bib_ref] Quality assessment of observational studies in a drugsafety systematic review, comparison of..., Margulis [/bib_ref]. To assess the certainty of the evidence, we adopted the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach (11), which classifies evidence as high, moderate, low, or very low certainty based on the following factors: risk of bias, inconsistency, indirectness, imprecision, and publication bias. Since all included studies were observational studies, the preliminary definition of the quality evidence was low, and other factors may then upgrade or downgrade the quality level. Any disagreements were resolved by consulting a third investigator (Jian-peng Du).
# Statistical analysis
In the analysis of LAAFV values in AF patients with and without recurrence after CA, means and SDs of LAAFV values were extracted, and standardized mean difference (SMD) and 95% CI were calculated for each study. In analyzing the association between LAAFV and the risk of Flow diagram of study selection and identification.
Frontiers in Cardiovascular Medicine 03 frontiersin.org AF recurrence after CA, univariate or multivariate ORs for AF recurrence reported by logistic regression analysis were extracted. For the study that only reported HRs, HRs was adopted as the best estimate of ORs. Since the studies included in this meta-analysis used LAAFV values as either a categorical or continuous variable to evaluate ORs, two separate meta-analyses were conducted for both variables. Heterogeneity was assessed by the Cochrane's Q test (P < 0.1 was considered statistical heterogeneity) and I 2 Statistics (25, 50, and 75% were considered to represent low, medium, and high heterogeneity, respectively). We adopted a randomeffect model for the meta-analysis because it incorporates the potential effects of heterogeneity and therefore allows for the retrieval of more generalizable results. Subgroup analyses were stratified by study location (Europe or Asia), study design (prospective or retrospective), sample size (≤100 or >100), AF type (persistent AF or paroxysmal AF), ablation procedure (circumferential pulmonary vein isolation or additional linear ablations), follow-up time (≤12 or >12 months), and ablation type (cryoballoon ablation or radiofrequency ablation). The inverted funnel plot and Egger's test were performed to assess publication bias. Sensitivity analyses by removing one individual study at a time to confirm the robustness of the results. All statistical analyses were carried out using STATA version 16 software.
# Results
## Study search
The database search and study identification procedure was presented in. A total of 322 records were retrieved, 208 of which were duplicates, and 79 studies were excluded after reading the title and abstract, primarily because they were irrelevant to the study purpose. The remaining 35 articles were evaluated for eligibility by full-text screening. Of these, 19 studies were further excluded because 10 did not report the AF recurrence outcomes, 4 did not report the LAAFV values, 3 did not use CA as an intervention, and the other 2 were reviews. Finally, 16 studies (12-27) were included in our systematic review and meta-analysis. [bib_ref] The prognostic significance of left atrial appendage peak flow velocity in the..., Yang [/bib_ref] [bib_ref] Association between left atrial appendage emptying velocity, N-terminal plasma brain natriuretic peptide..., Ma [/bib_ref] [bib_ref] Incremental predictive value of left atrial strain and left atrial appendage function..., Ma [/bib_ref] [bib_ref] Transesophageal echocardiography measures left atrial appendage volume and function and predicts recurrence..., He [/bib_ref] [bib_ref] The atrial fibrillation ablation pilot study: a European survey on methodology and..., Yang [/bib_ref] were conducted in China, 4 studies [bib_ref] Left atrial appendage flow velocity and time from P-wave onset to tissue..., Fukushima [/bib_ref] [bib_ref] Abutting left atrial appendage and left superior pulmonary vein predicts recurrence of..., Ariyama [/bib_ref] [bib_ref] Low left atrial appendage flow velocity predicts recurrence of atrial fibrillation after..., Kanda [/bib_ref] [bib_ref] Left atrial appendage volume and plasma docosahexaenoic acid levels are associated with..., Shiozawa [/bib_ref] in Japan, 2 studies [bib_ref] Left atrial appendage size is a marker of atrial fibrillation recurrence after..., Simon [/bib_ref] in Hungary, 1 study [bib_ref] Clinical and echocardiographic risk factors predict late recurrence after radiofrequency catheter ablation..., Kim [/bib_ref] in South Korea, 1 study (13) in Poland, 1 study (12) in Türkiye, and 1 studyin Romania. Sixteen studies with 5,006 AF patients after CA (1,479 patients with AF recurrence, 3,527 without AF recurrence) were included, and the proportion of AF patients with recurrence ranged from 24.1 to 41.5%. The proportion of males was higher than that of females, varying from 59.3 to 92.7%. The mean age ranged from 54.6 ± 10.4 to 67.5 ± 7.5 years, and the median follow-up time varied from 6 to 48 months. Nine (12-15, 17, [bib_ref] The prognostic significance of left atrial appendage peak flow velocity in the..., Yang [/bib_ref] [bib_ref] Association between left atrial appendage emptying velocity, N-terminal plasma brain natriuretic peptide..., Ma [/bib_ref] [bib_ref] Transesophageal echocardiography measures left atrial appendage volume and function and predicts recurrence..., He [/bib_ref] studies performed circumferential pulmonary vein isolation (CPVI) alone, whereas 7 studies conducted additional linear ablations. The LAAFV, which refers to the peak flow emptying velocity of the left atrial appendage at late diastole, was measured by transthoracic echocardiography (TEE) in all selected research. All studies used 24-h Holter and/or surface electrocardiogram recording to diagnose asymptomatic AF recurrence. All studies blanking period of post-CA procedure were 3 months, except for 1 study (15) was 2 months.
## Study characteristics
## Study quality
Based on NOS for observational studies, all examined studies had quality ratings ranging from 6 to 9 (mean score: 7.6), indicating moderate to high quality. Detailed quality assessment is presented in [fig_ref] TABLE 2: Quality assessment of the 16 included studies was assessed by the Newcastle-Ottawa... [/fig_ref].
According to the GRADE grade system, the evidence supporting the link between LAAFV and AF recurrence following CA was of low certainty. [fig_ref] TABLE 3: AF recurrence outcomes and GRADE classification in meta-analysis of observational studies [/fig_ref] displays the certainty assessment ratings and a description of the results.
# Results of meta-analysis
The difference in left atrial appendage flow velocity values between patients with and without atrial fibrillation recurrence after catheter ablation [fig_ref] TABLE 4: Subgroup analyses of difference in LAAFV values between patients with and without... [/fig_ref].. The subgroup analyses were summarized in [fig_ref] TABLE 5: Subgroup analyses of the risk of AF recurrence after CA based on... [/fig_ref] , and the effect sizes were consistent regardless of AF types and ablation procedure. When we
[formula] ⊕⊕- - Low crucial a. [/formula]
The score was downgraded because substantial heterogeneity between studies was detected and could not be fully explained downgraded. b. The score was upgraded because the magnitude of the effect was large (SMD < −0.5 and OR > 2) upgraded. c. The score was downgraded because all included studies in this meta-analysis were observational studies, we cannot rule out that some residual factors may reduce the demonstrated effect downgraded. d. The score was upgraded because there was evidence of significant dose-response association (every 1 cm/s rise in LAAFV values, the risk of AF recurrence decreased by 3%) upgraded. CI confidence interval, OR odds ratio, SMD standardized mean difference, AF atrial fibrillation, CA catheter ablation, LAAFV left atrial appendage flow velocity. Forest plots show the difference in LAAFV values between patients with and without AF recurrence after CA.
# Discussion
The aim of this meta-analysis and systematic review was to examine whether LAAFV is a reliable predictor of AF recurrence after CA. Our meta-analysis showed that patients with AF recurrence had lower mean LAAFV values than those without recurrence. Moreover, we evaluated the association between LAAFV and the risk of AF recurrence after CA. Every 1cm/s rise in LAAFV, the risk of AF recurrence after CA decreased by 3% in the pooled analysis of continuous factors, whereas lower LAAFV was associated with an increased risk of AF recurrence in the pooled analysis of categorical variables. The subgroup analyses showed that the association between LAAFV and AF recurrence after CA was not significantly affected by the AF type and ablation procedure.
Catheter ablation is the most frequently performed interventional electrophysiological therapy for AF. Ectopic pacing sites in patients with AF usually originate from pulmonary veins (PV), and pulmonary vein isolation (PVI) is the cornerstone of CA. Although the surgical effect is satisfactory, the long-term recurrence rate post-operative remains high, because non-PV areas other than PVI may be the source of initiation and maintenance of AF (28-30). The most common areas include the superior vena cava, the coronary sinus, the ligament of marshall, the crista terminalis, the LA posterior wall and the LAA (31-33). LAA is a finger-like projection extending from the main body of the LA and is primarily formed by the adsorption of the primordial PV and their branches [bib_ref] The left atrial appendage: anatomy, function, and noninvasive evaluation, Beigel [/bib_ref]. It is demonstrated that LAA is a significant source of AF and atrial tachycardia [bib_ref] Focal atrial tachycardia originating from the epicardial left atrial appendage, Yamada [/bib_ref]. A study [bib_ref] Difference in thermodynamics between two types of esophageal temperature probes: insights from..., Biase [/bib_ref] found that nearly 30 percent of AF triggers originate from non-pulmonary veins, especially the LAA. Di Biase et al. [bib_ref] Left atrial appendage: an underrecognized trigger site of atrial fibrillation, Biase [/bib_ref] analyzed 987 patients undergoing AF cablation, demonstrating that 27% of AF patients were triggered by LAA, and LAA electrical isolation can improve the success rate of AF cablation. The function of the LAA is most commonly determined by measuring emptying velocity with pulsed-wave Doppler [bib_ref] Left atrial appendage flow velocity and time from P-wave onset to tissue..., Fukushima [/bib_ref]. Previous studies have reported that lower LAAFV in AF patients was associated with a higher risk of thromboembolism (37) and a lower success rate of long-term cardioversion (38, 39). LAAFV, representing a hemodynamic feature of LA and the LAA (40), is widely acknowledged as a marker of LAA function (including contractility, stunning, and fibrosis) [bib_ref] Predicting favourable outcomes in the setting of radiofrequency catheter ablation of long-standing..., Combes [/bib_ref] [bib_ref] Usefulness of doppler assessment of pulmonary vein and left atrial appendage flow..., Donal [/bib_ref]. AF is associated with pathological changes such as remodeling, electrical intolerance changes, and atrial mass loss, which lead to enlargement and dysfunction of LAA, thus causing a decrease in LAA blood flow and ultimately a reduction in LAAFV values [bib_ref] Left atrial appendage emptying velocity as a predictor of recurrence of atrial..., Ezzeddine [/bib_ref].
Left atrial remodeling, including LA enlargement, hypertrophy, and/or fibrosis is the basis of AF recurrence [bib_ref] Atrial remodeling and atrial fibrillation recurrence after catheter ablation : past, present,..., Jalife [/bib_ref]. Moreover, LA size increases and LA voltages decrease due to LA remodeling, which are considered the surrogate for LA fibrosis and the significant predictor of AF recurrence after CA [bib_ref] Left and right atrial appendage functional features as predictors for voltage-defined left..., Lo [/bib_ref]. According to recent research, LAAFV is positively correlated with LA voltage and negatively correlated with LA volume (47, 48). As an indicator reflecting LA contractile and reserve function, LAAFV demonstrates the severity of LA functional remodeling, which may occur in the early stage of LA remodeling [bib_ref] The difference of predictors for recurrence after catheter ablation of non-paroxysmal atrial..., Kim [/bib_ref]. Paroxysmal AF patients are generally in the early stage of LA remodeling. Chronic pressure overload causes LA enlargement, while Impaired LA function precedes LA expansion. Therefore, LAAFV may be a more sensitive predictor for AF recurrence than LA size and volume, particularly in patients with paroxysmal AF. LAA has a stronger contraction and extension function than the LA, the distensible at a greater degree than the LA, as buffering effect on reducing LA pressure [bib_ref] Structure and function of the left atrium and left atrial appendage: AF..., Delgado [/bib_ref]. It provides a theoretical basis that flow velocity of LAA may be a more dependable parameter for AF recurrence than LA. LAAFV, which reflects the more comprehensive LAA dysfunction and atrial remodeling as mentioned above, mainly depends on the contraction of LAA, and therefore, would be a more reliable predictor of AF recurrence. The primary imaging method for assessing LAAFV is TEE [bib_ref] Evaluation of the volume and function of left atrial appendage and left..., Zhu [/bib_ref] , which provides a more accurate risk assessment because it allows the characterization of the AF substrates [bib_ref] Impact of anatomical features of the left atrial appendage on outcomes after..., Kocyigit [/bib_ref].
Although there have been many studies on the relationship between LAAFV and AF recurrence after CA, how to determine the cut-off value of LAAFV is a crucial question to be answered. Forest plots show the relationship between LAAFV (continuous variables) and the risk of AF recurrence after CA. In our meta-analysis, the cut-off values of the seven studies that defined LAAFV as categorical variables were displayed in [fig_ref] TABLE 7: The cut-off values of the seven studies defined LAAFV as categorical variables [/fig_ref]. The cut-off values of most studies are similar, and the differences across studies were probably caused by different research populations or methods, and in the largest related studies so far, the cut-off value of 40 cm/s has been proposed. To our knowledge, this is the first meta-analysis to summarize the association between the LAAFV and AF recurrence after CA. The advantages of the meta-analysis may include the following. First, the results of this study were relatively stable and reliable because the meta-analysis covered studies from different countries and had a large sample size. Second, the finding that LAAFV is associated with the risk of AF recurrence after CA was based on most adequately adjusted ORs, suggesting that the finding may not be affected by potential confounding factors. Third, studies with LAAFV analyzed as categorized and continuous data were summarized separately and derived consistent results, which further verified the stability of the results. Fourth, the sensitivity analyses by removing one individual study at a time had no significant impact on the results, suggesting the outcomes were credible. Fifth, multiple subgroup analyses were conducted to assess the potential study characteristics of the relationship between LAAFV and AF recurrence after CA.
However, this meta-analysis also had some limitations. First, as a meta-analysis of observational studies, it carries inherent limitations of the study design. Second, the heterogeneity of our study was significant. Even if sensitivity and subgroup analyses were adopted, the origin of heterogeneity could not be explored. Third, we cannot rule out that some residual factors may confuse Forest plots show the relationship between LAAFV (categorical variables) and the risk of AF recurrence after CA.
# Conclusion
Meta-analyses of observational studies show that patients with AF recurrence after CA have lower mean LAAFV values than patients without recurrence. Lower LAAFV was associated with an increased risk of AF recurrence after CA, and the assessment of LAAFV before CA could be used as a potential and feasible screening method to predict the risk of AF recurrence. Further studies with larger, well designed, and randomized studies with longer follow up periods for LAAFV should be conducted. In addition, the mechanism of LAAFV and AF recurrences remains to be further explored.
[fig] Fifteen: studies (12, 14-27) (16 data sets) reported the difference in LAAFV values between patients with (n = 1,349) and without (n = 3,200) AF recurrence after CA. Patients with recurrence exhibited lower LAAFV values than those without recurrence [SMD:−0.65, 95% CI: −0.88 to −0.42, P < 0.01; I 2 = 87.9%Figure 2]. The Funnel plot was symmetrical upon visual inspection(Supplementary Figure 1), and the P-value of Egger's test was 0.12(Supplementary Figure 2), indicating no significant publication bias. The sensitivity analysis results were consistent (SMD: −0.71 to −0.59, p all < 0.05,Supplementary Figure 3). The subgroup analyses were summarized in [/fig]
[fig] *: Represents one point, represents two points in the Newcastle-Ottawa Scale. The risk of atrial fibrillation recurrence after catheter ablation for the increment of left atrial appendage flow velocity values of 1 cm/s Fourteen studies (12-15, 17-27) reported the relationship between LAAFV values and the risk of AF recurrence after CA. Nine studies (14, 17-19, 21, 23-26) (11 data sets) defined LAAFV as continuous variables, and the pooled analysis showed that for every 1cm/s rise in LAAFV values, the risk of AF recurrence after CA decreased by 3% [OR:0.97, 95% CI: 0.95 to 0.99, P < 0.01; I 2 = 91.4% Figure 3]. The sensitivity analysis results were consistent (OR: 0.96 to 0.98, p all < 0.05, [/fig]
[fig] Figure 4 ]: The sensitivity analysis results were consistent (OR: 1.96 to 2.48, p all < 0.05, Supplementary Figure 5). The subgroup analyses were summarized in [/fig]
[table] TABLE 1: Characteristics of 16 studies included in the meta-analysis of difference in LAAFV between patients with and without post-CA AF recurrence. [/table]
[table] TABLE 2: Quality assessment of the 16 included studies was assessed by the Newcastle-Ottawa scale. [/table]
[table] TABLE 3: AF recurrence outcomes and GRADE classification in meta-analysis of observational studies.The difference in LAAFV values between patients with and without AF recurrence after CA The risk of AF recurrence after CA for the increment of LAAFV values of 1 cm/s [/table]
[table] TABLE 4: Subgroup analyses of difference in LAAFV values between patients with and without AF recurrence after CA.CPVI, circumferential pulmonary vein isolation; CPVI plus, includes CPVI with one or more of adjuvant ablations in cavotricuspid isthmus, mitral isthmus, left atrial roof, the basal posterior wall, superior vena cava or complex fractionate atrial electrograms; AF, atrial fibrillation. The pooled analysis showed that lower LAAFV was associated with an increased risk of AF recurrence after CA [OR:2.28, 95% CI: 1.46 to 3.57, P < 0.01; I 2 = 93.4% [/table]
[table] Table 6: When we stratified the studies by study location, sample size, and ablation type, the results were not statistically significant in the subgroup of Europe study [OR: 2.05, 95% CI: 0.99 to 4.22, P > 0.05], numbers < 100 study [OR: 2.93, 95% CI: 0.81 to 10.65, P > 0.05], and cryoballoon ablation study [OR: 1.27, 95% CI: 0.97 to 1.78, P > 0.05]. [/table]
[table] TABLE 5: Subgroup analyses of the risk of AF recurrence after CA based on LAAFV (continuous variable).CPVI, circumferential pulmonary vein isolation; CPVI plus, includes CPVI with one or more of adjuvant ablations in cavotricuspid isthmus, mitral isthmus, left atrial roof, the basal posterior wall, superior vena cava or complex fractionate atrial electrograms; AF, atrial fibrillation. [/table]
[table] TABLE 7: The cut-off values of the seven studies defined LAAFV as categorical variables.the link between LAAFV and AF recurrence. Forth, studies that defined LAAFV as categorical variables have different cut-off values, which would impact our study result. [/table]
|
HSV-1 Encephalitis in an Elderly Man Receiving Ibrutinib for Waldenstrom's Macroglobulinemia
Ibrutinib is a major new addition to the therapeutic armamentarium for chronic lymphocytic leukemia, mantle cell lymphoma, Waldenstrom's macroglobulinemia, and chronic graft versus host disease. ough ibrutinib has proven to be a revolutionary new small molecule agent, and has relatively minimal toxicity as compared to traditional chemotherapy, infections have emerged as a major complication of therapy. While fungal infections have been the most problematic (including CNS aspergillosis), zoster, hepatitis B reactivation, and chronic hepatitis E have been reported in association with ibrutinib therapy. is report describes a case of herpes encephalitis in an 86-year-old Waldenstrom's patient receiving ibrutinib and speculates as to whether this late life encephalitis may have been related to ibrutinib.
# Introduction
Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), has proven to be a major advance in the treatment of multiple malignancies and graft versus host disease. ough not as toxic as standard chemotherapy, this innovative agent has, not surprisingly, been associated with a growing number of infections. BTK inhibition affects not only B-cell function but also macrophage and monocyte responses, which are relevant to the control of fungal infections. Ibrutinib also irreversibly inhibits interleukin-2 inducible T-cell kinase (ITK) in CD4 cells. Absence of ITK in humans has been associated with severe herpesvirus infection. Invasive aspergillosis has been the most common serious infection, but other fungal pathogens (cryptococcosis, histoplasmosis, mucormycosis, and Pneumocystis jirovecii) have also been problematic. Viral infections have also been an issue with the occurrence of zosteras well as chronic hepatitis E and hepatitis B reactivation. is report details an unusual case of herpes encephalitis occurring in an 86-year-old man receiving ibrutinib for Waldenstrom's macroglobulinemia.
## Case
e patient, an 86-year-old man, had a ten-year history of Waldenstrom's macroglobulinemia and type 2 diabetes. His Waldenstrom had been treated with bendamustine and rituximab, but he developed progressive anemia and eventually, pancytopenia. Ibrutinib was started seven years into his Waldenstrom, with rapid and sustained normalization of his hematocrit, peripheral white blood cells, and platelet counts. During his three years of ibrutinib therapy, he had no evidence of toxicity and led a full and active life. No other Waldenstrom therapy was provided during this period.
ree days prior to his hospital admission, the patient reported abdominal pain, anorexia, and fatigue. He was also transiently confused and on occasion had subtle difficulty with word finding. After two days of symptoms, he was evaluated at an emergency room and found to be afebrile, with normal mental status and neurologic examination. Contrast-enhanced CT scans of both his brain and abdomen were normal, and standard laboratories (complete blood count and metabolic panel) were normal. He was sent home, but the next day he developed worsening difficulty with word finding, headache, and a feeling his "mind was not Hindawi Case Reports in Infectious Diseases Volume 2020, Article ID 6516037, 2 pages https://doi.org/10.1155/2020/6516037 working right." An MRI of the brain revealed subtle unilateral temporal lobe enhancement, and cerebral spinal fluid had normal glucose and protein levels with a white count of 5 cells per µL. Two red blood cells per µL were seen; no differential was done on the white blood cells. Qualitative PCR on the spinal fluid (FilmArray Meningitis/Encephalitis panel) was positive for HSV-1. e patient was started on intravenous acyclovir at 10 mg/kg dose every 8 hours. During the period around the MRI scan and lumbar puncture he, for the first time, exhibited overt confusion and his speech became garbled. After 3 days of acyclovir, his mental status had rebounded to nearly normal, his abdominal pain resolved, and he was ready for hospital discharge. He received 21 days of acyclovir at his home. A follow-up MRI showed resolution of the temporal lobe enhancement and a repeat lumbar puncture was PCR negative for HSV-1 at the conclusion of three weeks of intravenous acyclovir therapy. ere were still only 5 white cells per μL, though the CSF protein was now slightly elevated at 74 mg per dL. He suffered no sequalae except for subtle memory deficits.
# Discussion
is unusual case of herpes encephalitis occurred in an 86year-old man who was receiving ibrutinib for Waldenstrom's macroglobulinemia. is case was atypical in that the patient was greater than 85 years old, had an uncommon stuttering course, and did not have a CSF pleocytosis. Lack of CSF pleocytosis occurs in 3 to 26% of patients with herpes encephalitis.
Ibrutinib has primarily been associated with fungal infections, but reactivation of varicella zoster, hepatitis B, and chronic hepatitis E have also emerged as issues. A single case of West Nile encephalitis has also been reportedin an ibrutinib patient, suggesting that ibrutinib may predispose to severe viral infections, perhaps through irreversible inhibition of ITK. Whether this case is an isolated random occurrence, or represents the first report of herpes encephalitis as another ibrutinib related infectious risk remains to be seen. Clinicians using ibrutinib should be mindful of the risk of unusual viral as well as fungal processes and report novel infections as they occur.
## Conflicts of interest
e author declares that there are no conflicts of interest. |
Differential Expression of IL-36 Family Members and IL-38 by Immune and Nonimmune Cells in Patients with Active Inflammatory Bowel Disease
IL-1 family includes IL-38 (IL-1F10) and the subfamily of IL-36 and is the central mediators of inflammatory diseases, including pustular psoriasis, atopic dermatitis, rheumatoid arthritis, and gut inflammation. The purpose of the study was to evaluate on tissue of the patients with inflammatory bowel disease (IBD), the IL-36 , IL-36 , IL-36 , IL-36Ra, and IL-38 gene and cell expression and its correlation with clinical activity. Patients and Methods. A cross-sectional and comparative study was performed. Seventy patients with IBD and 30 noninflamed non-IBD controls were enrolled. Gene expression was measured by RT-PCR. Protein expression was detected by double-staining immunohistochemistry. Results. The mRNA expression of IL-36 family members but not IL-38 was increased in colonic mucosa from patients with active ulcerative colitis versus Crohn's disease group and noninflammatory control group (P<0.05). However, only gene expression of IL-38 was increased in tissue from patients with inactive ulcerative colitis versus active disease and control group (P<0.005). Conversely, gene expression of IL-36Ra was significantly higher in colonic tissue from patients with active versus inactive ulcerative colitis and noninflamed control group (P<0.05). A differential protein overexpression of IL-36 , IL-36 , IL-36 , IL-36Ra, and IL-38 by intestinal epithelial cells, macrophages, CD8+ T cells, and/or versus dendritic cells (pDCs) was found in patients with active inflammatory bowel disease compared with noninflamed controls. Conclusion. IL-38 and IL-36 family members' expression was increased by immune and nonimmune cells in patients with active inflammatory bowel disease. These cytokines and IL-36Ra might represent novel therapeutic targets in patients with gut inflammation.
# Introduction
Inflammatory Bowel Disease (IBD) is characterized by an imbalance between innate and adaptive immunity leading to the stimulation of T helper responses with preponderance of proinflammatory cytokines [bib_ref] Inflammatory bowel disease, Podolsky [/bib_ref]. An increase production of proinflammatory cytokines such as IL-1 and TNF-has been involved in the development of chronic inflammation of the gut, founded on the recent knowledge about the role of cytokine-driven pathways in intestinal immunity and it is based on animal models of acute and chronic intestinal injury and inflammation [bib_ref] New insights into the dichotomous role of innate cytokines in gut homeostasis..., Bamias [/bib_ref] The IL-1 family (IL-1F) includes IL-38 (IL-1F10) and the subfamily of IL-36 (IL-36Ra or IL-1F5; IL-36 or IL-1F6; IL-36 or IL-1F8; and IL-36 or IL-1F9). It has been considered one of the most important key regulators in the pathophysiology of inflammatory autoimmune diseases including Crohn's disease (CD), rheumatoid arthritis, and psoriasis [bib_ref] Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36 , IL-36 ,..., Towne [/bib_ref] [bib_ref] The novel cytokine interleukin-36 is expressed in psoriatic and rheumatoid arthritis synovium, Frey [/bib_ref] [bib_ref] Distinct expression of interleukin (IL)-36 , and , their antagonist IL-36Ra and..., Boutet [/bib_ref].
## Biomed research international
The IL-1 family overproduction but not from antagonist receptor (IL-36Ra) or IL-38 results in inflammation, in a robust immune response that acts as first line of defense against invasive pathogenic microorganisms and damage and when there is an aberrant immune response under appropriate genetic and environmental backgrounds in an autoimmune disease [bib_ref] Unprocessed interleukin-36 regulates psoriasis-like skin inflammation in cooperation with interleukin-1, Milora [/bib_ref].
IL38/IL-1F10 is a protein that in humans is encoded by the il1f10 gene [bib_ref] Identification of a novel human cytokine gene in the interleukin gene cluster..., Bensen [/bib_ref]. IL-38 is expressed in a range of tissues, including heart, placenta, fetal liver, skin, spleen, thymus, and tonsil. IL-38 is also expressed mostly in the skin and in proliferating B cells [bib_ref] Cloning and characterization of IL-1HY2, a novel interleukin-1 family member, Lin [/bib_ref]. This cytokine participates in a network of IL-1 family members to regulate adapted and innate immune responses by the inhibition of the production of T cell cytokines (IL-17 and IL-22). IL-38 also inhibits the production of IL-8 induced by IL-36 , thus regulating inflammatory responses [bib_ref] Role of Il-38 and its related cytokines in inflammation, Yuan [/bib_ref].
In addition, other secreted protein is IL-36 receptor antagonist (IL-36Ra) also known as IL-1F5, a natural inhibitor for IL-36 activity. The IL-36Ra is expressed by immune cells such as monocytes, B cells, dendritic cells/Langerhans cells, keratinocytes, and gastric parietal cells [bib_ref] IL-38 binds to the IL-36 receptor and has biological effects on immune..., Van De Veerdonk [/bib_ref].
Veerdonk et al. showed that IL-38 binds to the IL-36 receptor (IL-36R) and has similar biological effects to IL-36Ra on immune cells [bib_ref] IL-38 binds to the IL-36 receptor and has biological effects on immune..., Van De Veerdonk [/bib_ref]. According to its activity, low concentrations of IL-38 may have an anti-inflammatory function blocking IL-36R and IL-1R pathways, which suppressed IL-22 and IL-17 synthesis and secretion. Strikingly, IL-38 shares high sequence homology with IL-1Ra (41%) and IL-36Ra (43%) [bib_ref] Opposing functions of classic and novel IL-1 family members in gut health..., Lopetuso [/bib_ref] The distinct expression of IL-36 , IL-36 , and IL-36 , their antagonist (IL-36Ra), and IL-38 in autoimmune disease has been shown. They showed an increased expression of IL-36 and IL-38 only in patients with CD but did not show the expression of IL-36 family by producing effector immune cells regarding clinical activity in patients with ulcerative colitis (UC) [bib_ref] Distinct expression of interleukin (IL)-36 , and , their antagonist IL-36Ra and..., Boutet [/bib_ref].
Nonetheless, little is known about the presence of IL-38, IL-36Ra and IL-36 , IL-36 , and IL-36 producing intestinal effector immune cells (T cells, plasmacytoid dendritic cells, and monocytes) and nonimmune cells in Mexican Mestizo patients with IBD.
The purpose of the study was to evaluate on intestinal tissue IL-36 -, IL-36 -, IL-36 -, IL-36Ra-, and IL-38 producing cells as well as gene expression in immune (cytotoxic T cells, macrophages and plasmacytoid dendritic cells) and nonimmune cells from patients with IBD compared with noninflamed controls.
# Materials and methods
## Study subjects.
For the cross-sectional and comparative study, a total of 70 patients with IBD were recruited. Patients were categorized into the following groups: 30 active UC (aUC); 20 inactive UC (iUC); 10 active CD (aCD); and 10 inactive CD (iCD) patients. All patients were included between January 2014 and May 2015 from the Inflammatory Bowel Disease Clinic at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (a tertiary referral center in Mexico City, Mexico). The UC and CD diagnosis was done by the correlation of clinical, endoscopic, and histopathological findings. Colonic samples were obtained from IBD patients and noninflamed controls after a signed informed consent. Patients' clinical records were reviewed, and personal interviews were done, and the following information were collected for all IBD patients: age, sex, treatment (mesalazine, azathioprine, prednisone, mercaptopurine, etc.), and the presence of extraintestinal manifestations (present or absent). The disease severity was evaluated in colon biopsy by Mayo score disease activity index for UC [bib_ref] 5-Aminosalicylic acid enema in the treatment of distal ulcerative colitis, proctosigmoiditis, and..., Sutherland [/bib_ref] and Harvey-Bradshaw Index for CD patients [bib_ref] A simple index of Crohn'sdisease activity, Harvey [/bib_ref]. All colonoscopies were performed for cancer surveillance. Exclusion criteria included patients with indeterminate colitis, postradiation, infectious colitis, and other types of colitis.
The control group consisted of 30 noninflamed colonic biopsies (without endoscopic and histological evidence of any type of colitis, neoplasia, inflammatory disease, or other documented diseases). All participants underwent colonoscopy due to screening of polyps or the study of weight loss, for evaluation of anemia. Controls were matched by age and sex with IBD patients.
## Tissue samples
## Sample processing and gene expression analysis.
To study gene and in situ expression we followed the methods of Fonseca-Camarillo G et al. 2015 [bib_ref] Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and..., Fonseca-Camarillo [/bib_ref]. The colonic biopsies were taken by colonoscopy and immediately submerged in RNA later solution (Ambion, Austin, TX, USA) for storage. Then total RNA was isolated using High Pure RNA Tissue (Roche Diagnostics, Mannheim, Germany), following the manufacturer's guidelines.
Electrophoresis of one aliquot was made for each one of the RNA products in an agarose gel at 1%; it was then visualized by staining with ethidium bromide and then it was documented using an UV transilluminator.
Two hundred nanograms of total RNA was reversetranscribed into cDNA with random hexamer primers (Roche Diagnostics, Mannheim, Germany).
cDNA synthesis from total RNA through reverse transcription was made taking 20 L from total RNA of each of the products with the following protocol: preincubation: 25 ∘ C x 10 minutes, incubation: 55 ∘ C x 30 minutes, followed by denaturalization: 85 ∘ C x 5 minutes using a thermocycler (Perkin-Elmer).
Quantitative real-time PCR was used to measure the RNA transcription level of target genes. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as housekeeping was analyzed for normalization purposes.
PCR amplification was performed with a concentration of 20 ng of cDNA, 200 nM forward, reverse primer, and Taqman Master Mix (Roche Diagnostics, Mannheim, Germany Roche Diagnostics, Mannheim, Germany) in a final volume of 10 l. PCR reactions were run in a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany).
The mRNA relative quantification of target genes was conducted using the LightCycler software 4.1, according to the 2-delta-delta Ct method. [fig_ref] Table 1: Primers designs. [/fig_ref] shows the details of
## Patient samples for protein expression analysis.
A total of 10 surgical samples from patients with active severe UC refractory to conventional therapy were included for protein detection. Also, 10 patients with definitive diagnosis of active severe CD were enrolled in the study. Ten controls were obtained from noninflamed non-IBD intestinal tissue. All biopsies were obtained from whole colon in UC and right colon in CD patients. The Riley Index score was used for grading the severity of colonic inflammation [bib_ref] Comparison of delayed release 5 aminosalicyclic acid (mesazaline) and sulphalazine in the..., Riley [/bib_ref].
## Immunohistochemistry.
To determine the IL-38, IL-36Ra, and secondary IL-36 family expressing cells, 4 m thick formalin-fixed and paraffin-embedded tissue from patients and noninflamed/non-IBD controls were placed on positively charged slides. Sections were deparaffinized in xylene and rehydrated in water. Morphometric evaluation of the immune-stained sections was performed in a blinded manner. Tissues were counterstained with the nonalcoholic Mayer's hematoxylin (DAKO) and mounted in aqueous mounting medium (DAKO). Negative control staining was performed with the universal negative control (a reagent mixture of purified goat, rabbit, and mouse immunoglobulins) which was tittered to work with polymer-based secondary systems (IHCh universal negative control reagent, Enzo Life Sciences, Inc.) The reactive blank was incubated with phosphate buffer saline-and IHCh background blocker instead of the primary antibody. Both controls excluded nonspecific staining and endogenous enzymatic activities. IL-38, IL-36Ra, and IL-36 producing cells were reported as the single and double positive staining cells in at least two fields (×320). Histological analysis was performed by the program Image Pro Plus version 5.1.1.
## Double-staining
# Ethical considerations.
This work was performed according to the principles expressed in the Declaration of Helsinki, 1989. The study was carried out with approval by the ethical committee in our institution and a written informed consent was obtained from all patients recruited prior to their inclusion in the study.
## Statistical
Analysis. Statistical analysis was performed using the SPSS 19 program by the Kruskal-Wallis One-Way Analysis of Variance on Ranks Data expressed as the median, range, and mean ± SE. A P value ≤ 0.05 was considered as significant.
# Results
## Demographic and clinical characteristics.
A total of 100 individuals were recruited for the study. Patients were categorized in 4 groups: (1) active UC (n=30); (2) remission UC (n=20); (3) active CD (n=10); (4) remission CD (n=10). A fifth group consisted of 30 noninflamed/non-IBD control groups. All demographic and clinical characteristics from patients with IBD and noninflamed controls are depicted in [fig_ref] Table 2: Demographic and clinical characteristics of Crohn's disease and ulcerative colitis patients [/fig_ref]. Histochemical analysis showed that the intestinal tissue from IBD patients had higher expression of IL-36Ra and IL-38 throughout the mucosa, submucosa, muscular, and serosa layers when compared with noninflamed control group. IL-36Ra producing cells were by mucosal epithelial cells and cell from submucosa and perivascular mononuclear cells. CD14 + /IL-36Ra + double positive cells from intestinal tissue from noninflamed control tissue were practically undetectable versus aUC and CD [fig_ref] Figure 1: CD14 + /IL-36Ra-and CD123+/IL-38 expressing cells in intestinal tissue from IBD patients [/fig_ref].
## In situ
The IL-38 expression in tissue from patients with UC and CD was mostly by epithelial and parenchyma cells. Nevertheless, there were some perivascular inflammatory CD123 − cells that expressed this cytokine. In addition, there was a small subpopulation of CD123 + /IL-38 producing cells distributed along serosa, muscular, submucosa, and mucosa from IBD patients. The IL-38 expressing cells were plentiful in serosa, muscular, and submucosa from active UC compared to active CD and noninflamed control tissue [fig_ref] Figure 1: CD14 + /IL-36Ra-and CD123+/IL-38 expressing cells in intestinal tissue from IBD patients [/fig_ref].
The protein expression of IL-38 was scarce in mucosa from IBD patients.
## Il-36 .
The most important IL-36 production was detected in nonimmune cells including gut epithelial and parenchyma cells.
Immunohistochemical double staining revealed that both CD14+ macrophages and CD123+ plasmacytoid dendritic cells expressed IL-36 .
CD14/IL-36 cells and CD123/IL-36 pDCs were more abundant on muscular and serosa from active UC patients compared with active CD patients and noninflamed control tissue [fig_ref] Figure 2: IL-36 expression in immune and nonimmune intestinal cells from IBD patients [/fig_ref]. The IL-36 expressing cells in active UC tissue were mainly CD14+ cells [fig_ref] Figure 2: IL-36 expression in immune and nonimmune intestinal cells from IBD patients [/fig_ref] , a smaller pDC positive subpopulation CD123+ [fig_ref] Figure 2: IL-36 expression in immune and nonimmune intestinal cells from IBD patients [/fig_ref] , some plasma cells, and lymphocytes. A similar pattern of IL-36 expression was observed in samples from CD patients. There was no expression of IL-36 on intestinal biopsies from controls. Staining for IL-36 protein in inflamed gut correlated with the pattern of mRNA expression 3.2.3. IL-36 . CD14+/IL-36 + immunoreactive macrophages were noticeably higher in active CD patients compared with active UC and noninflamed control tissue [fig_ref] Figure 3: IL-36 production in immune and nonimmune intestinal cells from IBD patients [/fig_ref]. Double positive cells were localized mainly in the muscular and serosa. Furthermore, IL-36 producing cells, potentially lymphocytes, were increased in submucosa, muscular, serosa, and perivascular inflammatory infiltrates from active CD patients compared with noninflamed control tissue.
Biopsies from patients with active CD had a significant number of CD123 − /IL-36 expressing cells. A conspicuous number of IL-36 cells were observed in serosa, muscular, and submucosa; and smaller proportion was observed in mucosa. Furthermore, in patients with active UC a considerably lower number of CD123+/IL-36 + was detected in mucosa, submucosa, muscular, and serosa, although the most immunoreactive cells were CD123 − /IL-36 + immune and nonimmune cells. Tissue cells from noninflamed/non-IBD controls had a high number of IL-36 producing cells in serosa followed by muscular and submucosa [fig_ref] Figure 3: IL-36 production in immune and nonimmune intestinal cells from IBD patients [/fig_ref].
## Il-36 .
CD8 /IL-36 double positive cells from patients with active UC were found in a similar proportion to the CD8 − /IL-36 producing cells (potentially monocytes, lymphocytes, and plasma cells). Tissues from patients with active CD had lower number of IL-36 producing CD8 + T lymphocytes when compared with UC. These cells infiltrate primarily the mucosa, submucosa, and muscular. In biopsies from noninflamed non-IBD patients no positive cells were determined. In contrast, CD123 + /IL-36 + plasmacytoid dendritic cells were numerous on mucosa, submucosa, muscular, and serosa from active CD patients compared with active UC patients and noninflamed control tissue Inflammatory infiltrates from CD biopsies had a significant number of IL-36 expressing cells. They were distributed throughout all layers of the submucosa (and deeper layers), whereas in UC they were accentuated in the upper submucosa near the mucosal border (predominant mucosal inflammation in UC).
## Gene expression of il-38, il-36ra, and il-36 family in
Colonic Mucosa from UC and CD Patients. The IL-38, IL-36Ra, and IL-36 family gene expression were detected and quantified by RT-qPCR in colonic biopsies of UC and CD patients as well as noninflamed control tissue. Bars show mean ± standard error of the mean of transcript levels from patients with GAPDH as housekeeping gene determined by 2-ûûCt. * P value <0.05 was considered as significant.
The gene expression of IL-38 was increased in colonic tissue from noninflamed patients with UC when compared with inflamed UC and the control group (P= 0.009 and P= 0.008, respectively). No statistically significant difference was found among patients with active UC compared with noninflamed CD [fig_ref] Figure 5: IL-38 and IL-36Ra gene expression in IBD patients [/fig_ref].
The gene expression of IL-36Ra was significantly higher in inflamed colonic tissue from patients with active UC when compared with inactive UC and noninflamed control group (P= 0.006 and P= 0.007). [fig_ref] Figure 5: IL-38 and IL-36Ra gene expression in IBD patients [/fig_ref].
The gene expression of IL-36 was significantly increased in colonic inflamed tissue of patients with active UC when compared with inactive UC and noninflamed control group, respectively (P= 0.006 and P= 0.007; [fig_ref] Figure 6: IL-36 family relative gene expression quantified by RT-PCR [/fig_ref] ). We also found a significant difference among inactive UC versus inactive CD group as shown in [fig_ref] Figure 6: IL-36 family relative gene expression quantified by RT-PCR [/fig_ref] (P= 0.050).
IL-36 gene expression was higher in inflamed colonic mucosa from patients with active UC when compared with inactive UC and controls (P= 0.032 and P= 0.036; [fig_ref] Figure 6: IL-36 family relative gene expression quantified by RT-PCR [/fig_ref] and P= 0.03; [fig_ref] Figure 6: IL-36 family relative gene expression quantified by RT-PCR [/fig_ref] ). We also determined a significant difference among inactive UC versus CD group (P= 0.050; [fig_ref] Figure 6: IL-36 family relative gene expression quantified by RT-PCR [/fig_ref] ).
# Discussion
To the best of our knowledge, this study demonstrated the intestinal expression of IL-38 and IL-36 family members expression by immune and nonimmune cells in patients with inflammatory bowel disease.
The most recently identified IL-36 family members are widely expressed in inflammatory, epithelial, and other nonimmune cells. These cytokines combine with the cellsurface heterodimeric receptor IL-36R (IL1RAcP/IL-1Rrp2) and activate downstream nuclear transcripts such as nuclear factor-B (NF-B) and activator protein-1 (AP-1) and MAPKs pathways like IL-1. Therefore, most molecules involved in IL-36-induced signaling, such as MMPs, antimicrobial peptides, cytokines, chemokines, and adhesion molecules, all of them mediators of inflammatory diseases [bib_ref] Role of Il-38 and its related cytokines in inflammation, Yuan [/bib_ref]. IL-36 cytokine has significant in vivo effects on DCs and T cells in human immune responses via its role in the differentiation of inflammatory Th1 cells [bib_ref] Annotating genes with potential roles in the immune system: six new members..., Dunn [/bib_ref].
To identify the different cell populations of immune system we followed the methods of Fonseca-Camarillo G. et al. 2015 [bib_ref] Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and..., Fonseca-Camarillo [/bib_ref] and we detected different subpopulations such as CD8 + T cells, CD123 + pDCs and CD14 + monocytes, and nonimmune cells including epithelial and parenchyma cells that were remarkably increased in active UC patients compared with active CD and noninflamed controls.
Previously, IL-36 has been reported as an IL-1 family member expressed on monocytes, macrophages, and/or dendritic cells, T cells [bib_ref] Opposing functions of classic and novel IL-1 family members in gut health..., Lopetuso [/bib_ref] , but we also showed the protein expression of IL-36 by CD8 T cells and plasmacytoid dendritic cells on intestinal tissue from patients with IBD.
Russell et al. [bib_ref] IL-36 expression is elevated in ulcerative colitis and promotes colonic inflammation, Russell [/bib_ref] recently demonstrated that expression levels of IL-36 are specifically elevated in the colonic mucosa of UC pediatric patients and this finding was also reported in the inflamed colonic mucosa of mice, wherein Il36r −/− exhibited reduced disease severity in acute dextran sulfate sodium-(DSS-) induced model of colitis of IBD.
Kanda et al. demonstrated that IL-36 and IL-36 contribute to gut inflammation through the induction of proinflammatory mediators such as IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by human colonic subepithelial myofibroblasts [bib_ref] Interleukin(IL)-36 and IL-36 induce proinflammatory mediators from human colonic subepithelial myofibroblasts, Kanda [/bib_ref].
Moreover, Boutet et al. reported that IL-36 , IL-36 , and IL-38 were induced at low levels and correlated with IL-1 , M-CSF, and some chemokines but not with IL-17A in the colon of mice with DSS-induced colitis and in patients with CD when compared with psoriasis [bib_ref] Distinct expression of interleukin (IL)-36 , and , their antagonist IL-36Ra and..., Boutet [/bib_ref]. Besides, IL-36 and IL-38 share a common receptor, IL-36R, and exhibit dual proinflammatory effects on autoimmune diseases, particularly in psoriasis and rheumatoid arthritis. These are secreted by Langerhans cells, keratinocytes/stratified squamous epithelium, chief cells, and parietal cells [bib_ref] Cloning and characterization of IL-1HY2, a novel interleukin-1 family member, Lin [/bib_ref].
Medina Contreras showed that IL-36R-deficient (Il1rl2 (−/−) ) mice exhibited defective recovery following DSS-induced damage and an important reduction in IL-22 expression, a cytokine involved in tissue repair and regeneration, particularly by colonic neutrophils and suggest the important role of IL-36/IL-36R axis in the resolution of intestinal mucosal wounds [bib_ref] Cutting edge: IL-36 receptor promotes resolution of intestinal damage, Medina-Contreras [/bib_ref].
We also decided to explore the gene and protein expression of IL-38 and IL-36Ra receptor in intestinal tissue from patients with IBD. We found that IL-38 and IL-36Ra mRNA expression were increased in the tissue from active and remission IBD patients compared with noninflamed tissues.
Analysis of the whole samples showed that IL-38 mRNA levels were higher in colonic mucosa from patients in remission UC when compared with active UC. Interestingly, IL-38 gene expression was decreased in patients with active UC, and, conversely, percentage of IL-38 immunoreactive cells in active UC patients and active CD were increased compared with noninflamed tissues.
Increased IL-38 protein expression in mucosa from patients with active IBD suggests that the upregulation is a defense mechanism in the colonic epithelia in response to decreased bacterial invasion and inflammatory activity.
An increase of IL-36Ra mRNA expression was determined in active UC patients versus inactive CD patients.
Those results are relevant because previously it has been reported that the binding of IL-1Ra and IL-36Ra to their receptor reduces inflammation by blocking the binding of receptor ligands. Yuan et al. suggest the regulatory role of IL-38 by the production of fungal-induced IL-17, IL-22, and IL-36 -derived IL-8 was decreased by IL-38, which may play an important anti-inflammatory role in inflammatory diseases [bib_ref] Role of Il-38 and its related cytokines in inflammation, Yuan [/bib_ref].
This study described the expression of IL-36 family, IL-36Ra, and IL-38 in colonic cells and immune cells in patients with IBD.
Additional functional studies about IL-36 family and IL-38 in the gut mucosal immune response can confirm its role and support the proinflammatory role of this cytokines in patients with IBD.
There are some limitations of our study. As expected, it would be desirable to include a group of intestinal tissue from patients with remission IBD for immunohistochemistry analysis, but it is not possible since this set of patients do not need to be colectomized for treatment of the disease.
It is important to note that this study evaluated the presence of IL-36 family, IL-36Ra, and IL-38 in monocytes, CD8 T cell, and plasmacytoid dendritic cell subpopulations in IBD patients.
Summing up, current knowledge supports the concept that the pathophysiology of IBD is characterized by a robust elevation of IL-36 family members and IL-36Ra and IL-38 probably promoting agonist and/or healing activity, whose primary source is mononuclear cells and epithelial cells.
Our findings showed that clinical active IBD patients have an increased gene expression and production of IL-36Ra by macrophages. The IL-38 and IL-36Ra-related signaling pathway is poorly understood and certainly requires further studies to elucidate its role in patients with IBD.
In conclusion, our results suggest the role for IL-38 and IL-36R signaling in the colonic inflammation by effector immune cells and indicate that the IL-36Ra and IL-38 pathway may represent an innovative and rational target for therapeutic treatment in patients with IBD.
## Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
# Disclosure
This manuscript was presented as an abstract on "12th Congress of ECCO-European Crohn's and Colitis Organisation".
## Conflicts of interest
The authors declare that they have no conflicts of interest.
[fig] Figure 1: CD14 + /IL-36Ra-and CD123+/IL-38 expressing cells in intestinal tissue from IBD patients. CD14 + /IL-36Ra-and (b) CD123 + /IL-38 expressing cells. Representative immunoperoxidase in tissue from active ulcerative colitis patients (n=10; left panel), active Crohn's disease patients (n=10; middle panel), and noninflamed colonic tissue (n=10; right panel). Photomicrographs represent mucosa, submucosa, muscular, and serosa. Dotted arrows depict the cytokine expression, solid arrows show expression of cell-surface marker (leukocytes), double arrows indicate CD14 + or CD123 + (in brown)/IL-36Ra or IL-38 (in pink) double positive cells (burgundy), and circles highlight the positive cells from perivascular inflammatory infiltrates. Original magnification was X320. [/fig]
[fig] Figure 2: IL-36 expression in immune and nonimmune intestinal cells from IBD patients. (a) CD14 + /IL-36 expressing cells and (b) CD123 + /IL-36 pDCs. Representative immunoperoxidase in tissue from active UC patients (n=10; left panel), active CD patients (n=10; middle panel), and noninflamed colonic tissue (n=10; right panel). Photomicrographs represent mucosa, submucosa, muscular, and serosa. Dotted arrows depict the cytokine expression, solid arrows show expression of cell-surface marker (leukocytes), double arrows indicate CD14 + or CD123 + (in brown)/IL-36 (in pink) double positive cells (burgundy), and circles highlight the immunoreactive perivascular inflammatory infiltrates. Original magnification was X320. [/fig]
[fig] Figure 3: IL-36 production in immune and nonimmune intestinal cells from IBD patients. (a) CD14 + /IL-36 expressing cells and (b) CD123 + /IL-36 pDCs. Representative immunoperoxidase in inflamed tissue from active ulcerative colitis patients (n=10; left panel), active Crohn's disease patients (n=10; middle panel), and noninflamed colonic tissue (n=10; right panel). Photomicrographs represent mucosa, submucosa, muscular, and serosa. Dotted arrows depict the cytokine expression, solid arrows show expression of cell-surface marker (leukocytes), double arrows indicate CD14 + or CD123 + (in brown)/IL-36 (in pink) double positive cells (burgundy), and circles highlight the immunoreactive perivascular inflammatory infiltrates. Original magnification was X320. [/fig]
[fig] Figure 4: (b)) IL-36 gene expression was also significantly upregulated in colonic mucosa from patients with active UC in comparison with inactive UC and control group (P= 0.02 IL-36 production in intestinal tissue from IBD patients. (a) CD8 + /IL-36 expressing cells. (b) CD123 + /IL-36 pDC. Representative immunoperoxidase in tissue from active ulcerative colitis patients (n=10; left panel), active Crohn's disease patients (n=10; middle panel), and noninflamed colonic tissue (n=10; right panel). Photomicrographs represent mucosa, submucosa, muscular, and serosa. Dotted arrows depict the cytokine expression, solid arrows show expression of cell-surface marker (leukocytes), double arrows indicate CD8 + or CD123 + (in pink)/IL-36 (in brown) double positive cells (burgundy), and circles highlight the immunoreactive perivascular inflammatory infiltrates. Original magnification was X320. [/fig]
[fig] Figure 5: IL-38 and IL-36Ra gene expression in IBD patients. (a) IL-38 and (b) IL-36Ra relative gene expression quantified by RT-PCR. Bars show mean ± standard error of the mean of transcript levels in colonic mucosa from IBD patients with GAPDH as housekeeping gene determined by 2-ûûCt. [/fig]
[fig] Figure 6: IL-36 family relative gene expression quantified by RT-PCR. (a) IL-36 gene expression. (b) IL-36ß gene expression, (c) IL-36 gene expression. RT-qPCR was performed to assess mRNA levels in colonic mucosa biopsies from IBD patients, bars show means with standard error of the mean of IL-36 , IL-36ß and IL-36 transcript levels with GAPDH as housekeeping gene determined by 2 ûûCt, differences among groups were assessed by Kruskall Wallis test, and p values are presented in the figure. [/fig]
[table] Table 1: Primers designs. [/table]
[table] Table 2: Demographic and clinical characteristics of Crohn's disease and ulcerative colitis patients. CD: Crohn's Disease patient group; UC: ulcerative colitis patient group. ND: not determined. [/table]
|
Bedside Neonatal Intensive Care Unit Surgery- Myth or Reality!
Neonatal transport is associated with complications, more so in sick and unstable neonates who need immediate emergency surgery. To circumvent these problems, surgery in Neonatal intensive care unit (NICU) is proposed for these neonates. This article reviews the literature regarding feasibility of this novel concept and based on the generated evidence, suggest the NICU planners to always include infrastructure for this. Also neonatal surgical team can be developed that could be transported.
# Introduction
Neonatal surgery in neonatal intensive care unit (NICU) is a novel concept which has the potential to decrease the morbidity, mortality and cost of neonatal care. However, it is still not common because of management (infrastructure, manpower) issues. In this review, we have tried to find and compile the evidence for this in literature and based upon that suggest NICU planners to always include provision of same in their NICU.
# Materials and methods
The review of the literature was performed in November 2012 using the Medical Literature Analysis and Retrieval System Online (U.S. National Library of Medicine's life science database; MEDLINE), and Google© search. The MEDLINE search employed both ''MeSH'' (Medical Subject Heading) and ''free text'' protocols. Specifically, the MeSH search was conducted by combining the following terms retrieved from the MeSH browser provided by MEDLINE: Intensive Care Units; Neonatal, Neonatal Intensive Care Units; Newborn Intensive Care Units ; Infant, Newborn, Diseases/surgery. Multiple free-text searches were performed applying sin-gularly or in combination the following terms through all the fields of the records: neonatal intensive care unit, neonatal surgery, Laparotomy, patent ductus arteriosus. The related articles related to these searches generated by MEDLINE were also reviewed. Subsequently, the searches were pooled and all articles dealing with surgical intervention of neonates in NICU were included and duplicates were excluded. The authors individually reviewed all the abstracts of the retrieved studies in order to select the papers that were relevant to the review topic. In addition, the reference lists of the included papers were searched for any missing articles. After reviewing the studies, authors analyzed them for indications, results and authors recommendations regarding feasibility of the surgical procedures in NICU. Based upon these authors have also suggested future course of action for further development of this field.
# Results
Search of text 'Intensive Care Units, Neonatal, neonatal surgery, Patent ductus arteriosus' yielded 63 articles of which 18 were included. Search of Text 'Newborn Intensive Care Units, neonatal surgery, Laparotomy' yielded 17 articles of which 2 were included. After searching related articles and references from these articles, 26 articles dealing with surgical intervention in neonates in NICU were included in study , 2).
# Discussion
Surgery in neonates is required for a number of conditions, in emergency settings (necrotizing enterocolitis etc.), semi emergencies like congenital diaphragmatic hernia (CDH), esophageal atresia and routine cases like hernia. These neonates may be stable or unstable and many of them may be on mechanical ventilator. In non emergent cases, the neonates can be stabilized and then can be taken for surgery. However, in many emergency situations, surgery may be needed immediately. The surgery of these patients involves shifting to operation theater (OT), and then back from OT to Neonatal intensive care unit (NICU). This may mandate interhospital or intra-hospital transport. This needs neonatal transport. The adverse effects of interhospital transport of neonates are well documented [bib_ref] Surgical treatment of patent ductus arteriosus in preterm infants. Fouryear experience with..., Eggert [/bib_ref] [bib_ref] Quality evaluation of neonatal transports, Meberg [/bib_ref]. These include risk of deterioration especially in ventilated and unstable neonates.
Also, there are country wise differences in organization of neonatal transport units ranging from dedicated transport services to carry out all transfers to adhoc systems provided by big hospitals [bib_ref] Optimising neonatal transfer, Fenton [/bib_ref] [bib_ref] Neonatal Transport: time to change?, Field [/bib_ref]. This leads to a situation where unstable ventilated neonates are transferred in suboptimal conditions. This is more true for neonates requiring surgery, as all over the world there are stand-alone NICU with no surgical facilities and neonates who need surgery are transferred to centers with facility for neonatal surgery. Intra-hospital transport of critically ill patients is also associated with complications. Although there are no studies in neonates, in an Australian study for critically ill adult patients, serious adverse outcomes including major physiological derangement, patient/relative dissatisfaction, prolonged hospital stay, physical/psychological injury and death were reported [bib_ref] Incidents relating to the intra-hospital transfer of critically ill patients. An analysis..., Beckmann [/bib_ref]. To obviate these problems, operations of sick surgical neonates in NICU are proposed. The neonates can be operated in NICU where surgical facilities are available or a neonatal surgical team could be developed that could be transported for surgery. The major indications for surgery in NICU are the procedures which are needed in neonates on mechanical ventilator or emergently in an unstable patient. Most of these neonates are premature, very low birth weight (VLBW), may be on prolonged ventilator support [bib_ref] Surgical treatment of neonates with very low (VLBW) or extremely low (ELBW)..., Sawicka [/bib_ref] and may need surgery for congenital or acquired conditions. These neonates have high risk of transport related complication.
However this has problems related to infrastructure, manpower and patient outcome [bib_ref] Scope and feasibility of operating on the neonatal intensive care unit: 312..., Hall [/bib_ref]. In this review article, we have aimed to generate evidence in relation to surgery of neonates in NICU rather than OT.
## What neonatal surgeries had been done safely in nicu?
A review of the literature suggest that the since the publication of abdominal laparotomy drain for NEC in 1977 [bib_ref] Peritoneal drainage under local anesthesiafor perforations from nectrotizing enterocolitis, Ein [/bib_ref] and ligation of patent ductus arteriosus (PDA) in 1982 [bib_ref] Surgical treatment of patent ductus arteriosus in preterm infants. Fouryear experience with..., Eggert [/bib_ref] in NICU, a plethora of surgeries have been performed in NICU. PDA ligation is the commonest surgery that has been safely performed in NICU. The results of PDA surgery are reviewed in . Since first reported by Eggert et al [bib_ref] Surgical treatment of patent ductus arteriosus in preterm infants. Fouryear experience with..., Eggert [/bib_ref] in 1982, published studies from all around the world (USA, Netherlands, Germany, Taiwan, South Korea, etc.) confirm that a total of 669 neonates with PDA had successful ligation in NICU without any major complication. NICU PDA ligation has been known to be safe as well as cost effective [bib_ref] Surgical treatment of patent ductus arteriosus in preterm infants. Fouryear experience with..., Eggert [/bib_ref] [bib_ref] Operative closure of patent ductus arteriosus in premature infants in the neonatal..., Taylor [/bib_ref] [bib_ref] Surgical ligation of patent ductus arteriosus in a neonatal intensive care setting..., Shenassa [/bib_ref] [bib_ref] Surgical closure of the patent ductus arteriosus in the neonatal intensive care..., Coster [/bib_ref] [bib_ref] Operative closure of patent ductus arteriosus in the neonatal intensive care unit, Mortier [/bib_ref] [bib_ref] Use of neonatal intensive care unit as a safe place for neonatal..., Gavilanes [/bib_ref]. In fact, it is has been reported better than the medical treatment (Indomethacin) [bib_ref] Surgical ligation of patent ductus arteriosus in a neonatal intensive care setting..., Shenassa [/bib_ref] [bib_ref] Peritoneal drainage under local anesthesiafor perforations from nectrotizing enterocolitis, Ein [/bib_ref]. The pediatric cardiothoracic surgical team goes to NICU and performs the surgery avoiding any shifting of the sick neonate, while maintaining the continuity of care (same neonatologist) [bib_ref] A comparison of onsite and off-site patent ductus arteriosus ligation in premature..., Gould [/bib_ref]. In spite of such a large worldwide experience, NICU PDA ligation has not gained universal acceptance as most of the NICU planners do not incorporate the idea of neonatal surgery in intensive units; even basic surgical instruments and not always available in NICU.
## Laparotomy for necrotizing enterocolitis (nec)
is the other commonly reported surgery that has been performed in NICU [bib_ref] Laparotomy for necrotizing enterocolitis: intensive care nursery compared with operating theatre, Frawley [/bib_ref]. Most of these are sick unstable ventilated neonates.
Apart from this, CDH repair also has been described . Some NICUs have a well-established protocol for operative repair of CDH in NICU [bib_ref] Congenital diaphragmatic hernia: intensive care unit or operating room?, Lago [/bib_ref]. Newborns with CDH symptomatic at birth were sedated and paralyzed in the delivery room, and treated with elective high-frequency oscillatory ventilation (HFOV), surfactant, inhaled nitric oxide (iNO) and membrane oxygenation (ECMO) as necessary, delaying surgical repair until their clinical conditions were stable. Once the CDH newborn was stabilized, a trial on conventional ventilation was started at least 24 hours before surgery; however, if the patient was unstable, therapy was switched back to HFOV and surgery was performed in the NICU. They recommended a prolonged phase of pre-surgery stabilization and strict control of infection for the CDH newborns that might benefit from an exclusive HFOV and NICU surgery.
Besides, tracheostomy [bib_ref] Safety of pediatric bedside tracheostomy in the intensive care unit, Klotz [/bib_ref] , central line placement [bib_ref] Broviac catheter insertion: operating room or neonatal intensive care unit, Lally [/bib_ref] , tracheoesophageal fistula (TEF) repair [bib_ref] Scope and feasibility of operating on the neonatal intensive care unit: 312..., Hall [/bib_ref] , abdominal wall defect repair [bib_ref] Contemporary postnatal surgical management strategies for congenital abdominal wall defects, Marven [/bib_ref] , stoma closure [bib_ref] Scope and feasibility of operating on the neonatal intensive care unit: 312..., Hall [/bib_ref] , gastrostomy [bib_ref] Scope and feasibility of operating on the neonatal intensive care unit: 312..., Hall [/bib_ref] have been also done safely in NICU . A list of surgeries that could be performed in NICU has been tabulated .
## What are the infrastructures needed for surgery in nicu?
Almost all the NICU have certain basic instruments which are routinely used. These non consumable instruments can be used for surgery also . As apparent from the table, very few extra instruments/ infrastructural changes are needed to perform surgery in NICU. All future NICU planners must integrate OT, equipped with the common surgical consumables in NICU.
## What are the members needed for nicu operative team?
Every hospital should have a dedicated team to undertake these procedures. A sample team can consist of senior neonatal surgeon, two neonatal surgeons as assistants (one may be trainee), two trained surgical nurses (one scrub nurse and the other floor nurse), one technician to maintain the instruments and two neonatal anesthetists. Each neonatal NICU should have dedicated cupboard for surgical supplies that should be replenished after each surgery.
According to British Association of Perinatal Medicine Standards for Hospitals Providing Neonatal Intensive and High Dependency Care and Categories of Babies requiring Neonatal Care guidelines, level III Units should provide the whole range of medical neonatal care but not necessarily all specialist services such as neonatal surgery. However, they recommend defined lines of communication and access to specialist advice including Neonatal Surgery and Anaesthesia. As neonatal surgery is required only a few of the neonates, this policy of neonatal ICU operative team covering designated level III NICU within a geographical area can be utilized for optimal utilization and greater reach of resources. It has the advantages of maintaining the continuity of care and minimal inconvenience to mother. With more and more level II and level III NICU being developed all over the world, this policy can solve human resource problems for neonatal surgery.
# Conclusion
Neonatal surgery in NICU is a safe procedure and can be utilized in unstable or ventilated neonate. Every neonatal ICU planner should always create infrastructure for surgery in NICU. Surgical NICU operative team should be developed for optimal reach and utilization of resources.
Journal of Neonatal Surgery Vol. 2(2); 2013
Conflict of interest: NoneSource of Support: Nil |
Common DNA sequence variation influences 3-dimensional conformation of the human genome
[formula] HG00513_Rep1 HG00513_Rep2 GM12878_Rep1 GM12878_Rep2 GM19204_Rep2 GM18522_Rep2 GM18505_Rep2 GM19141_Rep2 GM19193_Rep1 GM19193_Rep2 GM18507_Rep1 GM18508_Rep2 GM18486_Rep1 GM18486_Rep2 GM19238_Rep1 GM19238_Rep2 GM18516_Rep2 GM18507_Rep2 GM19204_Rep1 GM19239_Rep1 GM19239_Rep2 GM19099_Rep1 GM19099_Rep2HG00512_Rep1 [/formula]
## Hi-c rep1
Hi-C Rep2 LCL-1 LCL-2
## Hi-c rep1
Hi-C Rep2
## Lcl-20
Hi-C Rep1
## Hi-c rep2
Shuffled Replicates:
2.0e-05 (w) 2.1e-04 (ks)
3.5e-12 (w) 2.8e-12 (ks) 6.5e-06 (w) 3.4e-05 (ks) 5.6e-09 (w) 3.2e-08 (ks)
3.1e-05 (w) 2.1e-04 (ks) 2.6e-04 (w) 8.9e-04 (ks)
6.7e-04 (w) 2.2e-03 (ks) . 3D chromatin variation among 20 LCLs and H1-derived lineages.
(a) Shuffling scheme used to assess biological variability in -d, and here in panels b-e. (b)-(e) Boxplots show Pearson correlation coefficient between biological replicates from the same cell line (Replicates = "True"), and between replicates from different cell lines (Replicates = "Shuff"; short for "shuffled"). The set of cell lines considered is indicated below each box. LCL = 20 LCL cell lines (40 replicates); H1 = H1-ES and the four derived lineages H1-ME, H1-NPC, H1-TB, H1-MSC (5 cell lines, 10 replicates); H1+LCL = 20 LCLs and 5 H1-derived lines considered together (50 replicates). All phenotypes examined here (DI, PC1, INS, or FIRE) show a signature of cell-type specificity whereby they are more similar across individuals when looking at the same cell type (i.e. LCLs), relative to comparing across cell types within an individual (i.e. same genetic background, H1s). Statistical significance calculated by two-sided Wilcoxon rank sum test (labeled w), or Kolmogorov-Smirnov test (labeled ks).
(f) Dendrograms from hierarchical clustering of 40 Hi-C replicates based on the Hi-Cderived phenotype indicated above each dendrogram (DI, PC1, INS, or FIRE). In most cases, replicates from the same cell lines cluster together. (g) Principal Component Analysis of 20 LCLs using one of four Hi-C-derived phenotypes, as indicated above each plot. Each population in our study is represented by a different color as indicated in the color key to the right. . Correlations between 3D genome phenotypes and other molecular phenotypes. Five subpanels show results for different types of variable regions: PC1, DI, INS, FIRE, contact matrix anchor bins (top to bottom). Density plots show the distribution of Spearman correlation coefficients (SCC) between 3D phenotype indicated to the left of each subpanel and molecular phenotypes as indicated above each plot. See Supplemental for schematic of calculations. Each subpanel is further organized into three columns as indicated at the top of the (left column) The ChIP-seq and RNA-seq data in this column are from . These data are the same as in , with the addition of histone modifications H3K4me1, H3K4me3, and H3K36me3, as well as DNase-seq data from . Plots include all individuals for which both Hi-C and the data type in question are available (N=8 individuals for ChIP-seq, N=7 for RNA-seq, and N=11 for DNase-seq). (middle column) SCC values with same ChIP-seq data as left column, but using a separate set of variable regions called in only 11 YRI individuals. We did not make these plots for variable contact matrix bins because there is only one variable bin call set. In each QQ plot, the Xaxis is the -log10 theoretical quantiles calculated from the uniform distribution. The Y-axis is the -log10 p-value calculated from linear mixed effects model for each type of QTL search. The grey area represents the 5% -95% confidence bands based on Beta distribution Beta(i, M-i+1), where i is the i-th order statistics and M is the total number of tested SNPs. (b) DI values by genotype for bins with positive DI (left), negative DI (right), and all QTLs (right). A Simpson's paradox is observed when all bins are considered together. (c) Genotype-phenotype relationship at QTLs using Hi-C data binned at different resolutions (25Kb, 50Kb, 100Kb), and normalized with different methods (HiCNorm, Knight-Ruiz matrix balancing). X axes represent beta values from the original QTL search. Y axes represent beta value from a linear model fit to the genotype-phenotype relationship using Hi-C data of the indicated resolution and normalization method.(d) Aggregate contact plots as in , but including several negative control sets. Top row shows C-QTL SNPs with 5 random shuffles of the genotypes. Bottom row shows 5 random sets of non-QTL SNPs selected from the opposite end of the FDR spectrum from actual C-QTLs (FDR>0.8). (e) Number of direct overlaps between QTL sets. |
Six Collective Challenges for Sustainability of Almería Greenhouse Horticulture
Globally, current food consumption and trade are placing unprecedented demand on agricultural systems and increasing pressure on natural resources, requiring tradeoffs between food security and environmental impacts especially given the tension between market-driven agriculture and agro-ecological goals. In order to illustrate the wicked social, economic and environmental challenges and processes to find transformative solutions, we focus on the largest concentration of greenhouses in the world located in the semi-arid coastal plain of South-east Spain. Almería family farming, predominantly cooperative, greenhouse intensive production, commenced after the 1960s and has resulted in very significant social and economic benefits for the region, while also having important negative environmental and biodiversity impacts, as well as creating new social challenges. The system currently finds itself in a crisis of diminishing economic benefits and increasing environmental and social dilemmas. Here, we present the outcomes of multi-actor, transdisciplinary research to review Int. and provide collective insights for solutions-oriented research on the sustainability of Almeria's agricultural sector. The multi-actor, transdisciplinary process implemented collectively, and supported by scientific literature, identified six fundamental challenges to transitioning to an agricultural model that aims to ameliorate risks and avoid a systemic collapse, whilst balancing a concern for profitability with sustainability: (1) Governance based on a culture of shared responsibility for sustainability, (2) Sustainable and efficient use of water, (3) Biodiversity conservation, (4) Implementing a circular economy plan, (5) Technology and knowledge transfer, and (6) Image and identity. We conclude that the multi-actor transdisciplinary approach successfully facilitated the creation of a culture of shared responsibility among public, private, academic, and civil society actors. Notwithstanding plural values, challenges and solutions identified by consensus point to a nascent acknowledgement of the strategic necessity to locate agricultural economic activity within social and environmental spheres.This paper demonstrates the need to establish transdisciplinary multi-actor work-schemes to continue collaboration and research for the transition to an agro-ecological model as a means to remain competitive and to create value.
## Global sustainability challenges of agricultural systems
Human population growth and food consumption and waste are placing unprecedented demands on agricultural systems and increasing pressure on natural resources. These demands are generating some of the most serious challenges faced by humanity, and are concurrently degrading land, water resources, biodiversity, and climate on a global scale. Current predictions for the future of agriculture point out that to meet surging demand, global food production must double by 2050. In this sense, many authors note that to meet the world's future food security and sustainability needs, food production must grow substantially while, at the same time, agriculture's environmental footprint must shrink dramatically. Despite the importance of addressing this global issue, it is receiving little urgency, and few quantitative targets are being developed by the global research and policy community.
Attaining sustainable global food systems implies, as a first step, understanding the trade-offs between food security (e.g., produce enough and healthy food) and environmental impacts (e.g., biodiversity loss, resource stresses). It has been extensively documented that impacts on biodiversity and natural resources are attributed to agricultural intensification (e.g., pesticides, fertilizers, tillage) and farmland abandonment (i.e. urbanization, job opportunities or aging populations). At a global scale, this environmental deterioration results in biodiversity loss and the decline of ecosystem services, placing the maintenance of human well-being at risk. In this adverse scenario, new farming initiatives and renewed practices are urgently required to promote a sustainable agriculture model. This implies the transformation of intensive systems into more complex agroecosystems based on a recoupling of farming practices and their ecological and social environments. Such farming transition should promote the adoption of farming initiatives and practices that preserve biodiversity, together with a bundle of agricultural-related ecosystem services such as pollination, erosion control, water management, cultural heritage and local identity. To advance in this direction, agroecology has emerged as one of the most promising initiatives to support the transition from conventional to sustainable farming, improving both environmental sustainability (biodiversity and ecosystem services) and socio-economic conditions (food accessibility, participation and empowerment, and fair prices). However, to instigate such transition globally, a better understanding of the interactions between the social, economic and environmental dimensions of agricultural systems as well as the interdependencies and feedbacks between these dimensions is required.
In this paper, we consider an example of intensive greenhouse horticulture in order to explore the tradeoffs and challenges in bringing about a transition to a more sustainable agricultural production system. The paper is organized in five sections. In Section 1 we set out the background to the socio-economicenvironmental challenges surrounding intensive greenhouse horticulture in Almería. Section 2 introduces the need for a new agro-ecological framework for sustainability. The transdisciplinary, multi-actor methodology is detailed in Section 3. Section 4 summarises the sustainability challenges identified in the workshop, and barriers and opportunities to address these challenges, and finally, actions to be taken. Section 5 sets out general conclusions and the importance of the six challenges identified herein as fundamental to transitioning into an agricultural model that ameliorates risks and avoids a systemic collapse, balances a concern for profitability with sustainability and provides crucial insights for the small family farming business model.
In Europe, a significant part of the greenhouse horticulture is concentrated in the semi-arid coastal plain of the province of Almeria, in south-east Spain. The greenhouse horticulture production in Almeria started after the 1960s and currently houses the largest concentration of greenhouses in the world. Since 1960, development strategies and the lack of land use planning resulted in socio-economic development in coastal areas and caused one of the most dramatic land use transformations in Europe, currently representing around 4% of the provincial surface area. The promotion of greenhouse horticulture has resulted in very significant social and economic benefits for the Almería Province, while also having important negative impacts on native biodiversity and natural resourcesas well as creating social challenges.
The economic contribution of greenhouse horticulture in Almería is approximately 1800 million Euros, and a related auxiliary business sector generates another 1.600 million Euros. In addition to the 15,000 family farmers engaged in production activity, 40,000 additional jobs are provided. Within the province of Almería, greenhouse production represents 13% of gross domestic product (GDP), as contrasted to the average of agricultural GDP in Spain of 2.5% . The total economic activity surrounding the farming system is 40% of the GDP of the province of Almería. However, this agricultural system has a relatively equitable distribution of wealth due to the fact that 95% of farms are family owned, and that their products are predominantly marketed by cooperatives or other social economy entities, as well as some investor-owned companies including a few auctions.
In Almería, small-holder family farm greenhouse horticulture started in earnest in the seventies, supported by local cooperative capital and knowledge transfer by way of the cooperative bank's experimental farm, and the founding of marketing cooperatives as well as the association of horticultural exporters. Greenhouse production is labour intensive and labour requirements were initially met by the local families of greenhouse owners. Since the end of the eighties, the increasing intensification of the family farming model has resulted in the need for family labour to be supported by immigrant labour, which mainly comes from different African countriesand Central-Eastern Europe. Currently, Almería's greenhouse sector has over 110 nationalities working and requires several types of labour. Highly skilled and qualified workers that support these technological and innovative greenhousesare necessary, as is manual unskilled labour, comprised of both family members and immigrant, who do the physical, routine work under difficult conditions inside the greenhouse. In addition, within the cooperatives and producer organisations, the handling and packaging utilises a mostly female workforce which also represents a wide range of nationalities. As precision farming increasingly requires higher levels of technology and ICT knowledge, both lack of skills and investment in technology present social and economic challenges, not to mention the anticipated tensions that will be created by the mechanisation and automatisation of tasks traditionally held by workers.
While the economic benefits and both positive and negative social consequences have been evident due to the rapid and dramatic land transformation, greenhouse horticulture expansion has also produced significant impacts on local biodiversity and the decline of important ecosystem services (e.g., aquifer recharge or erosion control). The uniqueness of the biodiversity of arid and semiarid environments of the Almería region has been documented since the early nineties. This region has been recently included among the 25 worldwide biodiversity hotspots and supports high levels of biodiversity, with numerous endemic species and habitats of priority interest at European levels. This unique diversity of species and ecosystems has led to the declaration of diverse protected areas. However, in this region, these conservation efforts have co-existed and co-evolved with intense human developments (e.g., urban and agricultural expansion) over recent decades. Historically, the conditions for human occupancy have been unfavourable, marked by scarce rainfall, rough land and frequent strong winds. The human development model was fundamentally limited by water scarcity, and it was dedicated to subsistence dryland agriculture characterized by low yields. As mentioned above, it was not until the 1970's that this socio-economic model changed, led by the development of intensive agriculture, the tourism sector and the construction industry. In particular, the rapid development of greenhouse agriculture along the Mediterranean coast has produced the alteration and fragmentation of habitat of numerous plant species and has affected the availability of groundwater resources. As a consequence, simultaneously with these intensive land transformations, there has been a tremendous effort by governments to protect natural areas, especially those sensitive to the impacts of climate change and desertification. As a result, several protected areas have been declared with different levels of protection status according to the International Union for Conservation of Nature (IUCN), including several new EU Sites of community importance of the "Natura-2000" network, the "Cabo de Gata Natural Park" in 1987 and the "Paraje Natural Desierto de Tabernas" in 1989. The convergence of particular ecological and socio-economic factors in the region has led to conflicts of interest between conservation and human development.
## Transitioning to a sustainable model for the almeria's agricultural sector.
There is a large body of literature that has advanced the conceptual models which contribute to understanding the basis of sustainability in socio-ecological systems. The Brundtland report "Our Common Future"introduced the three pillars of sustainable development: economic, social, and environmental, which have been widely used especially in the policy arena. In this approach, the three pillars are generally given equal weight and in many practical contexts sustainability is attributed to one of them at a time (e.g., sustainable economic The situation described above highlights the difficulty in determining tradeoffs and presents the "wicked" social and economic challenges implicit in making compatible market driven agriculture with agro-ecological goals. While the agricultural activity, largely based on social economy entities and family farms, has contributed greatly to the development of Almería and to the provision of healthy and safe food for Europe, serious social and environmental issues still persist, including informal contracting, lack of social integration and unregulated housing settlements, gender inequality, water aquifers/depletion and salinization, loss of biodiversity, and inadequate waste management (i.e., plastic and vegetable waste, fertilizers and pesticides). Whether small family farmers, their cooperatives and producer organizations, and related institutions will be in a position to carry out the necessary transformations in Almería in order to preserve positive social economic benefits, whilst meeting pressing agro-ecological challenges is an open question. We propose, as evidenced by this paper, that the academic community should play an important role in advancing solutions and closely work with all economic and social actors and local and regional administrations. Interactions between scientists and policymakers have often been limited to exchanges of scientific documents, and collaborative work between research and communities rarely occurs for addressing day-to-day social-economic-ecological problems. In this context, this paper is motivated by scientists concerned with aligning their research with real needs found in the local production system and the policy arena.
## Transitioning to a sustainable model for the almeria's agricultural sector
There is a large body of literature that has advanced the conceptual models which contribute to understanding the basis of sustainability in socio-ecological systems. The Brundtland report "Our Common Future"introduced the three pillars of sustainable development: economic, social, and environmental, which have been widely used especially in the policy arena. In this approach, the three pillars are generally given equal weight and in many practical contexts sustainability is attributed to one of them at a time (e.g., sustainable economic growth) to the exclusion of the other two. There is a need to shift this paradigm into a new sustainability framework, recognizing the significance of the life-support systems and the biosphere as the foundation for the economy, society, and the human dimension. From such a perspective, it is explicit that the economy is a subsystem of society, which is, in turn, a subsystem of the biosphere or environment. Within this sustainability framework, the long-term sustainability of greenhouse agriculture of Almería depends on the ability of the agricultural model to transition to a new sustainable framework which builds upon the biosphere as a precondition for socio-economic development. Therefore, a major goal for Almeria's greenhouse sector is to identify and understand the interactions and feedbacks among dynamic biophysical processes that regulate the biosphere (i.e., environment), and consequently its capacity for providing favourable conditions for complex human societies (i.e., society) and for long-term human prosperity (i.e., economy).
As in all other sectors of society, framing the agricultural sector in a new sustainability model based on recognition of the biosphere as the foundation upon which society and economy are basedis extremely challenging. Specifically, the current socio-ecological context of Almeria's agricultural sector illustrates complex realities resulting from balancing costs, benefits and trade-offs among social, economic and environmental dimensions that need to be analyzed with appropriate methods. For example, trade-offs are not straight forward when the inclusiveness of groups in processes would reduce cost effectiveness, or where business growth for one group of interest would lead to a loss in biodiversity affecting the common good. On the other hand, tradeoffs which favor increased biodiversity and protection of the environment may lead to the loss of traditional jobs or businesses. In carrying out our analysis of the challenges that face the sustainability of Almeria's agricultural sector, plural value-dimensions are present. These are often referred to as incommensurable valuesas opposed to commensurable values, where in the latter, trade-offs are more straightforward to determine and solutions can be chosen by deciding which solution is optimal, using agreed upon values. In contrast, the presence of plural values when values cannot be compared by means of a single scale (e.g., monetary scale) means that the estimation of trade-offs is problematic. An approach that allows for plural values to be considered is necessary in seeking solutions for the sustainability of Almería's agricultural sector. In addition, stakeholder identification of unintended benefits or disadvantages are needed to avoid the risks of simplifying complexities and to fully clarify and include plural value dimensions, transparently identify trade-offs, and address the real drivers and barriers to future developments of Almeria's agriculture sector. Therefore, an inclusive approach should take into account the development of common conceptual grounds through understanding and cooperationin order to address divergence of values in the analysis of complex systems, where disagreements in definition or solutions of a societal problem often exist. more straightforward to determine and solutions can be chosen by deciding which solution is optimal, using agreed upon values. In contrast, the presence of plural values when values cannot be compared by means of a single scale (e.g., monetary scale) means that the estimation of trade-offs is problematic. An approach that allows for plural values to be considered is necessary in seeking solutions for the sustainability of Almería's agricultural sector. In addition, stakeholder identification of unintended benefits or disadvantages are needed to avoid the risks of simplifying complexities and to fully clarify and include plural value dimensions, transparently identify trade-offs, and address the real drivers and barriers to future developments of Almeria's agriculture sector. Therefore, an inclusive approach should take into account the development of common conceptual grounds through understanding and cooperationin order to address divergence of values in the analysis of complex systems, where disagreements in definition or solutions of a societal problem often exist. Coupled with this plural value and multiple criteria approach is the inclusion of multi-actors. The multi-actor approach is an adequate scientific response to stimulate cooperation of scientists with practitioners, industry and other actors. This approach can be implemented by using tools and procedures of transdisciplinary research). Transdisciplinary research combined with the multi-actor approach integrates knowledge from various scientific and societal bodies of knowledgethrough co-learning and knowledge co-production processesand contributes to solutions or transitions for societal challenges. Three key arguments justify the adoption of this approach. First, scientific disciplines are generally hyper-specific and represent a reductionist view to understanding the complexity and uncertainty of human nature systems. Overcoming the barriers of such disciplinary specificity and fragmentation facilitates a holistic view of human nature interactions and allows one to combine and obtain different forms of knowledge to generate context-based knowledge. Second, research on workable solutions by scientists requires an understanding of the socioeconomic, cultural and political-administrative context to align scientific information with policy processes and societal demands. Finally, cooperation between academics and nonacademics is helpful in terms of legitimacy, ownership and shared responsibility for implementing problem-solving solutions. These approaches have been gradually increasing in multiple areas of sustainable governance such as water, agriculture and forests to help society transition towards sustainability. However, multi-actor and transdisciplinary approaches remain Coupled with this plural value and multiple criteria approach is the inclusion of multi-actors. The multi-actor approach is an adequate scientific response to stimulate cooperation of scientists with practitioners, industry and other actors. This approach can be implemented by using tools and procedures of transdisciplinary research). Transdisciplinary research combined with the multi-actor approach integrates knowledge from various scientific and societal bodies of knowledgethrough co-learning and knowledge co-production processesand contributes to solutions or transitions for societal challenges. Three key arguments justify the adoption of this approach. First, scientific disciplines are generally hyper-specific and represent a reductionist view to understanding the complexity and uncertainty of human nature systems. Overcoming the barriers of such disciplinary specificity and fragmentation facilitates a holistic view of human nature interactions and allows one to combine and obtain different forms of knowledge to generate context-based knowledge. Second, research on workable solutions by scientists requires an understanding of the socioeconomic, cultural and political-administrative context to align scientific information with policy processes and societal demands. Finally, cooperation between academics and non-academics is helpful in terms of legitimacy, ownership and shared responsibility for implementing problem-solving solutions. These approaches have been gradually increasing in multiple areas of sustainable governance such as water, agriculture and forests to help society transition towards sustainability. However, multi-actor and transdisciplinary approaches remain underutilized for developing strategies and actions for the sustainability of Almeria greenhouse horticulture and recent research suggests the need for transdisciplinary science.
Below, we report the outcomes of research based on the implementation of a multi-actor, transdisciplinary approach and methodology, including an initial questionnaire, multi-actor participation and evaluation, all supported by transdisciplinary scientific research and literature. The workshop is intended to serve as a starting point to provide collective insights for solutions-oriented research on the sustainability of Almeria's agricultural sector, in order to better identify and characterize challenges needed for long-term sustainability of the region.
## Methodology: a transdisciplinary process for identifying sustainability challenges
As part of the research methodology, a multi-actor structured workshop was conducted in April 2019 at the University of Almeria (Spain). The goal of the workshop was to collectively identify the key challenges for long-term sustainability of greenhouse horticulture of Almeria. A multi-actor community of practice was organizedthat included scientists and non-scientists with extensive experience in greenhouse agriculture from five knowledge domains (i.e., technology, environment, society and culture, economy, and governance). Non-scientists included public and private actors, including but not limited to cooperative presidents, public extension services, foundations, farmers associations, etc. Considering time and financial constraints a total of 15 multi-actor experts (three within each domain) were invited to participate in the workshop. To select these experts the 'reputational approach'was used, through which knowledgeable individuals in greenhouse agriculture in the study area were asked to select leading experts from diverse knowledge domains.
Prior to the structured workshop, the experts were invited to complete an online questionnaire(Appendix A). The purpose of this questionnaire was to individually, based on a review from different transdisciplinary expertise, identify three key challenges limiting sustainability of Almeria greenhouse agriculture. To ensure operability in the workshop, all experts were supplied with a timetable, goals pursued in the research, work schemes, and a full list of participants (Appendix B). The workshop was facilitated by two researchers with extensive experience and skills in multi-actor, transdisciplinary science. Both promoted knowledge transfer between the experts and facilitated communication and understanding between them. At the beginning of the workshop, presentations were given by the facilitators to provide a deeper understanding of the rationale, goals and methods of the workshop. Thereafter, each expert explained the challenges that she/he had identified from an individual perspective in the online questionnaire. All individual challenges were grouped according to their similarity by the facilitators using a card-writing system. By and through the brainstorming on challenges, the experts started to enrich their perspectives from other knowledge domains.
Thereafter, three collective dynamics were conducted. Methodologically, these dynamics were based on co-learning and knowledge co-production approaches in order to generate outcomes based on a balance of trade-offs among the experts from diverse knowledge domains. The collective dynamics were oriented specifically to (1) explore and define the sustainability challenges, (2) identify opportunities and barriers to address each challenge, and (3) establish collective actions to move from theory to practice in overcoming the identified challenges. All the collective dynamics shared the same structure: (1) introduction by the facilitators to explain goals and rules to develop the activity, (2) co-generation of outcomes through a multi-actor work group with mixed profiles, and (3) exposition of results by each work group in plenary to generate feedback and synergies between team groups. Each workgroup involved multi-actor experts of each knowledge domain to promote an integrated view of the sustainability of greenhouse agriculture from multiple perspectives. Each workgroup had a technical secretary who took minutes of the meeting. Upon completion of the three collective dynamics, all co-generated outcomes were collectively discussed and future directions to address the key challenges were identified by the experts. Throughout the dynamics the Mentimeter online application (https://www.mentimeter.com/) was used by the experts in order to prioritize the sustainability challenges and evaluate the workshop. This online software application allowed participants to vote in real-time and reach a consensus on the results. This provided transparency and credibility to the process. Once the workshop results were compiled by the facilitators, they were sent to the participants for their suggestions and corrections, ensuring that the results reflected consensus. This provided transparency and credibility to the process. Once the workshop results were compiled by the facilitators, they were sent to the participants for their suggestions and corrections, ensuring that the results reflected consensus.
## Sustainability challenges for the spanish greenhouse horticulture
As a result of the structured workshop methodology, participants collectively identified six challenges for ensuring the sustainability of the greenhouse horticulture in southeast Spain; The challenges corresponded to (1) Governance based on a culture of shared responsibility for sustainability, (2) Sustainable and efficient use of water, (3) Biodiversity conservation, (4) Implementing a circular economy plan, (5) Technology and knowledge transfer, and (6) Image and
## Sustainability challenges for the spanish greenhouse horticulture
As a result of the structured workshop methodology, participants collectively identified six challenges for ensuring the sustainability of the greenhouse horticulture in southeast Spain; The challenges corresponded to (1) Governance based on a culture of shared responsibility for sustainability, (2) Sustainable and efficient use of water, (3) Biodiversity conservation, (4) Implementing a circular economy plan, (5) Technology and knowledge transfer, and (6) Image and identity. Each challenge is introduced below, with its context and rationale, opportunities and barriers, related supporting scientific literature, and key collective actions to address it. identity. Each challenge is introduced below, with its context and rationale, opportunities and barriers, related supporting scientific literature, and key collective actions to address it.
## Sustainability challenge 1: governance based on a culture of shared responsibility for sustainability
The first sustainability challenge describes the need to shift to a new governance system that enables the Almería greenhouse horticulture to evolve towards a culture of shared responsibility for its long-term sustainability. A major governance issue of Almería agricultural sector identified in the workshop is the regulatory non-compliance tolerated by public local and regional administrations. It was observed that such non-compliance may benefit small farmers in the short term but harm Almería's society, including farmers, in the long term. We use the term "governance" in a rather limited sense in this article to refer to the overall aim to exercise public power (i.e., rules, procedures and practices) with the goal of deepening democracy through the active and direct participation of society in decision-making. In the context of environmental sustainability literature, governance refers to the set of regulatory processes, mechanisms and organizations through which political actors influence environmental actions and outcomes. According to, in the past, the role of civil society in creating public policies and governance was neglected. According to the United Nations Development Program, governance is defined as the set of mechanisms, processes, relationships and institutions through which citizens and groups articulate their interests, exercise their rights and obligations, and reconcile their differences. In addition, it is important to understand the different ways in which society relates to the government to define public policies. The diversity of governance interpretations and meanings, dependent as they are on plural values, as well as multidisciplinary traditions, results in a complex task in the attempt to define a common and generic governance strategy. As strategies for achieving adequate governance, the United Nations Development Programme and the European Union proposed key parameters that included promoting participation, making public decisions with transparency, building consensus on issues of general interest, the efficient use of public resources and accountability. This implies establishing a culture of shared responsibility through the involvement of all societal actors in decision making. In order to achieve this challenge, there is a need to facilitate new and practical forms of cooperation
## Sustainability challenge 1: governance based on a culture of shared responsibility for sustainability
The first sustainability challenge describes the need to shift to a new governance system that enables the Almería greenhouse horticulture to evolve towards a culture of shared responsibility for its long-term sustainability. A major governance issue of Almería agricultural sector identified in the workshop is the regulatory non-compliance tolerated by public local and regional administrations. It was observed that such non-compliance may benefit small farmers in the short term but harm Almería's society, including farmers, in the long term. We use the term "governance" in a rather limited sense in this article to refer to the overall aim to exercise public power (i.e., rules, procedures and practices) with the goal of deepening democracy through the active and direct participation of society in decision-making. In the context of environmental sustainability literature, governance refers to the set of regulatory processes, mechanisms and organizations through which political actors influence environmental actions and outcomes. According to, in the past, the role of civil society in creating public policies and governance was neglected. According to the United Nations Development Program, governance is defined as the set of mechanisms, processes, relationships and institutions through which citizens and groups articulate their interests, exercise their rights and obligations, and reconcile their differences. In addition, it is important to understand the different ways in which society relates to the government to define public policies. The diversity of governance interpretations and meanings, dependent as they are on plural values, as well as multidisciplinary traditions, results in a complex task in the attempt to define a common and generic governance strategy. As strategies for achieving adequate governance, the United Nations Development Programme and the European Union proposed key parameters that included promoting participation, making public decisions with transparency, building consensus on issues of general interest, the efficient use of public resources and accountability. This implies establishing a culture of shared responsibility through the involvement of all societal actors in decision making. In order to achieve this challenge, there is a need to facilitate new and practical forms of cooperation between governments and society, with a new view to carry out common tasks and negotiating and mobilizing coalitions of interest to achieve common purposes.
The analysis of the governance challenge provided the foundation for identifying opportunities and barriers to address it. Among the opportunities found was the existence of a legal framework that obligates public administrations to guarantee transparency and active participation in policies such as the Convenio de Aarhus 1998, Ley 26/2007, Real Decreto Legislativo 1/2001, Ley 19/2013 and Ley 1/2014, or the legal framework that requires economic agents to assume corporate social responsibility (Law 18/2018) and environmental responsibility (Law 26/2007). Among the barriers identified was the fact that the public administration has not developed an effective method by which to fulfil the obligation to include active public participation. In doing so, it is crucial to empower Almeria's society by offering training and experience in public participation for public policies, which requires that governments increase their legitimacy through greater engagement. More recently, the European Commission has emphasized the full engagement of public participation in governance and achievement of its research missions, where the public is not only involved in co-creation but also in co-implementation and co-evaluation.
On this basis, a key action to advance in a new governance model for the agricultural sector in Almeria is to improve internal and inter-institutional coordination for the integration of environmental goals in public policies. Moving to sustainable governance model requires the creation of new spaces based on social learning and collective construction for sustainability, instead of the negotiation of individual interests and preferences. For instance, the creation of public policies, at all levels, which acknowledge the creation of environmental goods and account for their value, which would benefit small farmers and society in general. By doing so, the economic risk of transitioning to a more sustainable agriculture would be compensated and not borne solely by small farmers, who may not be in a position to financially weather such change in the short term. Farmers could be seen to also be producers of environmental goods, and thus should be rewarded for contributing to sustainability. It was recently suggested to follow the protocol of the report of the Sustainable Development Goals 2030 ODS 17 "Partnership to Achieve the Goals", which states that a successful sustainable development program requires partnerships between governments, the private sector and society. These alliances must be inclusive, built on common principles and values, and a shared vision that understands that Almería's agricultural system must keep growth within the biophysical boundaries. Also, the costs and efforts of implementing such vision must be equitably distributed.
## Sustainability challenge 2: sustainable and efficient use of water
Unsustainable water management is one of the most important environmental issues worldwide in the twenty-first century. This global issue is especially relevant in Mediterranean semi-arid ecosystems of south-east Spain, where the scarcity and unpredictability of rainfall events and the lack of perennial rivers mean that water recharged by aquifer systems is the main source of water resources for irrigation and consumption. In addition, groundwater water resources play a key role not only in the conservation of unique ecosystems but also in sustaining the wellbeing of local communities through the supply of ecosystem services. This challenge corresponded to the urgent need of correcting the current trend of water use by greenhouse horticulture, which has caused over the last forty years severe deterioration of water quality and quantity. In particular, the main water issues associated to the greenhouse horticulture are: (a) aquifer overexploitation, which corresponds to about 80% of water sources for irrigation, and whose water deficit is currently estimated for the Campo de Dalias in 65 Hm 3 year −1 ; (b) water quality deterioration due to a progressive increase of water salinity in aquifers, which causes damage and losses of saline-sensitive horticultural crops. Water quality deterioration is caused by two major forces; marine intrusion processes and an unsustainable aquifer management; and (c) the nitrate (NO 3 − ) contamination of underlying aquifers and wetlands caused by the use of fertilizers the greenhouse-based vegetable production system. Much of the area where greenhouses are located has been declared Nitrate Vulnerable Zones (NVZ) in accordance with the Nitrate Directive of the European Union, which requires the adoption of improved management of both irrigation and nitrogen. The integrated use of alternative water sources from desalination, purification and rain harvesting and groundwater with the appropriate blending system to optimize cost, quality and water extraction rates can help to reduce the overexploitation of the aquifers. Several Almería farmers, cooperatives and associations are doing so and taking action. With the aim of achieving the sustainable and efficient use of water resources, diverse water authorities from regional to national scale (i.e., Andalusian Water Agency and ACUAMED), in coordination with the Poniente Water User's Board and the Geological and Mining Institute of Spain, launched the program for support the regeneration and recuperation of the aquifer system of Sur de Sierra de Gádor-Campo de Dalías in 2008. This program is based on a combination of measures and alternative water sources to restore the body of groundwater with the aim of achieving gradually its good status within 12 years. Whereas this plan has represented a successful initiative of coordination and joint efforts among governmental, societal and scientific entities, there remains uncertainty whether the planned goal will be achieved. In the relation to the higher costs of this alternative water source and according to, the incentives promoting the use of desalinated seawater for irrigation that most encourage farmers would be the implementation of tax relief, price reductions and the obligation to install rainwater collection systems.
Within this context, several opportunities for addressing this challenge were found. Such opportunities include (1) the compliance of the EU Water Framework Directive, which requires public administrations and economic agents to cooperate to achieve the good condition of all water bodies; (2) the existence of low-cost technologies developed and currently evaluated in the greenhouse industry as result of innovative research and knowledge transfer that would imply significant water savings and reduced nitrate leaching (see, for instance, the Fertinnowa project, https://www.fertinnowa.com/project/, the Internet of Food and Farm IoF2020, chain integrated greenhouse vegetable trial https://www. iof2020.eu/trials/vegetables/chain-integrated-greenhouse-production, the water and nutrient hubs established under peer-to-peer learning EU project NEFERTITI https://nefertiti-h2020.eu, and the recent founding of the Almería SmartAgriHub by the University of Almería, COEXPHAL and the Cajamar Foundation); (3) the existence of a very effective system for knowledge dissemination to greenhouse farmers, cooperatives and supply companies where the dissemination is conducted through technical advising by public and private organizations such as the Cajamar Foundation, COEXPHAL the Almería Association of F&V Producer, the Instituto de Investigación y Formación Agraria y Pesquera de Andalucía IFAPA, and the Centro de Investigación en Biotecnología Agroalimentaria (BITAL) at the University of Almería; (4) the existence of a market that is increasingly demanding more sustainable production processes, and which can be achieved by different certification systems (for example, GLOBAL GAP good agricultural practices certification); and (5) a very flexible and dynamic community of farmers and cooperatives capable of absorbing changes and promoting new strategies in the greenhouse industry. Barriers identified include the following: (1) a lack of public awareness by water users regarding the urgent need of a sustainable use of water resources and the implications for the long term sustainability of the greenhouse industry; (2) reticence by water users to pay for higher-cost irrigation water from alternatives strategies; (3) a lack of economic incentives by public administrations to change the current water management plans; and (4) a lack of control by public administrations over existing water regulations to prohibit illegal water practices such as the use of illegal wells.
Finally, actions identified amongst the participants include the following: (1) to adapt and control water use rights according to availability (Water Exploitation Index) and progress towards compliance with the water and regulatory framework in force (water use for agriculture and nitrates); (2) to provide the sector with regular information on the general condition of the groundwater bodies and create a panel of experts including both socio-economic agents (companies, unions, etc.) and the civil society (consumers, environmental organisations); (3) to promote the use of alternative water sources and efficient and sustainable irrigation and fertilisation management systems; and (4) to ensure the impact of the transfer of technologies and knowledge from local R&D organizations.
## Sustainability challenge 3: biodiversity conservation
The challenge of biodiversity conservation not only describes the need for protecting one of the most unique habitats of Europe, i.e., the Spanish drylands, but also the urgent need to communicate to society the interdependent relationship between the maintenance of the greenhouse horticulture and conservation of local natural resources. Although Almería greenhouses only occupies 4% of the province of Almeria, most are concentrated in the coastal platform where numerous habitats of priority interest are located. For instance, in the southern part of the coastal platform called "El Campo de Dalias", there are different Sites of Community Importance of the European Natura 2000 network, e.g., Paraje Natural of Punta Entinas-Sabinar (IUCN category III) and Natural Reserve of Punta Entinas-Sabinar (IUCN category Ia), that protect priority habitats and species of community interest that contribute to maintenance of biodiversity (e.g., bird species) and ecosystem services (e.g., erosion control and aquifer recharge). However, major changes occurred in recent decades in the economic activities of the Almeria province have led to severe impacts on these habitats. Among these habitats are the community of Ziziphus (i.e., Priority Habitat 5220, Directive 92/43/ECC), one of the priority habitats for conservation in Europe that has almost disappeareddue to the expansion of greenhouse industry, urbanization and beach tourism.
Among the opportunities to address this challenge, participants identified (1) the existence of local regulations that oblige the establishment of green infrastructures in the surroundings of new creation greenhouses (B.O.P. of Almería number 148 of 3 August 2017); and (2) the existence of incentives by regional administration to subsidize the establishment of green infrastructures (BOJA number 69 of 11 April 2017). In addition, new green infrastructures will be supported by the Strategy for Green Infrastructure and Ecological Connectivity and Restoration; (3) the dynamic and adaptive character of Almería's greenhouse industry, which shows a permanent capacity and flexibility to implement new measures and innovations; (4) new technologies and cultivation techniques to reduce harmful agricultural practices, such as integrated pest management, organic production, improved irrigation, decision support systems, internet of things, and digitisation in general could contribute to support biodiversity; and (5) better communication with consumers to promote sustainable agricultural products. Among the barriers, we found (1) the poor perception by all levels of society of the value of biodiversity and the direct and indirect benefits it provides to people, such as erosion control, maintenance of coastal and beach dynamics, flash-flooding control or the existence value of a unique biodiversity in Europe; (2) the restoration plant species of interest (e.g., Maytenus senegalensis subsp. europaea and Ziziphus lotus, Rosmarinus officinalis and Thymelaea hirsuta) is constrained by the greenhouse expansion; and (3) the globalised competitive horticultural markets which prioritise high, short-term economic performance, and the focus on technology to increase production at all costs. The latter has led to a socio-ecological vicious circle in which natural ecosystems are transformed into agricultural spaces that demand increasingly stressed ecosystem services to maintain their production.
Among the actions identified we found (1) increased public understanding of the need to preserve ecological processes that sustain the greenhouse industry (e.g., planting of autochthonous hedges between greenhouses); (2) implementation of effective measures for acknowledging the instrumental values of biodiversity. Examples of this instrumental value include for instance the crucial role of Sierra de Gádor mountain does in recharging aquifers that sustain the greenhouse industry and the importance of native flora species in the maintenance of insects for biological control; and (3) the restoration of local ephemeral streams (i.e., ramblas in Spanish) which will contribute to the maintenance of genetic diversity, and the habitat connectivity of the Nature 2000 Network. All these measures can contribute to ensuring the proper conservation of Natura 2000 sites in the region, as a basis for the population's understanding of the importance of conservation policies, and compliance with environmental regulations.
## Sustainability challenge 4: implementing a circular economy plan
The European Commission launched the Action Plan for promoting the Circular Economy in 2015. Subsequently, in 2018, both the Spanish and Andalusian governments adapted and designed their own circular economy plan. At the regional level, the government introduced circular bioeconomy as an "economic model based on the use of renewable biomass resources and their sustainable and efficient transformation into bioproducts, bioenergy and services for society". In this sense, the Almería production model lacks the non-closure of its material circuits. On one hand, plastics (both packaging and those used to protect crops) are a serious problem for which the solutions so far have been to use regulated disposal companies, who either recycle or transform into energy, or burn the plastics which cannot be recycled or utilised otherwise. On the other hand, accumulated crop residues represent a logistical, environmental and health issue, and are not yet solved, as there are not sufficient facilities which deal with this waste in a circular manner. Furthermore, leachate resulting from agricultural irrigation can end up in aquifers, worsening the quality of underground water resources.
A relevant opportunity was identified in the growing societal awareness associated with sustainability issues in general, and among consumers in particular. In this sense, the existence in Almería of a local productive system could facilitate the transfer of values, knowledge and technologies. In addition, the economic capital from agricultural industry surpluses and other sectors such as tourism, construction and industry could bring together the development of new sectors. Finally, the wide availability of sunlight in Almeria offers a parallel opportunity to the agricultural industry associated with new photovoltaic plant businesses. Among the obstacles that could hinder this challenge are the logistical difficulties related to the small farms agricultural model, where waste collection and recycling is complex and expensive. The sector is disadvantaged by a low level of research, development and innovation and research investment by the agricultural industry.
Key actions to achieve this challenge include the following: (1) the implementation by regional administration of promised initiatives that facilitate the sustainable use of resources by the agricultural industry; (2) the creation of knowledge and agricultural innovation system that facilitates sustainable strategies among all relevant public and private actors; (3) increased investment in research, development and innovation (R + D + i), through sustainable finance initiatives, venture capital and networks of business angles that invest in finding solutions for the local productive system; and (4) support for existing R + D + i initiatives between the research and business sectors, including but not limited to initiatives between the university and cooperative finance, the Almería SmartAgriHub, various operating groups that have been set up under EIP Agri to specifically focus on water use, plastics, vegetable waste, phytosanitary products, etc.
## Sustainability challenge 5: technology and knowledge transfer
In general, agricultural value chains are being transformed by digital technologies. The Almería agricultural sector is highly dependent on the use of adequate technologies adapted for Mediterranean greenhouse systems, yet it lags behind in adoption. Increasing consolidation and concentration in the agribusiness sector, particularly in the seed, fertiliser, chemical, genetics, and farm machinery industries, has been evident for decades, and it is expected that the new data technologies will drive the trend even more so. The retail sector is concentrated among five or six major actors. As a result, Almería farmers and farming community SMEs risk being squeezed in the middle between digitally savvy and powerful input suppliers and buyers and stuck in a production-oriented paradigm within traditional, sequential supply chains, where intermediaries enjoy the surplus value created. In addition, while agri-food supply chains and value ecosystems are converting into data-rich and data-defined sectors, the value of farmers' data is often unrealized by the farming community, and data ownership and control issues are neglected. Finally, while technologies and digitization may present opportunities to solve a range of agricultural challenges, including increasing sustainability, alternatively, they may create new social problems, inequalities and exclusion, extending the "power of the powerful" and allowing socio-economically advantaged actors, who are well connected and highly skilled, to scale up rapidly, and extend their social, economic, cultural and geographic influence. Successfully adopting appropriate technologies, knowledge systems and organisational approaches to address these factors and generate positive outcomes and mitigate negative impacts will determine whether Almería agriculture will become more sustainable and remain competitive in an increasingly globalized agri-food market.
Beginning with the Almería production process, the workshop experts noted that the introduction of technology in the greenhouse has two basic objectives related to sustainability: (1) to maximize production, improve quality and minimize the use of resources and improve their management (i.e., water, electricity, labor), (2) technologies are utilised for the integration of information from different sources that help the producer to make decisions or to implement decision-support systems in production processes: irrigation, climate, disease management, planning of workers' tasks, etc. Such technologies may also be used to improve and monitor regulatory compliance (e.g., nitrates, pesticides, water use, etc.).
While the greenhouse production system is implicitly connected to technologies, in Almería it may still be considered to be a "mid-tech" system. Marketing cooperatives have more sophisticated technology in place in their handling facilities particularly due to the demands of complying with strict food safety, traceability and certification requirements, and the intricacies of dealing with complex supply chains and exports, not to mention the requirements of complying with EU regulations pertaining to producer organizations. Yet at the greenhouse level, most farms are either low-tech or mid-tech. Less than 10% of farms utilise basic sensors which could contribute to increased efficiency in water use or control of climate conditions.
The results of the workshop indicated that the adoption of technology was seen to be most related to increasing economic benefits for the farmers, their cooperatives and also the surrounding auxiliary businesses, but such adoption was also seen to have an important role in making Almería agriculture more sustainable from an environmental point of view, with possibilities for social benefits as well. In the case of Almería, barriers to successful transformation include: (1) technologies are still in their early stages and untested so that applying them to the cooperatives' business ecosystem remains complex. Of particular concern is the risk and uncertainty of adopting and implementing technologies and whether they will prove beneficial from a cost/benefit perspective. Also, there has been an explosion of technical and digital "solutions" and guidance is lacking as to credible and tested devices and systems; (2) the lack of data standards and interoperability between devices and systems;
(3) people play a central role in digital technology and must adapt to the new working methods and skills required, particularly through the implementation of appropriate knowledge acquisition; (4) the laws and regulations applicable to data from the agricultural industry is a source of concern as it is unclear, insufficient and inconsistent; and (5) given its small-holding, family farming organizational structure, doubts arise as to whether the necessary investments may be made by such fragmented enterprises. With this in mind, experts found that technology adoption and digital transformation represented a significant challenge for farmers and cooperatives, but also a real opportunity to create value. Opportunities were found in the fact that technologies already exist which could be adopted and that producers had clear necessities to do so. Financing is available to a certain extent and examples of successful technology adoption and digitization already exist. Also, there has been an increase in public data made available (for example, climate and market data), thus providing the groundwork for innovations and allowing more effective solutions to be created.
In order to realise these opportunities, certain actions are required that mainly center around transfer of knowledge and technology, as well as training. Entities which can facilitate uptake and transformation in Almería include research and experimental centers, technology companies, cooperatives and the association of producer organisations (COEXPHAL) and a public administration concerned with knowledge transfer and uptake of technology and digitisation. Financing for such initiatives was also was seen to be available. Barriers to taking advantage of opportunities included the insufficient communication between business, university and producers, as well as the lack of technology for certain important agricultural tasks. The workshop participants also proposed that enterprises that generate and analyse data should come to an agreement on and elaborate data standards to exchange data and achieve interoperability. As a result, it was determined that Almería institutions such as research centers, technology companies, cooperatives and associations of producer organisations, as well as the administration, should collaborate and foster coordination in areas of research, whilst striving to improve transfer activities and create data standards. While technological innovation and the advancement of technology were seen to be important, the corresponding social innovation was seen to be equally important. Social innovation, which is closely aligned with processes of uptake of technologies, implies changes of attitudes, behaviours or perceptions within a network of people with "aligned interests that lead to new and improved ways of collaborative action within the group and beyond". Hence, technical and social innovation are closely tied to the socio-economic and ecosystem impact of technology advances in Almería. An organisational structure like that of cooperatives may prove to be a method to effectively and efficiently serve the farming community in an equitable and inclusive manner, and as an instrument to counteract the negative social and economic impacts of technology and digitisation of agriculture. Priorities include closing the "digital divide" and addressing the "rural penalty" in ICT where coverage and broadband are often sub-optimum, de-risking adoption and experimentation through cooperative testing facilities or university research centers. Almeria's cooperatives may support and provide a range of activities, from enhancing skills, encouraging adoption through a genuinely open approach to farmer needs and values, co-creation, and creating access through networks and platforms. Data generated by agricultural activities may be collected and analysed to provide valuable information to reduce environmental impacts. As part of the transformation, cooperatives may need to change their business models or create new cooperatives such as a data cooperative, to create economies of scale, and to function as coordination mechanisms. Their strategy should incorporate the opportunities for more sustainable practices that new tools have to offer.
## Sustainability challenge 6: image and identity
The challenge of improving the image and identity of Almeria's agricultural sector is structured into three components, (1) the improvement of rural hygiene in the surroundings of the greenhouse area, (2) promoting socially just and fair labor conditions, and (3) the need to publicize and inform the international community of the commitment of the agricultural sector to move forward in a sustainable model of agriculture where the economy operates within the limits of a sustainable environmental system.
Almeria's agricultural sector has shown in recent decades a great strength becoming an international recognised exporter of horticultural products. However, the urgent need to improve the visual impact and pollution produced by greenhouse horticulture is today a pending and urgent challenge. Examples of these impacts are, for example, the disturbing image of ephemeral streams (i.e., ramblas in Spanish) overflowing with the tide of garbage due to a deficient plan of rural hygiene, or the need to implement a management model for organic waste (e.g., plants) and inorganic waste (e.g., plastics). Recent research showed that the deterioration of the image has had an impact on the agricultural sector, including a reduction of demands among European consumers, ultimately leading to a decrease in imported goods, and affecting international consumers' perception, which are today increasingly inclined to make informed purchasing decisions concerning horticultural products. In addition, these findings indicate that European destination markets of the Spanish greenhouse horticulture could have a preconceived image; however, nationally the production system does not seem to influence the image of the same.
Another important issue related to the image of the agricultural sector is the need for fair labor conditions. Since conflicts concerning immigrants which occurred in 2000 in El Ejido, Almeria's image deteriorated and was associated by national, and especially international, media with poor treatment and living conditions of migrant workers, therefore turning it into a subject-matter of debate due to work tensions, ethnicity and gender. The rapid development of the agricultural sector favours the flow of migrants attracted by the need for labour. In this way, the so-called "Almeria miracle" is necessarily associated with important demographic and labor market transformations in the provinceand is based on the wage-earning work of immigrant workers. The demands of the Almeria agricultural labour market have mainly targeted and attracted migrants from Morocco and several West African countries whose living conditions have been defined by different authors as poor and precarious, with processes of labour segmentation, residential segregation, substandard housingand the existence of scattered settlements. Thus, social sustainability is questioneddespite the importance of family farms.
Finally, the image of the sector has also been affected by the negative impact of campaigns and news in both the national and international media on the Almería greenhouse model, mainly due to the sector's lack of capacity to address existing problems. However, this research indicated that some of the crises in the past were the result of unfounded accusations. All these challenges have burdened the image of Almeria's agriculture internationally without a proactive attitude to solve it by the agricultural sector and public administrations. The public administrations have developed integration programmes, such as the Integral Plan for Immigration of the Andalusian regional government, Junta de Andalucía, implemented by local administrations, and have granted access to education and health public services, despite the legal situation of immigrants. However, the natives' attitudes and discourses, the media and the emergence of a xenophobic political party in the province constitute barriers that diminishes the capacity of policies, responses or actions aimed to integrate at the full inclusion of the migrant collective, especially in sensitive issues as housing. The prevailing view in the agricultural sector, that many of those problems are not their responsibility or that they are too complex (both views were exposed during the workshop), has for long time hampered a deeper engagement to face this situation. Nevertheless, the growing importance of corporate social responsibility and the beginning of a demand by international distribution chains for controls on the living conditions of workers seems to be pushing a greater business awareness of these issues. Generalising and increasing the criteria of the social practice modules of certifications such as GRASPprovide an opportunity to improve working conditions and the image of the sector. Since its image is severely affected by these social issues, and since agriculture gives employment to more than half of legal foreign workers in the province and that they are the majority of their salaried workers, the sector should make an additional effort and play a more prominent role in encouraging or joining initiatives to promote social inclusion to clearly show its commitment to change.
Among the opportunities and barriers to address the above issues, workshop participants identified (1) the implementation of sanctions and creating the obligation for agrochemical companies to collect waste from their products and to pay for the packaging and recycling of each product, which will imply overcoming barriers related to costs and the need to convince private landowners; (2) improving the working and social conditions of immigrant workers by the eradication of shantytowns and the promotion of settlements with decent housing alternatives for seasonal workers, and the elaboration of an integration plan for immigrants, which implies knowledge of the staffing needs at each stage of the agricultural campaign; and (3) improvement of the national and international image through new awareness campaigns financed with private funds of the agricultural sector, which would imply a change of mentality in the agricultural sector on the need to recognize the consequences of this issue and the need to invest in initiatives to change it. Finally, the actions identified by workshop participants included (1) new regulations on greenhouse structures and installations, the use of plant screens in greenhouses and awareness-raising campaigns; (2) a closer collaboration with local administrations and entities concerned with the plight of immigrants that have a long experience of intervention and social integration; and (3) new local strategies, funded privately, to improve the image of the sector nationally and internationally. Identified actors who can play a pivotal role included the interprofessional entity HortyEspaña, the association of producer organisations, Coexphal, farm worker unions, and local and national authorities (given immigration is a national competence). Individual cooperatives also have a role to play, wherein leading and successful cooperatives do provide dignified housing and can serve as an example for others to follow suit.
# Conclusions
While the transdisciplinary multi-actor research and workshop activities provided a wide range of challenges and proposed solutions, as described above, this research paper concludes with two main observations, having to do with both methodology and strategy. The multi-actor transdisciplinary approach implemented herein facilitated the collective work among scientists and nonscientists to progress in finding solutions to sustainability issues of Almería greenhouse horticulture and to find pathways for the transition to an agro-ecological model, creating a culture of shared responsibility among public, private, academic, and civil society actors. This study should be considered as a starting point to encourage iterative, open and ongoing processes which are embedded within institutional settings and that evolve and change over time by strengthening collaboration and building trust and common understanding among science, policy and society. Whereas it is assumed that the study provided insights arising from the particular case chosen as an example, it is widely recognized that such insights provide a useful contextual orientation to design further transdisciplinary experiences in other contexts and sectors.
The six challenges identified herein as fundamental to transitioning into an agricultural model that ameliorates risks and avoids a systemic collapse balance a concern for profitability with sustainability and provides crucial insights for the small family farming business model. Originally organised around cooperatives which were founded to create economies of scale and avoid "market failure", Almería small farmers and their producer organisations have been attempting to survive and extract value according to the rules of the competitive, global agro-industrial business model, resulting in the aforementioned socio-ecological vicious circle based on an extractive logic. However, given decreasing benefits and increasing global competition which often depends on delocalized capital and production, the sector finds itself in crisis. Many of the challenges and opportunities identified herein are closely linked with finding added value in sustainability rather than in the standard market logic of efficiency (i.e., decreased inputs, increased outputs). The workshop represents a nascent awareness, notwithstanding the presence of plural values, of a new strategic business perspective: the necessity for the transition to an agro-ecological model in order to remain competitive and create value. Whereas as the agro-industrial model relies on the "corporate social responsibility" perspective to address environmental or social issues, the Almería model, embedded as it is within its territory and social environment (already largely based on the social economy of cooperative institutions), has the opportunity to realise a transition to a business model based on sustainability. However, this opportunity includes overcoming significant challenges, and the transition will by no means be an easy feat. Any solution will require collective action and significant innovation. |
Facet joint hypertrophy is a misnomer
One of the major causes of lumbar spinal canal stenosis (LSCS) has been considered facet joint hypertrophy (FJH). However, a previous study asserted that "FJH" is a misnomer because common facet joints are no smaller than degenerative facet joints; however, this hypothesis has not been effectively demonstrated. Therefore, in order to verify that FJH is a misnomer in patients with LSCS, we devised new morphological parameters that we called facet joint thickness (FJT) and facet joint cross-sectional area (FJA).We collected FJT and FJA data from 114 patients with LSCS. A total of 86 control subjects underwent lumbar magnetic resonance imaging (MRI) as part of routine medical examinations, and axial T2-weighted MRI images were obtained from all participants. We measured FJT by drawing a line along the facet area and then measuring the narrowest point at L4-L5. We measured FJA as the whole cross-sectional area of the facet joint at the stenotic L4-L5 level.The average FJT was 1.60 ± 0.36 mm in the control group and 1.11 ± 0.32 mm in the LSCS group. The average FJA was 14.46 ± 5.17 mm 2 in the control group and 9.31 ± 3.47 mm 2 in the LSCS group. Patients with LSCS had significantly lower FJTs (P < .001) and FJAs (P < .001).FJH, a misnomer, should be renamed facet joint area narrowing. Using this terminology would eliminate confusion in descriptions of the facet joint.Abbreviations: FJA = facet joint cross-sectional area, FJH = facet joint hypertrophy, FJT = facet joint thickness, LSCS = lumbar spinal canal stenosis, MRI = magnetic resonance imaging.
# Introduction
Lumbar spinal canal stenosis (LSCS) results from degenerative changes in the spinal canal and is one of the most common spinal disorders in elderly individuals. [bib_ref] Spinal stenosis prevalence and association with symptoms: the Framingham Study, Kalichman [/bib_ref] [bib_ref] Diagnosis and management of lumbar spinal stenosis, Haig [/bib_ref] [bib_ref] Lumbar spinal stenosis: therapeutic options review, Costandi [/bib_ref] It is characterized by narrowing of the spinal canal and is caused by hypertrophy of the ligamentum flavum, mechanical compression of the lumbar spinal nerve roots, and disc herniation combined with osteophytes. [bib_ref] Measuring spinal canal size in lumbar spinal stenosis: description of method and..., Hughes [/bib_ref] [bib_ref] Characterization of symptomatic lumbar foraminal stenosis by conventional imaging, Ohba [/bib_ref] Facet joint hypertrophy (FJH) is considered another major cause of LSCS. [bib_ref] Facet joint hypertrophy: the cross-sectional area of the superior articular process of..., Barry [/bib_ref] The facet joints play an important role in maintaining the stability of the spinal column. [bib_ref] Biomechanical response of lumbar facet joints under follower preload: a finite element..., Du [/bib_ref] Furthermore, changes in the mechanical facet joint environment have been associated with degeneration and osteoarthritis, either of which could eventually lead to LSCS. [bib_ref] Biomechanical response of lumbar facet joints under follower preload: a finite element..., Du [/bib_ref] The spinal canal can be narrowed by characteristic changes in the facet joints such as hypertrophy of articular processes, synovial cysts, or osteoarthritis. [bib_ref] The prevalence of lumbar spine facet joint osteoarthritis and its association with..., Ko [/bib_ref] [bib_ref] Epiduroscopic removal of a lumbar facet joint cyst, Jin [/bib_ref] However, Barry and Livesley [bib_ref] Facet joint hypertrophy: the cross-sectional area of the superior articular process of..., Barry [/bib_ref] asserted that "FJH" is a misnomer because normal facet joints are no smaller than degenerative facet joints. Their assertion has been hypothesized but has not been effectively demonstrated. Therefore, in order to verify that facet join hypertrophy is a misnomer in LSCS patients, we devised 2 new morphological parameters, facet joint thickness (FJT) and facet joint area (FJA). FJT and FJA have not yet been evaluated for their associations with LSCS. We hypothesized that both would be important morphologic parameters for identifying facet joints.
# Materials and methods
## Patients
The Catholic Kwandong University College of Medicine, Republic of Korea, Institutional Review Board (IRB) reviewed and approved the research project (IRB protocol number: IS17RISI0032). We retrospectively reviewed patients who had visited our pain clinic between March 2014 and June 2017 and had been diagnosed with LSCS. We included patients over age 60 if they had clinical manifestations compatible with LSCS (such as low back pain and/or neurogenic intermittent claudication), the most stenosis at L4-L5, and MRI performed within 12 months of the diagnosis that was available for review. We excluded patients if they had a history of previous lumbar surgery or spinal injury, congenital spine defects, history of spinal interventions such as kyphoplasty, or any anatomic anomalies.
We enrolled a total of 114 patients after the LSCS diagnosis was confirmed by 2 experienced, board-certified neuroradiolo-gists. The measurement analysis and data collection was performed in a double-blind fashion. In the LSCS group, there were 28 (24.56%) males and 86 (75.44%) females with a mean age of 68.15 ± 5.66 years (range: 60-87 years; [fig_ref] Table 1: Comparison of the characteristics of control and LSCS groups [/fig_ref]. To compare the FJAs and FJTs between patients with and without LSCS, we enrolled a group of control patients who had undergone lumbar MRI as part of routine medical examinations and who had no LSCS-related symptoms. The control group consisted of 86 participants [31 males (36.05%) and 55 females (63.95%)] with a mean age of 69.51 ± 7.72 years (range: 60-89 years; [fig_ref] Table 1: Comparison of the characteristics of control and LSCS groups [/fig_ref]. We also examined the FJAs and FJTs in the control group at the L4-L5 facet joint level.
## Imaging parameters
The MRI examinations had been performed with 3T scanners (Magnetom Skyra, Sonata, Biograph, Avanto, Siemens Healthcare, and Philips Ingenia [R4], Philips Medical Systems, Best, The Netherlands), and axial T2-weighted images with 4 mm thick slices had been obtained. The following other parameters were used as well: 0.4 mm intersection gap, 3000 ms/90 ms repetition time/echo time, 180 Â 180 cm field of view, 448 Â 270 matrix, and 15 echo train length (ETL). Sagittal T2-weighted images with 4 mm slice thickness were obtained. The following other parameters were used: 0.4 mm intersection gap, 2700 ms/95 ms repetition time)/echo time, 300 Â 300 cm field of view, 358 Â 512 matrix, and 15 ETL.
# Image analysis
The axial T2-weighted MR images had been acquired at the facet joint level for individual patients. We used a picture archiving and communications system to measure the FJAs and FJTs at the L4-L5 facet joints on MRI. We measured the FJA as the crosssectional area by outlining the facet joint at L4-L5 [fig_ref] Figure 1: Measurement of the facet joint area on MRI at the L4-L5 level [/fig_ref] and the FJT by drawing a line along the joint and then measuring the narrowest point at L4-L5 [fig_ref] Figure 2: Measurement of the facet joint thickness on MRI at the L4-L5 level [/fig_ref].
# Statistical analysis
We analyzed the data as means ± standard deviations (SD), and we used unpaired t tests to compare the FJTs and FJAs between the control and LSCS groups; we set significance at P < .05. We also analyzed the relationships between the FJT, the FJA, and agerelated changes using 1-way analysis of variance (ANOVA). We performed all statistical analyses with SPSS for Windows version 21 (IBM SPSS, IBM Corp., Armonk, NY).
# Results
The demographic data were not significantly different between the groups [fig_ref] Table 1: Comparison of the characteristics of control and LSCS groups [/fig_ref]. The average FJTs were 1.60 ± 0.36 mm in the control group and 1.11 ± 0.32 mm in the LSCS group, and the average FJAs were 14.46 ± 5.17 mm 2 in the control group and 9.31 ± 3.47 mm 2 in the LSCS group. The patients with LSCS had significantly lower FJTs (P < .001) and narrower FJAs (P < .001; see [fig_ref] Table 1: Comparison of the characteristics of control and LSCS groups [/fig_ref]. The mean FJTs and FJAs in the control group were 1.66 ± 0.37 mm and 13.33 ± 5.39 mm 2 in subjects aged 60 to 69 years, 1.48 ± 0.29 mm and 15.78 ± 3.44 mm 2 in those aged 70 to 79 years, and 1.73 ± 0.49 mm and 16.34 ± 7.77 mm 2 in those aged 80 to 89 years [fig_ref] Table 2: Age distribution of patients with mean FJT and FJA in the control... [/fig_ref]. In the control group, we found no statistically significant relationships between the FJT (F = 2.908; df = 2; P = .060), the FJA (F = 2.777; df = 2; P = .068), and agerelated changes on 1-way ANOVA. The mean FJTs and FJAs in the LSCS group were 1.13 ± 0.35 mm and 8.97 ± 3.61 mm 2 in those aged 60 to 69 years, 1.12 ± 0.24 mm and 9.91 ± 2.96 mm 2 in those aged 70 to 79 years, and 0.86 ± 0.26 mm and 10.72 ± 3.89 mm 2 in those aged 80 to 89 years [fig_ref] Table 3: Age distribution of patients with mean FJT and FJA in the LSCS... [/fig_ref]. In the LSCS group, no statistically significant relationships were evident between the FJT (F = 1.967; df = 2; P = .145), the FJA (F = 1.338; df = 2; P = .266), and age-related changes.
# Discussion
LSCS is a common pathologic condition in elderly individuals that causes intermittent neurogenic claudication and low back or buttock pain [bib_ref] Lumbar spinal stenosis, Genevay [/bib_ref] ; it results from a combination of pathogenic factors, including hypertrophy of the ligamentum flavum, a decrease in the area of the cauda equina, loss of intervertebral disk height, and hypertrophy of the facet joints. [bib_ref] Clinical correlation of a new practical MRI method for assessing central lumbar..., Park [/bib_ref] In addition, overgrowth of the facet joint capsule can lead to LSCS. [bib_ref] Characterization of symptomatic lumbar foraminal stenosis by conventional imaging, Ohba [/bib_ref] [bib_ref] Minimally invasive technique for decompression of lumbar foraminal stenosis using a spinal..., Yoshimoto [/bib_ref] Therefore, FJH has been considered a major cause in the development of LSCS. Degenerative changes in facet joints also include subchondral sclerosis, osteophytosis, joint surface irregularity, and apophyseal hypertrophy. [bib_ref] Grading osteoarthritic changes of the zygapophyseal joints from radiographs: a reliability study, Little [/bib_ref] [bib_ref] Radiographic indices for osteoarthritis, Lane [/bib_ref] [bib_ref] Atlas of individual radiographic features in osteoarthritis, Altman [/bib_ref] Many previous studies have investigated facet joints. Little et al [bib_ref] Grading osteoarthritic changes of the zygapophyseal joints from radiographs: a reliability study, Little [/bib_ref] investigated the reliability of a 5-point scale that grades the severity of degenerative facet joint changes: Grade 0 = absence of joint degeneration at the center of the radiograph, I = questionable osteophytes on the superior joint margin, II = subchondral sclerosis and definite joint osteophytes, III: subchondral sclerosis, some joint irregularity, and moderate osteophytes, and IV = severe sclerosis, irregularity of the articular joint surfaces, and many osteophytes. The authors asserted that this grading system may be useful for assessing facet joint osteoarthritis. [bib_ref] Grading osteoarthritic changes of the zygapophyseal joints from radiographs: a reliability study, Little [/bib_ref] Takashima et al [bib_ref] Investigation of intervertebral disc and facet joint in lumbar spondylolisthesis using T2..., Takashima [/bib_ref] demonstrated that facet joints are important for the segmentation and stability of the lumbar spinal column and that they possess articular cartilage. Therefore, osteoarthritis occurs in facet joints as it does in other synovial joints. Bajek et al [bib_ref] Histomorphological analysis of the osteophytic appositions in patients with lumbar lateral recess..., Bajek [/bib_ref] explained that osteophyte formation in the lumbar spine is an attempt to stabilize an unstable segment; this mechanism ultimately leads to FJH. Disc degeneration may also increase the stressful force on the facet joints. [bib_ref] The significance of facet joint cross-sectional area on magnetic resonance imaging in..., Chaput [/bib_ref] However, Barry and Livesley [bib_ref] Facet joint hypertrophy: the cross-sectional area of the superior articular process of..., Barry [/bib_ref] reported that "FJH" is a misnomer because normal facet joints are no smaller than degenerate facet joints. These authors also contended that there is no clear definition in the literature regarding lumbar FJH. [bib_ref] Facet joint hypertrophy: the cross-sectional area of the superior articular process of..., Barry [/bib_ref] But this hypothesis has not been confirmed. Therefore, in order to verify that FJH is a misnomer in patients with LSCS, we devised new morphological parameters we called the FJT and FJA. We believe that the FJT and FJA are the precise, objective measurement parameters to correct the mistaken terminology, and our results show that the patients with LSCS had significantly lower FJAs and narrower FJTs than did control subjects. It may be that any degenerative facet joint changes could be termed hypertrophic, but this imprecise term is not supported by the results of this study; in the present study, we measured both FJT and FJA. Although FJT can reflect significant facet joint space narrowing, the shape of the facet joint is not always regular and the direction of the axis of the facet surface cannot be determined. [bib_ref] In vivo topographic analysis of lumbar facet joint space width distribution in..., Simon [/bib_ref] To supplement these measurement errors, we also measured the whole cross-sectional area of the facet joint. Analyzing FJA is beneficial for comparing cartilage degeneration with facet joint structure. [bib_ref] In vivo topographic analysis of lumbar facet joint space width distribution in..., Simon [/bib_ref] Biomechanically, the function of facet joints is to limit and guide movement of that spinal column. [bib_ref] Biomechanical response of lumbar facet joints under follower preload: a finite element..., Du [/bib_ref] Our interpretation of these associations is that facet joint narrowing may be related to extensive loading during motion, which might contribute to facet joint osteoarthritis. [bib_ref] In vivo topographic analysis of lumbar facet joint space width distribution in..., Simon [/bib_ref] [bib_ref] Degenerative changes in the intervertebral discs of the lumbar spine and their..., Vernon-Roberts [/bib_ref] The process of facet joint narrowing begins with stress during lumbar flexion and rotation. These mechanical stressors put force on the facet joints, which leads to a high degree of abrasion, [bib_ref] Disc space narrowing and the lumbar facet joints, Dunlop [/bib_ref] and this etiology may alter the morphologic features of the facet joint area. If this is accurate, what is the way to correct this misnomer? Previously, authors have concluded that osteophytes and hypertrophy of the superior articular process were the main factors of facet joint narrowing. [bib_ref] Optimal cut-off value of the superior articular process area as a morphological..., Lim [/bib_ref] FJH in the LSCS refers to hypertrophy in the superior articular process and may be associated with facet joint narrowing. For simplicity, facet joint changes could be referred to as "facet joint narrowing." Using this terminology, descriptions of facet joints would not be confused with superior articular process hypertrophy.
Farrell et al [bib_ref] The anatomy and morphometry of cervical zygapophyseal joint meniscoids, Farrell [/bib_ref] described the morphological patterns of the zygapophyseal joint. These cross-sectional areas were analyzed from cadaveric hemi-spines. Simon et al [bib_ref] In vivo topographic analysis of lumbar facet joint space width distribution in..., Simon [/bib_ref] described the facet joint space width by measuring the cross-sectional area of the facet joint space using 3D computed tomography.
In this study, we measured the FJT and FJA from MRI images. Although MRI is the most important modality for characterizing LSCS and facet joint lesions, [bib_ref] Investigation of intervertebral disc and facet joint in lumbar spondylolisthesis using T2..., Takashima [/bib_ref] there are no previous reports of an association between LSCS and facet joints as a morphologic parameter on MRI. Therefore, we used MRI to compare the FJTs and FJAs between patients with LCSS and healthy controls; to our knowledge, these measurements have not been previously reported. This study only included individuals > 60 years old because previous studies have demonstrated that articular cartilage thinning, subarticular cortical bone hypertrophy, and narrowing of the facet joint gap are observed age-related changes. [bib_ref] In vivo topographic analysis of lumbar facet joint space width distribution in..., Simon [/bib_ref] This study has some limitations. First, although we measured the FJA and FJT in axial T2 images at the L4-5 facet joint, there may be errors associated with measuring these on MRI because these axial images may not be homogeneous due to differences in the cutting angle of the MRI resulting from individual anatomic variations and technical problems; in addition, the 4.0 mm slice of axial T2-weighted MR image is also thicker than an ideal slice. Second, the small sample sizes in some age groups can lead to less than ideal data analysis. Baseline demographic data of the patient population such as body weight and height vary widely. Third, we measured FJT at the narrowest distances between the inferior and superior facet joint surfaces; therefore, we could not estimate the cartilage widths at individual facet joints using this technique. Fourth, several different parameters are known to effectively discriminate LSCS, such as morphological grading and analysis of cauda equina. [bib_ref] The nerve root sedimentation sign for differential diagnosis of lumbar spinal stenosis:..., Zhang [/bib_ref] [bib_ref] Clinical validity of the nerve root sedimentation sign in patients with suspected..., Barz [/bib_ref] However, this study only investigated lumbar facet joint. Finally, another limitation of this study is its retrospective nature. Prospective researches are needed to validate and repeat our results. Despite these limitations, this is the first objective study to verify that FJH is a misnomer in patients with LSCS, and these results may be valuable information to analyze further exact diagnostic terminology when assessing LSCS.
# Conclusion
Our results demonstrate that FJH is a misnomer, and we suggest that it be renamed facet joint area narrowing. We believe that this renaming will help physicians in their evaluations of patients with LSCS. We also hope that pain physicians will no longer use the term "facet joint hypertrophy."
[fig] Figure 1: Measurement of the facet joint area on MRI at the L4-L5 level. [/fig]
[fig] Figure 2: Measurement of the facet joint thickness on MRI at the L4-L5 level. [/fig]
[table] Table 1: Comparison of the characteristics of control and LSCS groups.Data represent the mean ± standard deviation or the numbers of patients. FJA = facet joint cross-sectional area, FJT = facet joint thickness, LSCS = lumbar spinal canal stenosis, NS = not statistically significant (P > .05). [/table]
[table] Table 2: Age distribution of patients with mean FJT and FJA in the control group.FJT = facet joint thickness, N = number of patients. [/table]
[table] Table 3: Age distribution of patients with mean FJT and FJA in the LSCS group.FJA = facet joint cross-sectional area, FJT = facet joint thickness, LSCS = lumbar spinal canal stenosis, N = number of patients. [/table]
|
Multiple pancreatic metastases from malignant melanoma: Conclusive diagnosis with endoscopic ultrasound-guided fine needle aspiration
Pancreatic metastases are rare, ranging from 2% to 5% of pancreatic malignancies. Differentiating a primary pancreatic malignancy from a metastasis can be diffi cult due to similarities on imaging fi ndings, but is crucial to ensure proper treatment. Although transabdominal ultrasound, computed tomography, and magnetic resonance imaging provide useful images, endoscopic ultrasound (EUS) with fi ne needle aspiration (FNA) is often needed to provide a cytologic diagnosis. Here, we present a unique case of malignant melanoma with pancreatic metastases. It is important for clinicians to recognize the possibility of melanoma metastasizing to the pancreas and the role of EUS with FNA in providing cytological confi rmation.
# Introduction
Isolated pancreatic metastases are rare, accounting for approximately 2% of pancreatic tumors. [bib_ref] Surgical resection of malignant melanoma metastatic to the pancreas: Case series and..., Goyal [/bib_ref] The most common primary sites are renal, lung, breast, and colon cancer, with soft tissue sarcoma and melanoma observed less commonly. [bib_ref] Surgical resection of malignant melanoma metastatic to the pancreas: Case series and..., Goyal [/bib_ref] [bib_ref] Contrast harmonic endoscopic ultrasonography in the characterization of pancreatic metastases (with video), Fusaroli [/bib_ref] Differentiating metastases from a primary pancreatic malignancy, such as a ductal or neuroendocrine tumor, can be challenging. With endoscopic ultrasound, neuroendocrine tumors appear as well-defined masses in the pancreas, [bib_ref] Definitive diagnosis of neuroendocrine tumors using fine-needle aspiration-puncture guided by endoscopic ultrasonography, Gornals [/bib_ref] and can resemble pancreatic metastases. Endoscopic ultrasound (EUS) with fine-needle aspiration (FNA) plays an important role in providing cytological confirmation for diagnosis.
## Case report
A 75-year-old man with a history of metastatic melanoma presented for routine surveillance studies. His history was signifi cant for the diagnosis of malignant melanoma of the right chest with lymphatic spread to the right axilla 1 year prior. He had right mastectomy and sentinel node biopsy at that time, and there was no visceral organ involvement. At the time of our evaluation, he was asymptomatic, and there were no suspicious skin lesions or palpable lymphadenopathy. Vital signs were normal. Complete blood cell count, metabolic panel, and coagulation studies were within normal limits. Positron ENDOSCOPIC ULTRASOUND / APR-JUN 2015 / VOL 4 | ISSUE 2 emission tomography-computed tomography (CT) showed increased metabolic activity in the proximal pancreas, with standardized uptake value of 10.36. Magnetic resonance imaging (MRI) of the abdomen revealed a 1.7 cm focal pancreatic mass and a large left upper retroperitoneal lymph node. Before the patient was referred to us for EUS and possible FNA, there was concern from the patient's oncologist about a new pancreatic neoplasm. It was discussed that management would be dramatically different if this was a new primary pancreatic neoplasm versus a metastatic lesion to the pancreas from his melanoma that would be occurring approximately a year after the initial resection of the index melanoma lesion. EUS revealed several hypoechoic, rounded, well-defi ned masses. The dominant lesion was a 24.6 mm × 21.4 mm mass in the body of the pancreas [ ]. There was also a 6.9 mm × 6.6 mm nodule in the body [ ] and 14.1 mm × 11.7 mm and 10.6 mm × 7.2 mm masses [ ] in the pancreatic head. EUS-guided FNA of the pancreatic head [ ] lesions was performed with a 25-gauge needle. Aspirate smears showed a dispersed population of pleomorphic malignant cells with large hyperchromatic nuclei and prominent nucleoli [ ], consistent with a history of melanoma. The tumor cells were immunoreactive for Sox-10 [ ], confi rming the diagnosis. He subsequently underwent stereotactic gamma knife radiosurgery for a solitary brain metastasis and will be receiving immunotherapy to treat his pancreatic metastases.
# Discussion
Pancreatic tumors are commonly primary in origin (90% ductal and 5% neuroendocrine). [bib_ref] Unusual, metastatic, or neuroendocrine tumor of the pancreas: A diagnosis with endoscopic..., Eloubeidi [/bib_ref] Isolated pancreatic metastases are rare, accounting for approximately 2% of pancreatic tumors, [bib_ref] Surgical resection of malignant melanoma metastatic to the pancreas: Case series and..., Goyal [/bib_ref] with pancreatic metastases from melanoma being reported in 50% of cases of [1] The most common primary sites are renal, lung, breast, and colon cancer, with soft tissue sarcoma and melanoma observed less commonly. [bib_ref] Surgical resection of malignant melanoma metastatic to the pancreas: Case series and..., Goyal [/bib_ref] [bib_ref] Contrast harmonic endoscopic ultrasonography in the characterization of pancreatic metastases (with video), Fusaroli [/bib_ref] While patients with pancreatic metastases can present with jaundice, abdominal pain, and weight loss, [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] 50-83% have no organ-specifi c complaints when metastases are incidentally discovered on imaging studies. [bib_ref] Pancreatic parenchymal metastases: Observations on helical CT, Scatarige [/bib_ref] Differentiating pancreatic metastases from a primary pancreatic malignancy can be challenging. Accurate diagnosis is essential for optimal treatment and can infl uence whether surgical or non-operative management is pursued. [bib_ref] EUS-guided FNA of pancreatic metastases: A multicenter experience, Dewi [/bib_ref] Useful diagnostic tools include CT, MRI, and EUS with FNA biopsy, [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] but imaging alone cannot distinguish benign or primary pancreatic tumors from metastatic lesions. [bib_ref] EUS-guided FNA of pancreatic metastases: A multicenter experience, Dewi [/bib_ref] In the case of our patient, EUS showed hypoechoic, rounded, and well-defined masses in the pancreas. From this imaging finding, we were unable to make a conclusive diagnosis of neuroendocrine tumor versus pancreatic metastases, thus prompting use of EUS-FNA to obtain histological confi rmation.
Metastatic involvement of the pancreas may appear as a solitary lesion, which makes differentiation from a single primary tumor difficult. [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] CT imaging with evidence of a pancreatic mass with signifi cant peripheral enhancement of the lesion and a low attenuation on the central area suggests metastasis, [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] but may not be accurate for distinguishing from a neuroendocrine tumor, which is seen as an enhancing or hypervascular lesion on early and late arterial phase images. [bib_ref] Imaging of neuroendocrine tumors, Leung [/bib_ref] On MRI, neuroendocrine tumors generally appear hypointense on T1-weighted sequences and hyperintense on T2-weighted sequences, when compared with the liver parenchyma. [bib_ref] Imaging of neuroendocrine tumors, Leung [/bib_ref] Similarly, pancreatic metastases appear hypointense on T1-weighted images and demonstrate heterogeneous or moderate hyperintense signal on T2-images. [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] Compared with CT and MRI, EUS has the advantage of being able to detect small lesions (as small as 2-3 mm in diameter) within the pancreas and duodenal wall. [bib_ref] Imaging of neuroendocrine tumors, Leung [/bib_ref] Studies have shown that EUS has a sensitivity of 95% and specificity of 92% in the evaluation of solid pancreatic masses, [bib_ref] Unusual, metastatic, or neuroendocrine tumor of the pancreas: A diagnosis with endoscopic..., Eloubeidi [/bib_ref] although malignancy can still be missed, especially in the setting of acute or chronic pancreatitis. [bib_ref] The No Endosonographic Detection of Tumor (NEST) Study: A case series of..., Bhutani [/bib_ref] With endoscopic ultrasound, neuroendocrine tumors typically appear as homogenous, hypoechogenic, hypervascular solid lesions that have well-delimited borders. [bib_ref] Definitive diagnosis of neuroendocrine tumors using fine-needle aspiration-puncture guided by endoscopic ultrasonography, Gornals [/bib_ref] Pancreatic metastases have many similar characteristics. In general, pancreatic metastases on EUS have regular margins and appear as homogenous structures that are hypoechoic compared to the surrounding pancreas. [bib_ref] Contrast harmonic endoscopic ultrasonography in the characterization of pancreatic metastases (with video), Fusaroli [/bib_ref] EUS fi ndings of primary pancreatic cancer and malignancy are similar with regards to consistency, echogenicity, location, and tumor size. [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] [bib_ref] EUS-guided FNA of pancreatic metastases: A multicenter experience, Dewi [/bib_ref] However, in a study of 24 patients with pancreatic metastases and 80 with primary pancreatic malignancy, pancreatic metastases were more likely to have well-defined tumor margins than primary pancreatic cancer (46% vs. 4%). [bib_ref] EUS-guided FNA of pancreatic metastases: A multicenter experience, Dewi [/bib_ref] In previous case reports, demonstrated that pancreatic metastases from metastatic melanoma appear hypoechoic, heterogeneous, lobular, and round. In these cases, conclusive diagnosis was made with immunohistochemical staining. [bib_ref] Endoscopic ultrasound-guided fi ne-needle aspiration of melanoma metastatic to the pancreas: Report..., Dewi [/bib_ref] [bib_ref] Pancreatic metastasis of malignant melanoma diagnosed by EUS-guided fine needle aspiration (EUS-FNA), Minoguchi [/bib_ref] Our case and previous studies indicate that neuroendocrine tumors and pancreatic metastases may be indistinguishable based on initial imaging studies. In such cases, cytology with EUS-guided FNA can be instrumental in establishing a defi nitive diagnosis.
For EUS-guided FNA to be accurate in distinguishing pancreatic metastases from a primary carcinoma, effective sampling and immunocytochemistry are needed. [bib_ref] Metastatic disease to the pancreas: An imaging challenge, Triantopoulou [/bib_ref] [bib_ref] EUS-guided FNA of pancreatic metastases: A multicenter experience, Dewi [/bib_ref] While there are no dedicated published studies on the overall effi cacy of EUS FNA specifi cally for the diagnosis of pancreatic metastases from metastatic melanoma, Atiq et al. have reported a 91.3% diagnostic accuracy for pancreatic metastases, including one case of melanoma. [bib_ref] Role of endoscopic ultrasonography in evaluation of metastatic lesions to the pancreas:..., Atiq [/bib_ref] Sox-10 immunohistochemical staining has been shown to be useful in identifying melanoma metastases [bib_ref] Identifi cation of nodal metastases in melanoma using sox-10, Jennings [/bib_ref] and in our case, was used to establish a defi nitive cytologic diagnosis of malignant melanoma with pancreatic metastases.
Here, we present a unique case of pancreatic metastases from malignant melanoma that was conclusively proven with EUS-FNA. It is important for clinicians to consider a broad differential diagnosis when faced with inconclusive imaging studies of pancreatic tumors. In these cases, EUS with FNA may be useful in providing a defi nitive diagnosis.
[fig] Figure 1, Figure 2: Endoscopic ultrasound images revealing multiple well-demarcated, hypoechoic pancreatic lesions ((a) 24.6 mm × 21.4 mm mass in the body of the pancreas, (b) 6.9 mm × 6.6 mm nodule in the body, and (c) 14.1 mm × 11.7 mm and 10.6 mm × 7.2 mm masses in the pancreatic head) Endoscopic ultrasound-guided fi ne-needle aspiration of pancreatic head lesion [/fig]
[fig] Figure 3, Figure 4: Aspirate smear showing a dispersed population of pleomorphic tumor cells with prominent nucleoli and occasional multinucleation, consistent with metastatic melanoma (Papanicolaou stain) Aspirate tumor cells reactive for Sox-10, supporting a diagnosis of metastatic melanoma (immunohistochemical stain) ENDOSCOPIC ULTRASOUND / APR-JUN 2015 / VOL 4 | ISSUE 2 disseminated disease. [/fig]
|
Liquid crystals for organic thin-film transistors
Supplementary Figure 1 | Polarized optical microscope textures of Ph-BTBT-10 in a liquid crystal cell. (a) SmA and (b) crystal phases at 220 and 26 o C, respectively. White bars indicate a scale of 50μm in length. Supplementary Figure 2 | XRD patterns of Ph-BTBT-10 at several temperatures. In (a) small angel and (b) wide angel regions.
## Pfbt/au
## Supplementary note 1 | phase transtion behavior of ph-btbt-10
The phase transition temperatures were determined by differential scanning calorimetry (DSC:
Shimadzu 60). Their liquid crystal phases were identified by the textures observation under a polarized optical microscope (Nikon OPTIPHOT2-POL and Mettler Toledo FP82HT Hot stage).
Ph-BTBT-10, exhibited two mesophases from 143 to 223 o C as shown in the DSC chart of .
The polarized optical microcopy textures at 150, 220 and 26 o C showed disclination line, fan like patterns, and strip texture with cracks as shown in
## Supplementary note 2 | structure transition of ph-btbt-10 from sme to crystal phases
The liquid crystal and crystal structures of Ph-BTBT-10 are evaluated by X-ray diffraction (XRD) analysis using Rigaku RAD-2X diffractometer with CuKα radiation. The rectangular molecular alignment in SmE phase was very similar to that in crystal phase, and the d-spacing of the molecules within a layer in the wide angles continuously was changed without its abrupt shrinkage in the phase transition from the SmE to crystal phases, while the d-spacing between layers was shortened as shown in and . This result explained why no crake was formed in the polycrystalline thin films of Ph-BTBT-10.
## Supplementary note 3 | molecular alignment of polycrystalline thin films
The molecular alignments of the polycrystalline thin films on a SiO 2 /Si-substrate were evaluated by X-ray diffraction (XRD) analysis, using Rigaku RAD-2X diffractometer with CuKα radiation for out-of-plane measurement and Bruker AXS Co. Ltd. D8 Discover μHR for in-plane measurements.
XRD spectra of out-of-plane and in-plane of the polycrystalline thin films fabricated at 82 o C indicated that molecules are aligned perpendicular on the substrate as shown in .
## Supplementary note 4 | crystal structure of polycrystalline thin film after heating to sme phase
In order to clarify the mono-layer or bi-layer structures in a bi-layered film of Ph-BTBT-10 after heating at the temperature of over 143 o C, which is the phase transition temperature from crystal to SmE phases of Ph-BTBT-10 in heating process, we evaluated the thin films of Ph-BTBT-10 after thermal heating at 160 o C and fast cooling to room temperature by XRD measurement in small angle region, TOF-SIMS, and AFM images. XRD result showed a peak corresponding to mono-layer structure as shown in . Furthermore, TOF-SIMS result showed no structure in the depth profile of sulfur atoms as shown in . This indicates the film had the mono-layer structure as in the case of spin-coated films at a temperature of SmE phase. In fact, the films showed 2.5 nm steps for a mono-layer structure as shown in of the surface profile observed by AFM.
## Supplementary note 5 | contact resistance bottom-gate bottom-contact fet
The HOMO level of Ph-BTBT-10 was estimated to be 5.6eV by cyclic voltammetry which is the same as that of dialkyl-BTBT derivatives. In even bottom-contact FET, the contact resistance with gold electrodes was small, i.e., 3.8kΩcm as shown in , which is much smaller than the contact resistance of dialkyl-BTBT derivatives reported to be 10-100 kΩcm. 1
However, Ph-BTBT-10 FET has high mobility of over 10 cm 2 /Vs and high drain current, 10 -3 A (V DS =-100V, W=500μm) as shown in , and the total resistance of 5kΩcm, which was comparable to the contact resistance of several kΩcm. Because of these, FET showed non-ideal characteristics. In order to improve the FET performance, we adopted the bottom-gate bottom-contact FET with Au electrode treated with pentafluorobenzenethiol (PFBT) in order to improve the contact resistance. PFBT-treated Au electrodes gave a considerably small contact resistance of ca., 150 Ωcm as shown in , while bare Au electrodes gave a contact resistance of 3.8kΩcm, leading to improved FET performance as shown in .
# Supplementary methods
## Synthesis of 2-decyl-7-phenyl-[1]benzothieno[3,2-b][1]benzothiphene
2-decyl-7-phenyl-[1]benzothieno[3,2-b][1]benzothiphene was synthesized by the coupling reaction of 2-decyl-7-iodo-BTBT with either the corresponding boronic acid or triphenylstannane in the presence of palladium catalyst as described below in detail as shown in Supplemetary Scheme 1.
2-decyl-7-iodo-BTBT was prepared by the modified Sandmeier reaction of 7-decyl-BTBT-2-amine, 2 which was prepared by nitration of 2-decyl-BTBT and successive reduction with tin power and hydrochloric acid.
## 2-decyl-7-phenyl-[1]benzothieno[3,2-b][1]benzothiophene (ph-btbt-10) (i)
2M potassium phosphate aqueous solution (0.45ml) and phenyl boronic acid (Tokyokasei, 110mg, 0.9 mmol) was added to 8ml dioxane solution containing 2-decyl-7-iodo-BTBT (228mg, 0.45mmol),
[fig] Figure 3 |Supplementary, Figure 5 |Supplementary, Figure 6 |: XRD patterns of polycrystalline thin film of Ph-BTBT-10. The polycrystalline thin films were fabricated in SmE phase at ca. 80 o C. XRD patterns were measured in (a) out-of-plane and (b) in-plane. Supplementary Figure 4 | Molecular alignment of Ph-BTBT-10 after heating at SmE phase. The polycrystalline thin film of Ph-BTBT-10 after heating at 160 o C in SmE phase. (a) The XRD pattern in small angle, (b) depth profile of sulfur atom observed by TOF-SIMS, and (Contact resistance in a transfer line method for a bottom-gate bottom-contact FET. (a,b) bare gold electrode and (c,d) PFBT-modified Au electrodes. Synthesis of 2-decyl-7-phenyl-[1]benzothieno[3,2-b][1]benzothiphene. [/fig]
|
COVID-19 Knowledge/Practices, Mental Status, and Return-To-Work Concerns Among Healthcare Workers in Huangzhou District, Huanggang City, China
# Introduction
Following the outbreak of novel coronavirus disease in Wuhan, China, in December 2019, COVID-19 spread rapidly around the world and was declared a pandemic by the WHO on March 11, 2020 [bib_ref] Factors associated with mental health outcomes among health care workers exposed to..., Lai [/bib_ref]. Over the ensuing months, COVID-19 caused severe damage to human life and to the global economy. As of, there were 16,550,892 confirmed cases and 4,321,451 deaths. Traditional public health measures achieved good results in China (3), and many provinces were quickly able to contain the number of new confirmed cases. Once the epidemic is under control, economic recovery will be a major agenda, and the resumption of work and production will be a key factor in this process. However, the resumption of work will inevitably increase the risk of infection, with the increased flow of people escalating exposure.
Health care workers (HCWs) have played a key role in the control of COVID-19, and they suffer both tremendous risk of infection and psychological pressure. As of February 11, 2020, front-line medical staff accounted for 3.8% of all confirmed cases, of which 63% were from Wuhan, and more than 70% of the population are estimated to have experienced psychological distress [bib_ref] Psychological impact of healthcare workers in China during COVID-19 pneumonia epidemic: a..., Xiao [/bib_ref] [bib_ref] Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak..., Wu [/bib_ref]. Thus, it is important that HCWs understand and practice preventive measures for COVID-19 and maintain their mental health. With society beginning to resume work and production, throat swab testing of batches of returning workers, and the resumption of medical activities, HCWs are facing uncertain risks, including asymptomatic infected individuals and unknown transmission routes. In this context, we conducted an investigation to learn about HCWs' COVID-19 prevention knowledge and practices, and HCWs' psychological state regarding their return to work. In addition, we investigated their concerns and requirements around returning to normal work practices, with the goals of establishing the problems that HCWs are most concerned about and the conditions that HCWs need, and thus hopefully provide more targeted policies to ensure an orderly return to work.
Traditional Chinese Medicine (TCM) incorporates thousands of years' experience in the prevention and treatment of pandemic and endemic diseases. In China, over 85% of patients with COVID-19 have received TCM treatment on top of conventional therapies [bib_ref] Traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus..., Yang [/bib_ref]. This combination of TCM and Western medicine (WM) has been established to be superior to WM alone in improving symptoms, shortening the course of disease and the length of hospital stay, reducing moderate-severe conversion rate, and attenuating side effects associated with conventional therapeutics [bib_ref] Clinical retrospective study on the efficacy of Qingfei Paidu decoction combined with..., Xin [/bib_ref]. TCM prevention and treatment remedies have been included in China's official diagnosis and treatment guidelines for COVID-19 from the third to the eighth version. Based on historical records and direct evidence of SARS and H1N1 influenza prevention, TCM could be an alternative approach for the prevention of COVID-19 in highrisk populations [bib_ref] Can chinese medicine be used for prevention of corona virus disease 2019..., Luo [/bib_ref]. While we were carrying out this study, the COVID-19 vaccine was being developed, and had not yet been applied. In addition to routine prevention methods, such as mask protection and traditional disinfection, TCM has been demonstrated to be one of the most unique and promising prevention methods in China. Hence, we also investigated the attitudes of HCWs to the prevention and treatment of COVID-19 with TCM, and their demand for and preferred form of COVID-19 related TCM prevention products. After determining these needs, we will explore the feasibility of promoting Chinese medicine as a preventive measure for COVID-19. The overall aim of our study is to understand the readiness of HCWs for future resumption of work and production, and to provide a reference for formulating prevention strategies to apply once work and production have resumed.
# Methods
## Study design and population
This study was conducted using a quantitative cross-sectional online survey research design. Purposive sampling was used to recruit HCWs working in public hospitals in Huangzhou District, Huanggang City, Hubei Province, China. Huanggang is a prefecture-level city located in the eastern part of Hubei Province, adjacent to Wuhan, which was one of the cities hardest hit by COVID-19 [fig_ref] FIGURE 1 |: Geographical location of Huanggang, where the respondents were located [/fig_ref]. Huangzhou District is the political and economic center of Huanggang City. The 13 public hospitals under Huanggang City jurisdiction (acting in consortium) played a major role in the fight against the epidemic. On March 30, 2020, the Huanggang municipal government issued a notice of resumption of work and production, and HCWs on secondment started returning to their units to resume normal medical work. Over the following 2 months, each unit in the area arranged for their HCWs to gradually return to work after undergoing a throat swab test (according to unit conditions). At this point, cluster sampling was used to recruit all HCWs in all public hospitals to avoid sampling errors.
## Questionnaire design
The questionnaire consisted of five main components: demographic characteristics; knowledge and practice of preventative COVID-19 measures; concerns about resuming work; anxiety symptoms; and trust of and demand for TCM-based COVID-19 prevention.
Baseline demographic characteristics such as gender, age, education, marital status, occupation, and work unit were recorded. A self-made questionnaire was used to assess participants' knowledge and practice of preventative COVID-19 measures. Concerning the knowledge part, respondents were asked about their familiarity with preventative measures, and responses were recorded on a scale ranging from 1 ("very familiar") to 5 ("unfamiliar"). Using the question "What do you know about basic preventive measures against COVID-19?, " participants were provided with 10 options to assess their knowledge of preventive measures. To assess preventative practices, respondents were asked "Do you practice these preventive measures in daily life?, " and responses were recorded on a scale ranging from 1 ("absolutely practice") to 5 ("Practice none at all").
For the question assessing whether participants were concerned about resuming work, participants could answer "Yes" or "No." "Yes" respondents could subsequently choose possible reasons from a list of options provided. Anxiety was assessed using the Self-Rating Anxiety Scale (SAS) [bib_ref] Rating instrument for anxiety disorders, Zung [/bib_ref] [bib_ref] Dimensionality of somatic complaints: factor structure and psychometric properties of the self-rating..., Olatunji [/bib_ref] , which is a validated screening instrument for use among adults. The SAS is a self-reported 20-item scale that asks participants to rate their level of distress for each component during the past seven days. Scores are calculated by summing the scores for each item and multiplying by 1.25 to get the final score. The scores are graded as follows to assess the severity of psychological impact: normal (<50); mild (50-59); moderate (60-69); and severe (>70).
In the final component, the questions were designed to assess participants' trust in using TCM against COVID-19, whether they believed TCM preventative methods are needed to deal with the risks of resuming work, what types of methods they prefer, and what information about TCM preventative measures they would like to receive. TCM constitution, guided by TCM theories, classifies humans into nine constitution types according to physiological and psychological features and adaptive capacity, i.e., balanced constitution, qi-deficiency constitution, yangdeficiency constitution, yin-deficiency constitution, phlegmdampness constitution, damp-heat constitution, blood stasis constitution, qi stagnation constitution, and an inherited special constitution. The TCMC results were obtained using the adult edition of the Rating Scale for 9 TCM Constitutions which has 60 evaluation items for a self-rating scale (14, 15) (Additional File 3).
## Data collection
From April 23 to May 14, 2020, we distributed the electronic questionnaires to all 13 public hospitals in Huangzhou District, Huanggang City, including one tertiary hospital, five secondary hospitals, and seven primary hospitals. Huangzhou District People's Hospital had the additional responsibility of providing throat swab tests to people returning to work in Huanggang (providing more than 800 examinations a day, and reaching more than 1,200 during peak periods). Because of this added burden, Huangzhou District People's Hospital was considered separately as a high-risk hospital. HCWs in all hospitals were grouped into occupational hierarchies based on the risk of COVID-19. The two main groups were medical staff (doctors and nurses) and administrative staff (including logistics workers). Complete questionnaires concluded from April 23 to May 14, 2020 were considered qualified and were included in the analysis. Incomplete questionnaires and participants who had been quarantined due to diagnosed COVID-19 or a positive swab test were excluded. All respondents provided informed consent.
Statistical Analysis SPSS 21.0 was used for data analysis (SPSS, Inc., Chicago, IL, USA). Significance was set at α = 0.05, and all tests were two-tailed. Qualitative variables are described by frequency distribution. A two-tailed chi-squared test was used to compare the distribution of nominal data. The non-parametric Mann-Whitney U-test and Kruskal-Wallis test were used to compare ordinal data between two or more groups. We analyzed the relationships between participants' COVID-19 knowledge, their symptoms of anxiety and worry, and their characteristics using log-binomial models to estimate relative risk (RR) and corresponding 95% confidence intervals (CIs). The response variables were all binary categorical variables. A five-level Likert scale questionnaire (good to bad) was used to subjectively evaluate participants' knowledge and practice of COVID-19 preventive measures. The responses were then classified as "Good knowledge" or "Common knowledge" using the option settings and the capacity of the results for division. Anxiety and worry were also transformed into binary variables, with 1 denoting yes and 0 denoting no. The lower value binary variable was chosen as the reference category.
# Results
## Demographic characteristics
For this cross-sectional survey, we retrieved a total of 2,079 questionnaires from 13 hospitals. After 29 questionnaires were excluded as incomplete, 2,050 questionnaires were used in our analyses, giving an overall response rate of 94.6%. Most participants were female (1557, 76.0%) in the 31-40 years old age range. Most had completed junior college or above (1758, 85.8%), and 68.6% were unmarried. A total of 550 participants worked at Huangzhou District General Hospital (high risk of infection), and 1,500 worked at the other hospitals (lower risk of infection). The majority were medical staff (1,941, 94.7%), while 109 were administrative staff (5.3%). Secondary hospitals accounted for the highest proportion of participants (1,095, 53.4%), followed by tertiary (602, 29.4%), and primary hospitals (353, 17.2%) [fig_ref] TABLE 1 |: Baseline characteristics of study participants [/fig_ref].
## Knowledge and practice of preventative covid-19 measures
Overall, 47.9% of the participants chose "very familiar" and 46.6% chose "relatively familiar." Since the capacities of the next three levels were small (5.3, 0.2, and 0%), the lower three levels were merged with Level 2 into one category, yielding a "Good knowledge" category (Level 1) and a "Common knowledge" category (Levels 2-5). On chi-squared tests, the variables significantly related with knowledge levels were gender (χ 2 = 20.797, P < 0.001), job category (χ 2 = 14.255, P < 0.001), hospital grade (χ 2 = 13.598, P = 0.001), and education level (χ 2 = 33.244, P = < 0.001). Age, different risk levels of hospital, and marital status were not statistically significantly related with self-reported knowledge level [fig_ref] TABLE 2 |: Association between HCWs' COVID-19 knowledge a and their characteristics [/fig_ref].
With knowledge status as the dependent variable (good knowledge = 1, common knowledge = 0), a log-binomial model and multivariable regressions were performed to identify the main factors affecting HCWs' knowledge. In the adjusted analyses, tertiary hospital (RR: 1.216; 95% CI: 1.043-1.416; P = 0.012), male gender (RR: 1.242; 95% CI: 1.128-1.368; P < 0.001), medical staff (RR: 1.568; 95% CI: 1.173-2.095; P = 0.002), and undergraduate/postgraduate qualification were associated with good knowledge [fig_ref] TABLE 3 |: Association between participant characteristics and COVID-19 prevention knowledge [/fig_ref].
Among the preventive measures, wearing masks was known to everyone, followed by washing hands frequently (2033, 99.2%), and reducing the numbers of people gathering (1958, 95.5%) (Additional File 2; Supplementary [fig_ref] TABLE 1 |: Baseline characteristics of study participants [/fig_ref]. Participants' information sources were mainly official channels such as TV news, official online platforms, and government notices (97.2%) (Additional File 2; Supplementary [fig_ref] TABLE 2 |: Association between HCWs' COVID-19 knowledge a and their characteristics [/fig_ref].
Regarding practices, the survey assessed the degree of implementation of preventive measures in daily life. Overall, 46.5% chose "absolutely practice, " and 49.3% chose "practice more" (the other three levels accounted for 3.8, 0.1, and 0.3%). Using the analogy knowledge classification method, we divided practice into two categories (combining the last three levels into Level 2). We then conducted a chi-square test between knowledge and practice. The results reveal that the higher the HCWs' knowledge of COVID-19 prevention, the more measures they practiced (OR: 3.277; 95% CI: 2.734-3.928; P < 0.01).
## Psychological status concerning resuming work and the influencing factors
Overall, 59.8% (1,226) of participants reported worries about resuming work. Asymptomatic infection was the main cause of concern (89.2%, 1,093), followed closely by gathering of. Compared to their own concerns about resuming work, HCWs were more worried about their children returning to school (χ 2 = 115.647, P < 0.01). In addition, we used the SAS scale to investigate HCWs' anxiety states, 10.8% of HCWs had mild anxiety, 1.5% moderate anxiety, and 0.1% severe anxiety. Next, we analyzed the factors that might have influenced worry and anxiety among the participants [fig_ref] TABLE 5 |: Multivariable analysis of anxiety and worry based on different characteristics [/fig_ref]. The adjusted results indicate that females were more likely to have anxiety and worry. Marital status was also an influencing factor on mental status. Compared with divorced/widowed HCWs, single HCWs (RR = 0.573, 95% CI: 0.388-0.845) had a lower risk of worry. As a designated throat swab screening hospital for returning to work, the People's Hospital of Huangzhou District bears a higher infection risk. This factor affected two psychological characteristics of the participants. As shown in [fig_ref] FIGURE 2 |: Comparison of psychological features between two groups [/fig_ref] and [fig_ref] TABLE 6 |: Different severity levels of anxiety and worry among the participants [/fig_ref] , HCWs working at a high-risk hospital (Huangzhou District People's Hospital) had higher anxiety levels than HCWs working at low-risk hospitals (16.1 vs. 10.0%, respectively). Among HCWs at the high-risk hospital, 13.5% had mild anxiety, and 2.5% moderate to severe anxiety; the corresponding proportions were 9.8 and 1.2%, respectively, for those at low-risk hospitals. The differences in the severity of anxiety between the two groups were significant (P = 0.002). Furthermore, 64.5% of HCWs at the high-risk hospital reported worries about resuming work, while only 58.1% of HCWs at low-risk hospitals expressed such worries; again, the differences between the two groups were significant (P = 0.008).
## Knowledge of preventive measures was also an influencing factor
Frontiers in Public Health | www.frontiersin.org TCMC is a unique concept in TCM. TCMC provides a systematic method that includes physiology and psychology to divide people into nine different types. In the sense of modern medicine, a balanced constitution is a healthy person, while the other eight constitutions, representing unbalanced states of Qi, blood, Yin, Yang, phlegm, or dampness (the viewpoint of TCM philosophy), are similar to the sub-healthy state of modern medicine. According to TCM theories, disease occurrence is due to an imbalance in the body caused by external factors. To address this imbalance, corresponding intervention methods are adopted for different physical types to restore body balance and help prevent diseases. Our results indicate that those with an unbalanced constitution were more likely to have anxiety (except in the case of damp-heat constitution). Several imbalanced constitutions, including qi-deficiency (RR: 7.057; 95% CI: 5.309-9.381), qi-stagnation (RR: 6.445; 95% CI: 4.613-9.005), and yindeficiency (RR: 6.067; 95% CI: 4.084-9.012), posed higher risks for anxiety than a balanced constitution.
## Hcws' requirements for resuming work and attitudes regarding tcm covid-19 prevention
We next investigated HCWs' requirements for resuming work. Thorough workplace disinfection and the provision of adequate protective tools (e.g., masks, alcohol) had the highest positive response rates (92.7 and 93.3%, respectively). Taking temperature measurements as people enter the workplace was also high on HCWs' requirements (85.6%). Canceling activities that require crowd gathering and reviewing whether staff meet the [fig_ref] TABLE 5 |: Multivariable analysis of anxiety and worry based on different characteristics [/fig_ref]. Regarding students returning to school, the abovementioned items accounted for a higher proportion (Additional File 2; [fig_ref] TABLE 6 |: Different severity levels of anxiety and worry among the participants [/fig_ref].
Finally, we investigated HCWs' trust in using TCM to treat and prevent COVID-19. [fig_ref] FIGURE 3 |: Trust in using TCM to prevent and treat COVID-19 [/fig_ref] shows that trust in using TCM to treat COVID-19 was higher than trust in using it for prevention (P < 0.001, Wilcoxon signed-rank test); 38.4% believed TCM could effectively treat COVID-19, while 25.2% believed TCM could prevent COVID-19. Overall, only 1.3% expressed little or no trust in the ability of TCM to treat COVID-19. In comparison, 13.5% had little or no trust in the ability of TCM to prevent COVID-19. We next investigated HCWs' TCM dosage preferences, and the information they would like to receive about preventative TCM products.
# Discussion
This study was conducted at a time when the epidemic was under control and society was facing the resumption of work and production. Our study focused on HCWs' knowledge and practice of COVID-19 preventive measures, their psychological status, and their concerns and requirements for resuming work. Our aim is to inform training and policies for people returning to work and to guide HCWs in prioritizing protection and avoiding occupational exposure. Our study also provides a basis for developing strategies to improve the mental health of HCWs, thus relieving concerns and ensuring an orderly and efficient return to work. This analysis of HCWs' knowledge and the factors affecting their practices should provide a reference for formulating targeted prevention and public policies for HCWs, especially in high-risk areas like Huanggang. Of the HCWs surveyed in this study, 94.5% were familiar with COVID-19 prevention procedures, although medical staff demonstrated a higher knowledge level than administrative staff. Moreover, HCWs from tertiary hospitals and HCWs with undergraduate/postgraduate qualifications also showed higher knowledge levels. Our results are in line with multiple previous findings. For example, Xu et al. and Saqlain et al. reported that 89 and 93.2% of participants, respectively, have sufficient knowledge regarding COVID-19 [bib_ref] Knowledge, awareness, and attitudes relating to the COVID-19 pandemic among different populations..., Xu [/bib_ref] [bib_ref] Knowledge, attitude, practice and perceived barriers among healthcare workers regarding COVID-19: a..., Saqlain [/bib_ref]. A number of factors are known to influence COVID-19 prevention knowledge, including gender, age, education, and occupation, etc. [bib_ref] Patterns and associated factors of COVID-19 knowledge, attitude, and practice among general..., Bekele [/bib_ref]. While previous studies have reported that gender and good knowledge are not clearly correlated [bib_ref] Public knowledge, attitudes and practices towards COVID-19: a cross-sectional study in Malaysia, Azlan [/bib_ref] [bib_ref] Knowledge, attitudes, and practices towards COVID-19 among Chinese residents during the rapid..., Zhong [/bib_ref] , male participants in our study demonstrated higher levels of knowledge.
Knowledge is a prerequisite for establishing prevention understanding and promoting positive behaviors, and an individuals' cognition toward disease affects their behaviors to a certain extent [bib_ref] Meta-analysis of the reasoned action approach (RAA) to understanding health behaviors, Mceachan [/bib_ref]. Our investigation revealed a positive correlation between knowledge and practice of preventive measures, which is consistent with previous research. Thus, a good understanding of preventive measures is the main premise for promoting practice. Our results indicate that health education measures should target groups of different cultural levels, occupations, and genders to improve their prevention knowledge and their practice of preventive measures. These classes could be conducted through hospital organized training, as 23 interviewees specifically mentioned. HCWs' information sources were mainly official channels (97.2%) and community notifications (89.9%), followed by portals/news clients (65.4%), and online social tools (52.5%). Although health authorities have been consistently disseminating COVID-19 information since the disease was first detected, there has also been a surge in false and inaccurate information [bib_ref] Available online at, People [/bib_ref]. This overload of information is likely to have caused confusion and difficulties in ascertaining the correct information. In this regard, official institutions can make use of social media tools to provide more targeted and authoritative information about COVID-19, thus combating the spread of unreliable information.
Overall, 59.8% of HCWs were concerned about resuming work, and 12.4% of HCWs suffered anxiety symptoms (including moderate to extremely-severe anxiety symptoms in 1.6% of HCWs). Our results suggest that the psychological states of HCWs is a problem that needs attention and guidance. Asymptomatic infection (89.2%) and crowd gathering (84.6%) were the main causes of concerns, consistent with the frequent occurrence of asymptomatic infections at the time (23). In the early and peak stages of the COVID-19 outbreak, HCWs faced enormous psychological pressures, and many demonstrated symptoms of anxiety, depression, insomnia, and Burnout Syndrome; more than 70% were reported to experience psychological distress [bib_ref] SARS/MERS/SARS-CoV-2 outbreaks and burnout syndrome among healthcare workers. An umbrella systematic review, Magnavita [/bib_ref]. Compared to these figures, our cohort reported lower prevalence rates of anxiety, especially moderate to severe anxiety [bib_ref] A multinational, multicentre study on the psychological outcomes and associated physical symptoms..., Chew [/bib_ref] [bib_ref] Psychological symptoms among frontline healthcare workers during COVID-19 outbreak in Wuhan, Du [/bib_ref] [bib_ref] Frontline nurses' burnout, anxiety, depression, and fear statuses and their associated factors..., Hu [/bib_ref] [bib_ref] Psychological distress, coping behaviors, and preferences for support among New York healthcare..., Shechter [/bib_ref]. Thus, although reduced, psychological pressures concerning the resumption of work during the pandemic remained. Being female and working in a high-risk hospital were risk factors for concerns about resuming work and anxiety symptoms. Some studies have reported a higher prevalence of mental health problems among women during the COVID-19 outbreak. For example, women have reported a higher fear of COVID-19 beyond infection [bib_ref] Perceived fear of COVID-19 infection according to sex, age and occupational risk..., Andrade [/bib_ref] , and a higher likelihood of anxiety/depression/PTSD symptoms [bib_ref] COVID-19 impacts mental health outcomes and ability/desire to participate in research among..., Cardel [/bib_ref].
HCWs in close contact with COVID-19 patients (and at a higher risk of infection) also experience more psychological disorders, exhibiting almost twice the risk (compared to nonclinical staff) of suffering anxiety and depression [bib_ref] Psychological status of medical workforce during the COVID-19 pandemic: a cross-sectional study, Lu [/bib_ref]. These reports are consistent with our own results. Compared to HCWs at the other hospitals, HCWs at Huangzhou District People's Hospital reported more severe anxiety and worry. As noted above, Huangzhou District People's Hospital screened a large number of people for throat swab tests to assist their return to work. Thus, HCWs at this hospital faced a greater flow of people, and higher exposure to infectious patients [bib_ref] Intolerance of uncertainty, depression, and anxiety: examining the indirect and moderating effects..., Dar [/bib_ref]. The reasons for more severe anxiety may be related to "hypochondriac concerns" (i.e., excessive worry about being infected), and fear that the epidemic was hard to control [bib_ref] Hypochondriacal concerns and somatization in panic disorder, Furer [/bib_ref]. In terms of job attributes, one previous study investigated differences in the psychological status of medical staff and administrative staff during the COVID-19 pandemic, with medical staff reporting greater fear, anxiety, and depression than administrative staff [bib_ref] Psychological status of medical workforce during the COVID-19 pandemic: a cross-sectional study, Lu [/bib_ref]. Medical staff may also suffer from higher risk of anxiety (compared to administrative staff) during the period of work resumption. In general, the pressures experienced by medical staff have been greater. (1) Medical staff have shouldered and continue to shoulder the majority of the work of epidemic prevention in hospitals. In particular, medical staff have been responsible for screening and collecting nasopharyngeal swabs and have been in close contact with patients' secretions, resulting in higher exposure risks and greater psychological pressures. (2) Medical staff are also subjected to increased work intensities and work stresses (compared to administrative staff). In our present study, medical and administrative staff did not exhibit significant differences in their mental health. This could be related to the large difference in sample size between the two groups-there were only 109 administrative staff. In addition, family factors also affected the psychological states of the interviewees, with divorced/widowed HCWs at higher risk of concern. This outcome is also consistent with the results of a large number of studies. Isolation during difficult periods may affect mental health, and HCWs with high work stresses undoubtedly benefit from family support at this time.
During the COVID-19 outbreak, the government issued a number of policy documents addressing the mental and physical health needs of HCWs, including provision of a place for adequate rest with food and supplies, and proper provision of protective equipment [bib_ref] Emergency management of the prevention and control of novel coronavirus pneumonia in..., Chen [/bib_ref]. We devised the present study, because we believed it was similarly important to help HCWs reduce their concerns about returning to work and to reduce their anxiety symptoms after the peak of the COVID-19 epidemic. We propose that all HCWs, but especially females, divorced/widowed, and those working in high-risk hospitals, receive adequate support, including psychological counseling, long-term mental health management, and flexible policies for work resumption.
In our survey of HCWs, we found that good knowledge of COVID-19 prevention measures reduced anxiety symptoms. This is consistent with previous research indicating that useful and trustworthy information encouraged perceptions that the outbreak could be controlled by protective behaviors and lowered the prevalence of depression and anxiety [bib_ref] Knowledge, attitudes, and practices towards COVID-19 among Chinese residents during the rapid..., Zhong [/bib_ref]. Positive preventive measures such as the frequent practice of hand hygiene, the wearing of face masks, and improvements in workplace hygiene could decrease the likelihood that people would experience stress, anxiety, depression, and insomnia [bib_ref] Is returning to work during the COVID-19 pandemic stressful? A study on..., Tan [/bib_ref]. These findings further highlight the need for the promotion of the knowledge and practice of preventive measures.
Because of a lack of specific antiviral therapeutics and vaccines for COVID-19, conventional medicines (including broad-spectrum antibiotics, antivirals, and corticosteroids) and TCM are both used for the treatment of COVID-19 patients in China. The combination of TCM and Western medicine (WM) was superior to WM alone in improving symptoms, shortening the course of disease and the length of hospital stay, reducing moderate-severe conversion rate, and attenuating side effects associated with conventional therapeutics [bib_ref] Clinical retrospective study on the efficacy of Qingfei Paidu decoction combined with..., Xin [/bib_ref]. Furthermore, TCM has a long history of epidemic prevention [bib_ref] Can chinese medicine be used for prevention of corona virus disease 2019..., Luo [/bib_ref] , and has achieved relatively good results for SARS (a related coronavirus that causes pneumonia) [bib_ref] A single-arm study on the preventive effect of chinese medicine decoction on..., Zhu [/bib_ref] [bib_ref] The use of an herbal formula by hospital care workers during the..., Lau [/bib_ref]. TCM prevention and treatment remedies have been included China's official diagnosis and treatment guidelines for COVID-19 from the third to the eighth version. However, clinical evidence for the use of TCM to prevent COVID-19 is generally inadequate. A single arm study revealed good preventive effects for TCM decoctions on close contacts of patients with COVID-19 [bib_ref] A single-arm study on the preventive effect of chinese medicine decoction on..., Zhu [/bib_ref]. A large prospective cohort study using proprietary Chinese medicines provided evidence that TCM intervention could reduce the occurrence of flu symptoms, although neither the test group nor the control group included confirmed cases of COVID-19 [bib_ref] Largescale prospective clinical study on prophylactic intervention of COVID-19 in community population..., Yan [/bib_ref]. In our survey, 77.1% of HCWs held a positive attitude toward TCM prevention of COVID-19 (15.4% remained neutral and 13.5% held a negative attitude). Overall, HCWs reported a high degree of trust in TCM prevention, which is consistent with a previous study conducted among medical professionals in Sichuan, China [bib_ref] Knowledge of medical professionals, their practices, and their attitudes toward traditional Chinese..., Pu [/bib_ref]. Likewise, Chinese immigrants to Canada also reported their belief that TCM was an effective means of preventing COVID-19, and they reported that they would use it if they were experiencing COVID-19 symptoms [bib_ref] Attitudes of Chinese immigrants in Canada towards the use of traditional Chinese..., Kong [/bib_ref].
Concerning their requirement for preventive TCM products developed by an authority, 93.7% of HCWs indicated such a need (with 52.5% indicating a large need). These results remind us that TCM can be used to supplement conventional COVID-19 prevention methods to facilitate a resumption of work and a return to normal life. Even after an efficient vaccine has been made available, TCM remains a very worthy prevention method to choose (taking into account the medical burden). HCWs did express the need for a focus on safety and efficacy in preventive TCMs. Thus, attention should be paid to the evaluation of the safety and superiority of TCM, and to the disclosure of this data. Concerning TCM delivery, 62.4% preferred oral granules; this is related to the perception that TCM involves drinking decoction. Moreover, 88.2% of HCWs expressed a more positive attitude toward TCM treatment of COVID-19 compared with TCM prevention (77.1%). Ostensibly, this may be because TCM prevention lags behind TCM treatment in terms of popularization and publicity. However, the good clinical efficacy of TCM for the treatment of COVID-19 may be a more critical reason. Although most provinces in China have issued preventive programs recommending Chinese herbal formulae (11), TCM is not routinely used in COVID-19 prevention. To address this shortfall, pharmacokinetic studies should be conducted to obtain the pharmacokinetic profile of TCM products, including their absorption, distribution, metabolism, and excretion parameters. Clinical trials should then be conducted to test their preventive efficacy and safety on human subjects. Finally, we also investigated the constitution type of HCWs. Constitution is a TCM concept that focuses on individual differences in people, of which there are nine basic constitution types [bib_ref] Classification and diagnosis basis of nine basic constitutions in Chinese medicine, Qi [/bib_ref]. People with different constitutions may have different psychological characteristics. Previous studies have found that those with a "phlegm-dampness" constitution and a "qi-deficiency" constitution suffer more from the effects of COVID-19 [bib_ref] Analysis on traditional chinese medicine syndrome and constitutions of 90 patients with..., Jiayao [/bib_ref]. Our studies reveal that an imbalanced constitution increased the risk of anxiety (especially with qi-deficiency, qi-stagnation, and yin-deficiency constitutions). A dysfunction in the hypothalamic-pituitary-adrenal (HPA) axis could be related to the connection between constitution and anxiety [bib_ref] Effect of hypothalamus pituitary adrenal axis disorder on hippocampal formation in rats..., Jia [/bib_ref] [bib_ref] Relationship between anxiety, depression and TCM constitution, Xiaoyan [/bib_ref]. Therefore, HCWs' TCMC types could be used as a reference for the development of COVID-19 related products.
This study has several limitations. First, and foremost, we used a cross-sectional research method to assess COVID-19 knowledge/practices and mental status during the return to work, and work and production have now largely been resumed. In our study, we reveal the important needs and psychology of HCWs during the process of resumption of work and production. The provision of policies addressing these needs is a very important measure performed by the Chinese government, which has thus far managed the resumption of work and production to great effect. TCM has played an important role in COVID-19 prevention and control during this period, and this study is of great value in this context. Second, the self-evaluation method and absence of objective test questions in the survey may have influenced HCWs' knowledge. Third, the SAS was used to investigate anxiety symptoms, and the information provided on symptoms was not verified by a medical professional. The data analysis parameter using "worry" as a psychological symptom may have been unreliable, since the only options were "yes" or "no." Fourth, the questionnaire did not subdivide medical staff, such as doctors and nurses, by department category. Lastly, the study was limited in scope. Societies that experienced the COVID-19 outbreak and are facing the resumption of work are represented here by Huanggang. Although sporadic cases sometimes occur, the epidemic in China has been mostly contained. However, it may be assumed that our research remains meaningful, because of the constant threat of COVID-19 throughout the globe.
# Conclusion
HCWs from public hospitals in Huanggang demonstrated a good knowledge of COVID-19 preventative measures during the period covering the resumption of work after the epidemic was contained. This was especially true for male HCWs, HCWs in tertiary hospitals, medical staff, and HCWs with a higher degree. However, good knowledge of COVID-19 preventative measures did not completely eliminate the psychological impact of work resumption. Thus, 59.8% of HCWs reported worries about resuming work, and 12.4% reported anxiety symptoms. Supporting HCWs is an important public health measure for ensuring an orderly and efficient return to work, and in helping to prevent the further spread of COVID-19. It is especially important that public health measures include measures to avoid infection among HCWs and to support their mental health. Special training on COVID-19 prevention measures is recommended to reduce the risk of infection and to relieve mental problems. The provision of adequate protective equipment and the thorough disinfection of the workplace are other vital measures. In addition, humanistic care and psychological counseling should be provided to HCWs, in particular female HCWs, divorced/widowed HCWs, HCWs with insufficient knowledge, and HCWs in high-risk hospitals. Although TCM demonstrates potential for COVID-19 protection, there is currently a lack of reliable evidence. To address this, preventative TCM products should be developed further, and in vivo and in vitro trials should be conducted. Only when reliable evidence is provided, can TCM be confidently used to supplement conventional COVID-19 prevention methods. It is hoped that this study can serve as a reference for policy making during the resumption of work in countries or regions facing similar situations.
# Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: This can be found at https://doi.org/10.6084/m9.figshare.15087891.v1.
# Ethics statement
The studies involving human participants were reviewed and approved by Beijing University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.
# Author contributions
LL and JW: study design and data collection. TL: data analysis and writing. MB, YanfZ, GD, HX, YiZ, ZZ, HC, and JH: data collection. YoZ, HL, and BL: data analysis guidance. YangZ, LL, ZL, and QW: study design and give direction to the paper. All authors contributed to the article and approved the submitted version.
[fig] FIGURE 1 |: Geographical location of Huanggang, where the respondents were located. [/fig]
[fig] FIGURE 2 |: Comparison of psychological features between two groups. (A) Proportion of anxiety. (B) Proportion of worry. [/fig]
[fig] FIGURE 3 |: Trust in using TCM to prevent and treat COVID-19 (N = 2,050). requirements for resuming work yielded 82.8 and 79.7% positive response rates, respectively. Receiving training on COVID-19 prevention was a requirement of 75.1% of HCWs, while 65.7% indicated a need for preventive products. Among all requirements for resuming work, providing dedicated buses had the lowest positive response rate (40.7%) (Additional File 2; Supplementary [/fig]
[fig] FUNDING: This work was supported by the Key Research and development projects of the Ministry of Science and Technology (2020YFC0845200). [/fig]
[table] TABLE 1 |: Baseline characteristics of study participants (N = 2,050). [/table]
[table] TABLE 2 |: Association between HCWs' COVID-19 knowledge a and their characteristics. **p < 0.01; a Knowledge based on participants' self-evaluation; "very familiar" was considered "good knowledge"; the rest were considered "Common knowledge." b 12 other public hospitals. c Divorced or widowed. [/table]
[table] TABLE 3 |: Association between participant characteristics and COVID-19 prevention knowledge. **p < 0.01; *p < 0.05; Log-binomial model adjusted for all independent variables listed in the table simultaneously. a divorced or widowed. [/table]
[table] TABLE 5 |: Multivariable analysis of anxiety and worry based on different characteristics. Frontiers in Public Health | www.frontiersin.org [/table]
[table] TABLE 6 |: Different severity levels of anxiety and worry among the participants. [/table]
|
Current perspectives on autoimmune pancreatitis and IgG4-related disease
IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder recognized as a novel clinical entity with either synchronous or metachronous multi-organ involvement. Patients with IgG4-RD show diffuse or focal organ enlargement and mass-forming or nodular/thickened lesions with abundant infiltration of IgG4-positive plasmacytes and fibrosis, and such patients respond well to steroid treatment. It should be differentiated from mimics by a combination of serum IgG4 level, imaging features, and histopathological findings. The current first-line drug is corticosteroids, or rituximab in high-risk patients for steroid intolerance. Although relapse rates are high, standardized protocols for relapsed cases have not been approved yet. Based on genetic factors, disease-specific or -related antigens, abnormal innate and adaptive immunity may be involved, although the precise pathogenic mechanism and long-term outcome still remain unclear.
IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder recognized as a novel clinical entity with multi-organ involvement and unknown origin, associated with abundant infiltration of IgG4-positive cells and fibrosis. 1)-3) Involvement of the central nervous system, thyroid, lacrimal glands, salivary glands, breast, lung, liver, bile duct, pancreas, gastrointestinal tract, kidney, prostate, retro-peritoneum, arteries, lymph nodes, and skin, and good responses to steroid treatment have been reported. 1)-3) However, before establishment of the concept of IgG4-RD, each organ lesion has been described independently. [bib_ref] Diagnosis and classification of autoimmune pancreatitis, Okazaki [/bib_ref] In 1892, Mikulicz et al. first observed a patient with symmetrical swelling of the lachrymal, parotid and submandibular glands, with massive infiltration of mononuclear cells, and the condition was called Mikulicz's disease (MD); however, it has been classified as an atypical type of Sjögren's syndrome (SjS), which also presents with bilateral, painless, and symmetrical swelling of the lacrimal, parotid, and submandibular glands. Küttner reported a tumor-like enlargement of the submandibular gland of unknown origin that was sometimes a result of stones in the Wharton duct. In 1961, Sarles et al. first observed a case of particular pancreatitis with hypergammaglobulinemia, a prototype of autoimmune pancreatitis (AIP). [bib_ref] Diagnosis and classification of autoimmune pancreatitis, Okazaki [/bib_ref] In 1991, proposed a histopathological description of lymphoplasmacytic sclerosing pancreatitis (LPSP) from the resected pancreas of tumor-like appearing pancreatitis, which are clinically difficult to distinguish from pancreatic cancer, and is now regarded as a characteristic histopathological finding of IgG4-related AIP (type 1 AIP). [bib_ref] Lymphoplasmacytic sclerosing pancreatitis with cholangitis: a variant of primary sclerosing cholangitis extensively..., Kawaguchi [/bib_ref] In 1995, proposed the concept of AIP. [bib_ref] Chronic pancreatitis caused by an autoimmune abnormality. Proposal of the concept of..., Yoshida [/bib_ref] In 1967, Comings et al. [bib_ref] Familial multifocal fibrosclerosis, Comings [/bib_ref] reported the first familiar case of multifocal fibrosclerosis with retroperitoneal fibrosis, mediastinal fibrosis, sclerosing cholangitis, Riedel's thyroiditis, and pseudotumor of the orbit, which is now regarded as the syndrome of IgG4-RD. 1)- [bib_ref] Recommendations for the nomenclature of IgG4-related disease and its individual organ system..., Stone [/bib_ref] In 2001, Hamano et al. reported increased serum levels of IgG4 in Japanese patients with AIP, [bib_ref] High serum IgG4 concentrations in patients with sclerosing pancreatitis, Hamano [/bib_ref] an epoch-making discovery in the history of IgG4-RD. Thereafter, many studies of AIP have been reported, mainly by Japanese investigators. The histopathological findings of LPSP are characterized by the periductal localization of predominantly CD4 positive T-cells, IgG4-positive plasma cells, storiform fibrosis with acinar cell atrophy frequently resulting in stenosis of the main pancreatic duct, and obliterative fibrosis. 1) Approximately 60-80% of patients with AIP show obstructive jaundice with sclerosing cholangitis (IgG4-related sclerosing cholangitis; IgG4-SC) and other organ involvements (OOIs), in which cholangiographic features are similar to those of primary sclerosing cholangitis (PSC), pancreatic cancer, or cholangiocarcinoma. The steroid responses and the prognoses of sclerosing cholangitis associated with AIP differ from patients with PSC, which suggests different pathological conditions. In 2006, Kamisawa et al. [bib_ref] Autoimmune pancreatitis: proposal of IgG4-related sclerosing disease, Kamisawa [/bib_ref] suggested a concept of a systemic sclerosing disease with fibrosis and abundant infiltration of IgG4-positive plasma cells based on multifocal fibrosclerosis proposed by Comings et al. Further histological and clinical profiling of patients with "AIP" reveals two distinct subtypes, type 1 and type 2. 9) Type 1 AIP is now regarded as a pancreatic manifestation of IgG4-RD, and type 2 AIP is supposed to be a distinctive pancreatic disease with granulocytic epithelial lesion (GEL) and occasional association with ulcerative colitis. [bib_ref] International consensus diagnostic criteria for autoimmune pancreatitis: guidelines of the international association..., Shimosegawa [/bib_ref] Conversely, most patients with MD show elevated serum levels of IgG4, negative anti-SS-A/ Ro or anti-SS-B/La antibodies, infiltration of IgG4positive plasma cells into the glands, and recovery of secretion with steroid treatment. Patients with MD often show steroid-responsive OOIs such as AIP, sclerosing cholangitis, retroperitoneal fibrosis, enlarged celiac and hilar lymph nodes, chronic thyroiditis, or interstitial nephritis, which suggests that MD is completely different from Sjögren's syndrome. 1)- [bib_ref] Recommendations for the nomenclature of IgG4-related disease and its individual organ system..., Stone [/bib_ref] In addition to the original concept of multifocal idiopathic fibrosclerosis, recent studies led us to develop a novel concept of a systemic disease such as IgG4-related systemic sclerosing disease, [bib_ref] Autoimmune pancreatitis: proposal of IgG4-related sclerosing disease, Kamisawa [/bib_ref] systemic IgG4-related plasmacytic syndrome (SIPS),or IgG4-positive multiorgan lymphoproliferative syndrome (IgG4-MOLPS), [bib_ref] Proposal for a new clinical entity, IgG4-positive multi-organ lymphoproliferative syndrome: Analysis of..., Masaki [/bib_ref] all of which may refer to the same conditions. Based on these findings, the Japanese Research Committees for "Systemic IgG4related Sclerosing Disease" (chaired by Okazaki, K.) and "IgG4-MOLPS" (chaired by Umehara, M.) supported by the Research for Intractable Disease program from the Ministry of Health, Labour, and Welfare of Japan, have proposed the comprehensive term 2) and diagnostic criteria for "IgG4-related disease (IgG4-RD)". [bib_ref] Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), Umehara [/bib_ref] The first International Symposium on IgG4-RD held in Boston (chaired by Stone, J.) endorsed the Japanese concept and proposed nomenclatures and pathological criteria for individual organ lesions. [bib_ref] Recommendations for the nomenclature of IgG4-related disease and its individual organ system..., Stone [/bib_ref]
## ),13)
## Current concepts of igg4-rd
Patients with IgG4-RD, either synchronously or metachronously, show diffuse or focal organ enlargement and mass-forming or nodular/thickened lesions in various organs with abundant infiltration of IgG4-positive plasmacytes with fibrosis. 1)-3) IgG4-RD includes a wide variety of diseases, including MD, AIP, hypophysitis, Riedel thyroiditis, interstitial pneumonitis, interstitial nephritis, prostatitis, lymphadenopathy, retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pseudotumor [fig_ref] Figure 1: Current concept of IgG4-related disease [/fig_ref]. Approximately 10-20% of the patients have a single organ involvement. Although it is unclear whether the pathogenic mechanism is the same among individual organs or not, recent studies have suggested possible multi-pathogenic factors in the development of AIP similar to other immunogenic diseases. Based on genetic factors, disease-specific or -related antigens, abnormal innate and adaptive immunity may be involved. IgG4-RD mainly affects middle-aged to elderly men except for MD, in which previous epidemiological studies did not show a gender difference. Clinical symptoms vary depending on the individual organs involved, but these are dramatically relieved by steroid therapy in many cases; 1)-3) however, long-term prognosis still remains unclear. Some patients develop serious complications such as obstructive jaundice due to hepatic, gallbladder, or pancreatic disease; hydronephrosis due to retroperitoneal fibrosis; or respiratory symptoms due to pulmonary disease. The infiltration of IgG4-positive cells, increased serum levels of IgG4, storiform fibrosis and obliterative phlebitis are characteristic in most organ involve-ments of IgG4-RD including pancreatic, biliary tract, retroperitoneal, and renal lesions, but storiform fibrosis and obliterative phlebitis are rarely observed in lymph nodes, salivary, or lacrimal glands.
In the International Symposium on IgG4-RD, the nomenclature of individual organ manifestations of IgG4-RD were proposed [fig_ref] Table 1: Comprehensive diagnostic criteria for IgG4-related disease, 2011 [/fig_ref] using "IgG4related" as a modifier, except for the pancreatic manifestation. [bib_ref] Recommendations for the nomenclature of IgG4-related disease and its individual organ system..., Stone [/bib_ref] Formerly called Mikulicz's disease, Riedel thyroiditis, or Küttner tumor, Ormand's disease is replaced by IgG4-related dacryoadenitis and sialoadenitis, IgG4-related thyroid disease, IgG4-related submandibular gland disease, and IgG4related retroperitoneal fibrosis, respectively. The pancreatic manifestation of IgG4-RD is termed "type 1 autoimmune pancreatitis (IgG4-related pancreatitis)", because it is widely accepted among gastroenterologists and pancreatic surgeons, and helps to discriminate between type 1 and type 2 AIP, which are not a part of the IgG4-RD spectrum. When the pathogenesis of type 2 AIP is clarified, the term "type 1 AIP" may be replaced by "IgG4-related pancreatitis". [bib_ref] Recommendations for the nomenclature of IgG4-related disease and its individual organ system..., Stone [/bib_ref] (1) Marked lymphocyte and plasmacyte infiltration and fibrosis.
(2) Infiltration of IgG4D plasma cells: ratio of IgG4D/IgGD cells > 40% and >10 IgG4D plasma cells/HPF.
[formula] Definite: 1) D 2) D 3) Probable: 1) D 3) Possible: 1) D 2) [/formula]
However, it is important to differentiate IgG4-RD from malignant tumors of each organ (e.g., cancer, lymphoma) and similar diseases (e.g., Sjögren's syndrome, primary sclerosing cholangitis, Castleman's disease, secondary retroperitoneal fibrosis, Wegener's granulomatosis, sarcoidosis, and Churg-Strauss syndrome) by additional histopathological examination. Even when patients cannot be diagnosed using the CCD criteria, they may be diagnosed using organ-specific diagnostic criteria for IgG4-RD. Recent studies have suggested that AIP is classified as two distinct subtypes, types 1 and type 2, and the International Consensus Diagnostic Criteria (ICDC) for AIP, which enabled the differentiation of type 1 from type 2 AIP and is the most widely accepted major diagnostic criteria. 9) Clinically, type 1 AIP (IgG4-related pancreatitis) seems to be the pancreatic manifestation of IgG4-RD, characterized by: (i) mild abdominal symptoms, usually without acute attacks of pancreatitis; (ii) occasional occurrence of obstructive jaundice; (iii) increased serum gammaglobulin, IgG, and/or IgG4 concentrations; (iv) presence of autoantibodies; (v) diffuse, segmental, or focal enlargement of the pancreas with a capsule-like low-density rim on dynamic computed tomography/magnetic resonance imaging (CT/MRI) images; (vi) irregular narrowing of the main pancreatic duct on endoscopic retrograde cholangiopancreatography images; (vii) histopathologically, LPSP: abundant infiltration of lymphocytes and IgG4-positive plasmacytes and fibrosis, and obliterative phlebitis; and (viii) occasional association with OOIs (Figs. 2 and 3). Patients with type 1 AIP often have obstructive jaundice, with both pancreatic and extrapancreatic manifestations responding to steroid therapy. 1),8), [bib_ref] International consensus diagnostic criteria for autoimmune pancreatitis: guidelines of the international association..., Shimosegawa [/bib_ref] Histological examination by American and European pathologists of the resected pancreases of patients with chronic non-alcoholic pancreatitis revealed another histopathological pattern, called idiopathic duct-centric pancreatitis (IDCP) [bib_ref] Idiopathic chronic pancreatitis with periductal lymphoplasmacytic infiltration: clinicopathologic features of 35 cases, Notohara [/bib_ref] or AIP with GELs, [bib_ref] Histopathological features of diagnostic and clinical relevance in autoimmune pancreatitis: a study..., Zamboni [/bib_ref] later named as type 2 AIP. 9) Although patients with type 2 AIP show similar pancreas images to type 1 AIP, they show different clinicopathological features from type 1 AIP such as no elevation of serum IgG4, occasional infiltration of IgG4-positive plasma cells, no autoantibodies, or no involvement of other organs, except for inflammatory bowel disease (Figs. 4 and 5). The most characteristic feature of type 2 AIP is GEL, commonly accompanied by destruction and obliteration of the pancreatic duct. 9) Although type 1 AIP (LPSP type) often occurs in older men and is accompanied by a variety of extrapancreatic lesions, type 2 AIP (IDCP/GEL type), has no gender difference, younger age at onset (often <40 years), and is frequently associated with inflammatory bowel disease (about 30%). Thus, after a worldwide debate over the diagnostic criteria for AIP, LPSP has been defined as type 1 (IgG4-related pancreatitis) and IDCP/AIP with GEL has been defined as type 2. 9)
## Diagnosis of igg4-related disease
Patients with IgG4-related disease show organ enlargement or nodular/hyperplastic lesions in or- gans in the entire body, synchronously or metachronously, due to the prominent infiltration and fibrosis of lymphocytes and plasmacytes. The organs known to be affected include the central nervous system, lacrimal/salivary glands, thyroid gland, lungs, pancreas, biliary duct, liver, gastrointestinal tracts, kidneys, prostate gland, retroperitoneum, skin, arteries, and lymph nodes. Clinical symptoms vary depending on the organ in which the lesions are located, which suggests that it is hard to establish criteria covering all patients with IgG4-RD. Therefore, some specific diagnostic criteria have been proposed for each involved organ, such as for IgG4related Mikulicz's disease (IgG4-related dacryoadenitis/sialadenitis), 12),16) type 1 AIP (IgG4-related pancreatitis), [bib_ref] International consensus diagnostic criteria for autoimmune pancreatitis: guidelines of the international association..., Shimosegawa [/bib_ref] and IgG4-related kidney disease. [bib_ref] Proposal for diagnostic criteria for IgG4-related kidney disease, Kawano [/bib_ref] However, these organ specific criteria do not cover other organs or are not familiar to general clinicians and non-specialists. Therefore, Japanese investigators have proposed comprehensive diagnostic criteria (CDC) for IgG4-RD, containing three major criteria (clinical, hematological, and histopathological examinations) for practical use by general physicians and non-specialists [fig_ref] Table 2: Preferred nomenclature for individual organ manifestations of IgG4-related disease [/fig_ref]. [bib_ref] Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), Umehara [/bib_ref] Clinical examination. Physical examinations and imaging by ultrasound/CT/MRI can show the characteristic diffuse/localized swelling, masses, or thickness in single or multiple organs.
Hematological examination. The cutoff value for serum IgG4 concentration, 135 mg/dL, was based on receiver operating characteristic (ROC) curves, [bib_ref] High serum IgG4 concentrations in patients with sclerosing pancreatitis, Hamano [/bib_ref] and its validity was confirmed. [bib_ref] Are classification criteria for IgG4-RD now possible? The concept of IgG4-related disease..., Okazaki [/bib_ref] In patients with single-organ involvement and serum IgG4 concentration <135 mg/dL, the IgG4/IgG ratio may be helpful in making a diagnosis. However, elevated IgG4 may also be observed in other diseases (e.g., atopic dermatitis, pemphigus, asthma, and multicentric Castleman's disease), and especially in about 10% of malignancies, which suggests that high serum IgG4 is not necessarily a specific marker of IgG4-RD. [bib_ref] Are classification criteria for IgG4-RD now possible? The concept of IgG4-related disease..., Okazaki [/bib_ref] Therefore, at present, the significance of elevated IgG4 in the pathogenesis/pathophysiology of IgG4-RD still remains unknown.
## Histopathologic examination.
Although tissue biopsies are difficult to obtain from some organs, including the pancreas, retroperitoneum, and ocular cavity, histopathological examination is important. Marked lymphocyte and plasmacyte infiltration with storiform fibrosis or obliterative phlebitis is characteristic of IgG4-RD. IgG4 IgG4-positive plasma cells are recommended in the diagnosis of type 1 AIP. 9) Based on these findings, the CDC for IgG4-RD recommend both the ratio of IgG4/IgG-positive cells >40% and infiltration of >10 IgG4-positive plasma cells/HPF for definitive diagnosis. [bib_ref] Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), Umehara [/bib_ref] Eosinophilic infiltration is often observed along with infiltration of IgG4-positive cells.
Although the sensitivity of the CDC for definitive IgG4-RD is low in organs from which it is difficult to take biopsy specimens, it can detect possible cases of IgG4-RD. In probable or possible cases, organ-specific criteria should be used concurrently.
## Treatment
Although some studies of the treatment for IgG4-RD have been proposed, [bib_ref] Second international symposium on IgG4-related disease. International consensus guidance statement on the..., Khosroshahi [/bib_ref] , [bib_ref] Factors in glucocorticoid regimens associated with treatment response and relapses of IgG4-related..., Shirakashi [/bib_ref] comprehensive management for IgG4-RD has not yet been established. Because the clinical features of patients with IgG4-RD depend on characteristics of the organs involved, the current consensus for the treatment of type 1 AIP, [bib_ref] International consensus for the treatment of autoimmune pancreatitis, Okazaki [/bib_ref] as a representative lesion of IgG4-RD, is described [fig_ref] Figure 6: Therapeutic algorithm for type 1 AIP [/fig_ref].
## Induction for remission.
For induction of remission, steroids can successfully achieve induction in most patients with type 1 AIP (990%). 21),22) The [bib_ref] Amendment of the Japanese Consensus Guidelines for Autoimmune Pancreatitis, 2013 III. Treatment..., Kamisawa [/bib_ref] and international consensus [bib_ref] International consensus for the treatment of autoimmune pancreatitis, Okazaki [/bib_ref] have recommended that steroids are the first-line agent in all active and symptomatic patients with untreated AIP, who have obstructive jaundice or abdominal and/or back pain due to pancreatic or other organ involvements, unless patients have any contraindications or high risks for steroid use. They have also recommended steroid treatment even in asymptomatic patients with persistent pancreatic mass on imaging, or those with associated IgG4related sclerosing cholangitis (IgG4-SC) who have persistent liver test abnormalities. [bib_ref] International consensus for the treatment of autoimmune pancreatitis, Okazaki [/bib_ref] In patients with contraindications to steroid treatment, rituximab can induce remission as a single agent. 21),24) Except for rituximab, however, other steroid-sparing agents such as thiopurines are poorly effective as a single agent for the induction of remission. [bib_ref] Treatment of relapsing autoimmune pancreatitis with immunomodulators and rituximab: the Mayo Clinic..., Hart [/bib_ref] Before any initial steroid therapy, management of blood glucose in diabetic patients or biliary drainage in patients with obstructive jaundice are recommended, [bib_ref] Amendment of the Japanese Consensus Guidelines for Autoimmune Pancreatitis, 2013 III. Treatment..., Kamisawa [/bib_ref] because biliary drainage can prevent biliary infection, and brushing or bile juice cytology can differentiate IgG4-SC from biliary malignancy. In some cases of mild jaundice without signs of infection, however, steroid treatment alone can be performed safely without biliary stenting. [bib_ref] Obstructive jaundice in autoimmune pancreatitis can be safely treated with corticosteroids alone..., Bi [/bib_ref] Relapse and second-line treatment. According to international or Japanese multicenter studies, 27) 30-50% of the patients with type 1 AIP experienced relapse. Most risk factors for relapsing remain poorly understood and require further study. A history of relapse seems to be a strong risk factor for future relapse. Although an international study [bib_ref] Longterm outcomes of autoimmune pancreatitis: a multicentre, international analysis, Hart [/bib_ref] could not confirm the finding, Japanese national retrospective 27)-29) studies have suggested that diffuse pancreatic enlargement type, and persistently high, low decreased rate or re-elevation of serum IgG4 levels after steroid therapy, may be predictors of AIP relapse. Association with IgG4-related SC, especially the proximal type of IgG4-SC seems to be a risk factor for relapse. In addition to serum IgG4 levels, circulating immune complexes have been reported as useful early predictors of relapse. [bib_ref] Autoimmune pancreatitis and complement activation system, Muraki [/bib_ref] Although the usefulness of steroids as an initial treatment is agreed upon, there is no "gold standard" for treatment in relapse cases. The international consensus recommends re-administration or and increased dose of steroid or steroid-sparing agent, such as immunosuppressive agents or rituximab. [bib_ref] Second international symposium on IgG4-related disease. International consensus guidance statement on the..., Khosroshahi [/bib_ref] Maintenance treatment. Although there is no high-level evidence regarding maintenance therapy, certain patients benefit from maintenance therapy after a successful course of induction therapy. [bib_ref] Second international symposium on IgG4-related disease. International consensus guidance statement on the..., Khosroshahi [/bib_ref] ,23), [bib_ref] The clinical course of patients with IgG4-related kidney disease, Saeki [/bib_ref] Whereas some patients do not relapse after withdrawal of steroids after remission is achieved, some patients relapse during steroid taper or require relatively high-dose maintenance therapy. Therefore, to determine the indications for maintenance therapy, it is important to evaluate disease activity during induction therapy.
Maintenance therapy may consist of low-dose glucocorticoids or any of the steroid-sparing agents. Retrospective studies from Asian countries, mainly Japan [bib_ref] Standard steroid therapy for autoimmune pancreatitis, Kamisawa [/bib_ref] and South Korea, [bib_ref] Substitution of aspartic acid at position 57 of the DQb1 affects relapse..., Park [/bib_ref] have suggested that steroid maintenance monotherapy may prevent relapse after remission. In addition to retrospective studies, a recent prospective Japanese multicenter study has shown that steroid maintenance therapy provides beneficial outcomes after remission. [bib_ref] Randomised controlled trial of long-term maintenance corticosteroid therapy in patients with autoimmune..., Masamune [/bib_ref] In cases of low disease activity such as involvement in the pancreas alone with segmental/focal lesions without any OOIs and complete radiological remission with normalized IgG/IgG4 in rapid response to steroid, steroids can be tapered off within 3 months followed by no steroid maintenance. [bib_ref] International consensus for the treatment of autoimmune pancreatitis, Okazaki [/bib_ref] On the other hand, maintenance therapy using a low dose of steroid, immunomodulators, or rituximab is recommended in patients with type 1 AIP showing diffuse enlargement of the pancreas, delayed radiological remission or persistently high serum IgG4 (>2# upper limit of normal [ULN]) after treatment, or 62 OOIs, or association with proximal IgG4-SC before treatment. [bib_ref] International consensus for the treatment of autoimmune pancreatitis, Okazaki [/bib_ref] Outcome of type 1 AIP and IgG4-RD Clinical symptoms vary depending on the individual organs involved and are dramatically relieved by steroid therapy in many cases; 19)-21), [bib_ref] Amendment of the Japanese Consensus Guidelines for Autoimmune Pancreatitis, 2013 III. Treatment..., Kamisawa [/bib_ref] however, long-term prognosis still remains unclear. Corticosteroids improve pancreatic exocrine and endocrine function by reducing inflammation, fibrosis, and regeneration through correct aberrant cystic fibrosis transmembrane conductance regulator localization in the duct and regenerate acinar cells in AIP; [bib_ref] Corticosteroids correct aberrant CFTR localization in the duct and regenerate acinar cells..., Ko [/bib_ref] therefore, the short-term prognosis of AIP appears to be positive with steroid therapy. On the other hand, it is unclear whether the long-term outcome is positive, because there are many unknown factors, such as relapse, pancreatic exocrine or endocrine dysfunction, and associated malignancy.
Although not commonly observed, approximately 10% (7-40%) of the patients with type 1 AIP may develop pancreatic calcification or chronic pancreatitis. 35)-39) Maruyama et al. [bib_ref] Type 1 autoimmune pancreatitis can transform into chronic pancreatitis: a long-term follow-up..., Maruyama [/bib_ref] ,39) proposed a sequential progression mechanism for the transition of AIP to chronic pancreatitis. They identified two risk factors, pancreatic head swelling and nonnarrowing main pancreatic duct in the pancreatic body, which may cause pancreatic juice stasis in the upstream pancreatic duct and increased intrapancreatic duct pressure that is resistant to typical AIPspecific main pancreatic duct narrowing in the pancreatic body.
Although the association of malignant tumors with type 1 AIP or IgG4-RD is controversial, several cases of pancreatic cancer or other malignancies have been described in patients with AIP or IgG4-RD. [bib_ref] Longterm outcomes of autoimmune pancreatitis: a multicentre, international analysis, Hart [/bib_ref] ,40)-44) In recent reports, 22),40)-44) in about 1,000 patients, 8.5% (10.1-13.9%) of IgG4-RD cases and 1% of AIP cases (2.1-13.9%) exhibited malignant tumors. Therefore, it is noted that pancreatic cancer or other malignant tumors may be complicated with AIP or IgG4-RD. A Japanese multicenter study of 108 cases of AIP reported that the highest risk for cancer is in the first year after AIP diagnosis. The absence of a relapse after successful treatment of the coexisting cancers suggests that AIP may develop as a paraneoplastic syndrome in some patients. [bib_ref] Risk of cancer in patients with autoimmune pancreatitis, Shiokawa [/bib_ref] Conversely, in an international multicenter study, the most frequently occurring cancers during followup were gastric, lung, and prostate, but not pancreatic, cancer. 22) Kamisawa et al. [bib_ref] Frequent and significant K-ras mutation in the pancreas, the bile duct, and..., Kamisawa [/bib_ref] investigated K-ras mutations in gallbladder and pancreas tissues obtained from AIP patients, and demonstrated significant K-ras mutation in the pancreatic and biliary regions, which suggested the possibility of AIP as a risk factor for pancreatic and bile duct cancer.
## Recent advances in the pathogenic mechanisms of aip and igg4-rd
Although it is unclear whether the pathogenic mechanism is the same among individual organs or not, recent studies have suggested possible multipathogenic factors in the development of AIP similar to other immunogenic diseases. Based on genetic factors, disease-specific or -related antigens, abnormal innate and adaptive immunity may be involved [bib_ref] Autoimmune pancreatitis: the past, present, and future, Okazaki [/bib_ref].
# Immunogenic backgrounds.
Although immunogenic backgrounds are unknown in IgG4-RD, the susceptibility to AIP in Japanese patients, most of whom exhibit IgG4-RD, may be associated with the class II antigen haplotype of the major histocompatibility complex (HLA-DRB1*0405-DQB1*0401), [bib_ref] HLA DRB10405-DQB10401 haplotype is associated with autoimmune pancreatitis in the Japanese population, Kawa [/bib_ref] polymorphism of nuclear factor-5B, and Fc-receptor-like 3 genes expressed on B cells. [bib_ref] Genetic association of Fc receptor-like 3 polymorphisms with autoimmune pancreatitis in Japanese..., Umemura [/bib_ref] An inhibitory molecule, cytotoxic T lymphocyte antigen-4 (CTLA-4; CD152), expressed on the activated memory T cells or CD4 D CD25 D regulatory T cells (Tregs), was independently reported as a susceptibility factor. 48),49) Based on immunogenic backgrounds, abnormal conditions of immune responses may be involved in the development of type 1 AIP, although the precise pathogenic mechanisms remain unclear.
Innate immunity. Recently, abnormal innate immunity has been demonstrated in some patients with IgG4-RD. [bib_ref] Involvement of activation of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors in..., Watanabe [/bib_ref] Activation of NOD-2 and Toll-like receptor (TLR) ligands on monocytes or basophils from patients with IgG4-related AIP enhances IgG4 responses via B-cell activating factor and IL-13, although the specific pathogens still remain unclear. [bib_ref] Involvement of activation of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors in..., Watanabe [/bib_ref] ,51) Moreover, abundant infiltration of TLR-7 positive M2-macrophages was observed in pancreatic tissues from type 1 AIP cases. [bib_ref] Possible involvement of Toll-like receptor 7 in the development of type 1..., Fukui [/bib_ref] In animal models, activation of TLR3 (polyinosinic:polycytidylic acid) or TLR4 (lipopolysaccharide) can induce immune-mediated cholangitis, pancreatitis, and sialadenitis similar to human IgG4-RD. [bib_ref] Comparative study on experimental autoimmune pancreatitis and its extrapancreatic involvement in mice, Yamashina [/bib_ref] Recently, possible roles of basophils, which are activated via TLR signaling, may be involved in the development of type 1 AIP. [bib_ref] Basophils activated via TLR signaling may contribute to pathophysiology of type 1..., Yanagawa [/bib_ref] Possible roles of IgG4 in IgG4-RD. Although the association of IgE-mediated allergy and IgG4 antibodies is well known, IgG4 characteristics are still poorly understood. IgG4 is involved in an immune process referred to as 'Fab-arm exchange', which involves the swapping of a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule. This usually results in asymmetric antibodies with two different antigencombining sites. [bib_ref] Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange, Van Der Neut Kolfschoten [/bib_ref] Although these modified antibodies are hetero-bivalent, they behave as monovalent antibodies. Another aspect of IgG4 is that it mimics IgG rheumatoid factor activity by interacting with IgG, namely through Fc-mediated aggregation. [bib_ref] A novel immunoglobulin-immunoglobulin interaction in autoimmunity, Kawa [/bib_ref] IgG4 seems to be associated with a pathogenic effect in a few situations. In pemphigus, recognition of skin autoantigens (desmogleins) by IgG4 is at the origin of the disease process. [bib_ref] Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed..., Ishii [/bib_ref] molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen. [bib_ref] Structural determinants of unique properties of human IgG4-Fc, Davies [/bib_ref] Another recent study of the regulation of IgG4 showed that IgG4-RD may reflect an excessive production of anti-inflammatory cytokines such as IL-10, which triggers an overwhelming expansion of IgG4-producing plasma cells. 59)-63) Increased peripheral inducible-memory Tregs are positively correlated with serum levels of IgG4. [bib_ref] Circulating naïve and CD4 D CD25 high regulatory T cells in patients..., Miyoshi [/bib_ref] In addition, prominent infiltration of Tregs upregulated IL-10 in the liver of patients with IgG4-SC. [bib_ref] Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis..., Zen [/bib_ref] These findings suggested that IgG4 does not act as a pathogenic factor, nor is it an anti-inflammatory factor in IgG4-RD. Further studies are necessary to clarify the precise role of IgG4 in IgG4-RD.
## The complement system.
Patients in the active stages of AIP occasionally show decreased complement (C3, C4) with elevated circulating immune complex, as well as elevated serum levels of IgG4 and the IgG4 subclass of immune complexes. [bib_ref] Autoimmune pancreatitis and complement activation system, Muraki [/bib_ref] However, a previous study showed that the classical pathway of complement activation through IgG1 may be involved in the development of AIP, as opposed to mannose-binding lectin or alternative pathways through IgG4. [bib_ref] Autoimmune pancreatitis and complement activation system, Muraki [/bib_ref] Autoantibodies and candidate of target antigens. Although some patients with IgG4-RD have non-specific antibodies such as an anti-nuclear antibody, this is rare. From the viewpoint of IgG4 function, it remains unclear whether IgG4-RD is an autoimmune or an allergic disease. Although diseasespecific targets are unknown, the occasional coexistence of OOIs leads us to consider that there may be common target antigens in the organs involved, especially in the pancreas, which exhibit a high coincidence. Among candidate antigens previously reported, lactoferrin, 65),66) carbonic anhydrase (CA)-II, 65)-68) CA-IV, [bib_ref] Serum antibodies to carbonic anhydrase IV in patients with autoimmune pancreatitis, Nishimori [/bib_ref] and pancreatic secretory trypsin inhibitor, [bib_ref] Identification of a novel autoantibody against pancreatic secretory trypsin inhibitor in patients..., Asada [/bib_ref] are distributed in the pancreas, salivary glands, biliary duct, lungs, and renal tubules, among others. Immunization with CA-II or lactoferrininduced systemic lesions such as pancreatitis, sialadenitis, cholangitis, or interstitial nephritis in mice models is similar to human IgG4-RD. [bib_ref] Experimental immunemediated pancreatitis in neonatally thymectomized mice immunized with carbonic anhydrase II..., Uchida [/bib_ref] Amylase ,-2A, [bib_ref] Amylase ,-2A autoantibodies: novel marker of autoimmune pancreatitis and fulminant type 1..., Endo [/bib_ref] heat shock 10 kDa protein 1, [bib_ref] HSP 10 is a new autoantigen in both autoimmune pancreatitis and fulminant..., Takizawa [/bib_ref] and Helicobacter pylori 74),75) are also disease-associated antigen candidates. Among the organs involved in IgG4-RD, recent studies suggest an extremely high association of pancreatic and biliary lesions. [bib_ref] Treatment of relapsing autoimmune pancreatitis with immunomodulators and rituximab: the Mayo Clinic..., Hart [/bib_ref] , [bib_ref] Obstructive jaundice in autoimmune pancreatitis can be safely treated with corticosteroids alone..., Bi [/bib_ref] Peribiliary glands in the biliary tract and pancreatic duct glands associated with pancreatic ducts in humans are intermingled with small amounts of pancreatic exocrine acini [bib_ref] A novel approach to biliary tract pathology based on similarities to pancreatic..., Nakanuma [/bib_ref] and biliary tree-derived stem cells constitute pancreatic organogenesis in mice. [bib_ref] Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible..., Wang [/bib_ref] Thus, Nakanuma et al. proposed a new concept of the "biliary diseases with pancreatic counterparts", [bib_ref] A novel approach to biliary tract pathology based on similarities to pancreatic..., Nakanuma [/bib_ref] in which targets of type 1 AIP and IgG4-SC may be periductal glands around the bile and pancreatic ducts. Further studies of the pathophysiology of the biliary tract based on its similarity to pancreatic counterparts are warranted.
Recently, two novel candidates of target antigens related to connective tissues in some patients with type 1 AIP have been reported; one is annex-in12 [bib_ref] Annexin A11 is targeted by IgG4 and IgG1 autoantibodies in IgG4-related disease, Hubers [/bib_ref] and the other is laminin 511. 79),80) Antiannexin A11 IgG4-antibodies were positive in 9/50 patients, and IgG1-Abs in 7/97 patients, and antilaminin 511 IgG4-and IgG1-antibodies were positive in a half of the patients studied.
Role of B cells. In addition to steroid and immunomodulators, the B-cell depletion by rituximab, which reduces only IgG4, but not IgG1, IgG2, or IgG3, is useful as a therapeutic strategy in IgG4-RD. 81),82) A recent study showed expansion of IgG4 D B-cell receptor clones in blood and tissue from patients with active IgG4-cholangiopathy, and disappearance of the clones with corticosteroid treatment. [bib_ref] Immunoglobulin G4D clones identified by next-generation sequencing dominate the B cell receptor..., Maillette De Buy [/bib_ref] A recent study showed that increased CD19 D CD24 high CD38 high Bregs may suppress the disease activity of type 1 AIP, whereas decreased CD19 D CD24 high CD27 D Bregs might be involved in the development of type 1 AIP. [bib_ref] The role of CD19 D CD24 high CD38 high and CD19 D..., Sumimoto [/bib_ref] These findings suggested that specific B-cell responses may play a pivotal role in the pathogenesis of IgG4-RD.
Th1 and Th2 immune balance. The effector cells in IgG4-RDs are poorly understood. CD4 D Tcells differentiate from naïve T-cells (Th0) into Th1, Th2, Th17, and Treg cells. In the livers of IgG4-SC patients, a Th2-type immune reaction 59), [bib_ref] Th2 and regulatory immune reactions contribute to IgG4 production and the initiation..., Tanaka [/bib_ref] is induced in addition to Th1 responses. 66),71) Th2 cytokines may be involved in progression of the disease process, especially through the maturation and proliferation of local B-cells and plasmacytes.
## Regulatory t cells.
Forkhead box P3 is a member of the forkhead/winged-helix family of transcriptional regulators, and functions as the master regulator in the development and function of CD4 D CD25 D Tregs. Forkhead box P3 is classified as a naturally occurring CD4 D CD25 D Treg (nTregs) that originates in the thymus, whereas adaptive Tregs are induced in the periphery by different antigens.In type 1 AIP, circulatory naïve (CD45RA D ) Tregs are significantly decreased in the peripheral blood, whereas memory (CD45RA ! )-Tregs are significantly increased. [bib_ref] Circulating naïve and CD4 D CD25 high regulatory T cells in patients..., Miyoshi [/bib_ref] In addition, prominent infiltration of Tregs with upregulation of IL-10 is observed in the liver of type 1 AIP and IgG4-SC patients. 59),62) These findings suggest that increased memory-Tregs in the periphery and local tissues may be an inhibitory immune response against inflammation, although decreased naïve Tregs may be pathogenic.
## Concluding remarks
Recent advances support the concept of IgG4-RD, a unique clinical entity, as a systemic disease, and the classification of two distinctive subtypes of AIP. Type 1 AIP is proposed as a pancreatic manifestation of IgG4-RD and type 2 AIP is associated with GELs and inflammatory bowel disease. To improve the diagnostic accuracy and monitoring of disease activity, novel biomarkers more specific for each subtype of AIP, other than serum IgG4 in type 1 and histological findings in type 2, should be established in the near future. In relapse or steroid refractory cases of type 1 AIP and IgG4-RD, alternative treatment should be established as well as factors for predicting relapse. In the pathogenesis of AIP/IgG4-RD, multi-pathogenic factors including genetic backgrounds, disease-specific antigens, and the role of IgG4 must be clarified.
[fig] Figure 1: Current concept of IgG4-related disease (cited from ref. 8 with permission). IgG4-RD contains multi-organ involvement simultaneously or metachronously. [/fig]
[fig] Figure 2: Pancreatic images of type 1 AIP (cited from ref. 1 with permission). a. Dynamic CT shows diffuse swelling of the pancreas with late phase enhancement and low-density rim. b. Endoscopic pancreatography shows diffusely irregular narrowing of the main pancreatic duct. c. Endoscopic cholangiography shows beaded like and long stenotic biliary duct similar to primary sclerosing cholangitis. [/fig]
[fig] Figure 3, Figure 4, Figure 5: /IgG-positive cells >40% have been reported in lymph nodes of patients with IgG4-RD. On the other hand, Lymphoplasmacytic sclerosing pancreatitis (LPSP) (cited from ref. 1 with permission). a. Abundant infiltration of plasma cells and lymphocytes around the pancreatic duct with fibrosis (H&E, #40). b. Abundant infiltration of plasma cells and lymphocytes around the pancreatic duct with fibrosis (H&E, #200). c. Storiform fibrosis (H&E, #400). d. Abundant infiltration of IgG4 positive plasma cells anti-IgG4 antibody staining (H&E, #400). e. Obliterative phlebitis (Evans staining, #400). Pancreatic images of type 2 AIP (cited from ref. 1 with permission). a. Dynamic CT shows diffuse swelling of the pancreas with late-phase enhancement and low-density rim (arrow) similar to type 1 AIP. b. Positron emission tomography-computed tomography (PET-CT) shows diffusely abnormal accumulation of FDG. c. Endoscopic pancreatography shows irregular narrowing of the main pancreatic duct. d. Colonoscopy shows proctitis type of ulcerative colitis. Granulocytic epithelial lesion (cited from ref. 1 with permission). a. Abundant infiltration of granulocytes into and around medium-sized pancreatic duct with destruction of epithelia (H&E, #40). b. Abundant infiltration of granulocytes into and around medium-sized pancreatic duct with destruction of epithelia (H&E, #200). [/fig]
[fig] Figure 6: Therapeutic algorithm for type 1 AIP (cited from ref. 26 with permission). This algorithm is based on Eastern and Western experts' opinions to recommend standard treatment of AIP worldwide. ICDC: International Consensus Diagnostic Criteria for autoimmune pancreatitis. OOI: other organ involvement. ST: steroid treatment. *: In some cases of mild jaundice without signs of infection, steroid treatment alone can be performed safely without biliary drainage. [/fig]
[table] Table 1: Comprehensive diagnostic criteria for IgG4-related disease, 2011 (cited from ref. 12 with permission) [/table]
[table] Table 2: Preferred nomenclature for individual organ manifestations of IgG4-related disease (cited from ref.3 with permission)IgG4-related kidney disease. The specific renal pattern should be termed IgG4-related tubulointerstitial nephritis and membranous glomerulonephritis secondary to IgG4-RD. Involvement of the renal pelvis should be termed IgG4-related renal pyelitis. [/table]
|
Intravenous Iron Isomaltoside 1000 Reduces Postoperative Anemia in Patients Undergoing Elective Urologic Surgery and Those with Urosepsis
Purpose: Postoperative anemia is associated with increased morbidity and mortality in patients undergoing surgery. Anemia is also a common feature during sepsis. Therefore, here, we aimed to investigate the safety and efficacy of intravenous iron isomaltoside 1000 (Monofer ® ) in patients undergoing elective urologic surgery and in those with urosepsis. Materials and Methods: This multicenter study was conducted through the review of the medical records of patients with postoperative anemia undergoing elective urologic surgery or with urosepsis in a multicenter hospital. Patients received a single intravenous iron isomaltoside (IIM), and their hemoglobin (Hb) level was evaluated before and after administration of IIM. Safety data included adverse effects and hypersensitivity reactions. In addition, the patients were divided into three groups (200 mg, 400 mg, and 600 mg IIM) to compare Hb changes before and after the administration of IIM. Results: The study analyzed 52 men and 30 women with a mean age of 67 years. There was a significant difference between pre-treatment Hb and post-treatment Hb according to the type of the surgery after administration (p=0.01) of IIM in patients with postoperative anemia, and the mean preoperative Hb before IIM administration was 8.5 g/dL and that after IIM administration was 9.9 g/dL (p=0.006) in patients with urosepsis. The mean preoperative Hb changed from 10 g/ dL to 11 g/dL after administration (p<0.001) of IIM in the whole cohort. There were no side effects due to the administration of intravenous IIM. Conclusion: A single perioperative intravenous injection of IIM 1000 significantly increased the Hb level in patients with anemia who underwent urologic elective surgery. Moreover, this treatment can be considered to have potential clinical benefits for anemia caused by sepsis.
# Introduction
Anemia is very common in patients with acute illness, including those in the intensive care unit. In 3534 ICU patients, Vincent et al observed mean hemoglobin (Hb) concentrations of 11.3 g/dL at admission, with 29% of these patients having a concentration of less than 10 g/dL. [bib_ref] Anemia and blood transfusion in critically ill patients, Vincent [/bib_ref] Many factors are known to influence the Hb level in critically ill patients; some of the important factors among these are sepsis, overt or occult blood loss, decreased production of endogenous erythropoietin, and functional iron deficiency. [bib_ref] Anemia in surgical intensive care patients, Viljoen [/bib_ref] Preoperative anemia was independently associated with increased morbidity and mortality in patients who underwent surgery. [bib_ref] the Reducing Bleeding in Cardiac surgery (RBC) investigators. Risk associated with preoperative..., Karkouti [/bib_ref] [bib_ref] Preoperative Hemoglobin level as a predictor of survival after coronary artery bypass..., Van Straten [/bib_ref] Untreated preoperative anemia and acute perioperative blood loss may increase surgical risk. Beattie et al [bib_ref] Risk associated with preoperative anemia in noncardiac surgery: a single-center cohort study, Beattie [/bib_ref] reported that preoperative anemia was associated with a nearly five-fold increase in the odds of postoperative mortality in noncardiac surgery.
Sepsis is the culmination of complex interactions between the infecting microorganism and the host immune, inflammatory, and coagulation responses. [bib_ref] The pathophysiology and treatment of sepsis, Hotchkiss [/bib_ref] Anemia is common in sepsis, in part because it is mediated by TNF-α and interleukin-1β and decreases the expression of the erythropoietin gene and protein. [bib_ref] Proinflammatory cytokines lowering erythropoietin production, Jelkmann [/bib_ref] Sepsis due to urological problems, including obstructive uropathy and acute prostatitis, is common and fatal. The mortality ranges from 28% to 50% in patients with severe sepsis. Transfusion is worthwhile if needed at the emergency stage of sepsis; Rivers et al observed a marked decrease in mortality due to sepsis when transfusion was provided early. [bib_ref] Early goal-directed therapy in the treatment of severe sepsis and septic shock, Rivers [/bib_ref] However, recent evidence has emerged to indicate that red blood cell transfusion may adversely impact patients who underwent surgery, causing higher mortality, increased risk of infection, and longer hospital and intensive care unit stays. [bib_ref] Perioperative anemia: an independent risk factor for infection, mortality, and resource utilization..., Dunne [/bib_ref] [bib_ref] Intraoperative transfusion of 1 U to 2 U packed red blood cells..., Bernard [/bib_ref] In addition, religious concerns, such as those posed by the Jehovah's Witnesses, may require a substitute for blood transfusion after elective surgery or sepsis. The use of erythropoiesis-stimulating agents and intravenous iron repletion have played a central role in the optimal correction of this anemia.
Therefore, here, we investigated the efficacy and safety of iron isomaltoside 1000 (Monofer ® ) in urologic sepsis and elective urologic surgery.
# Materials and methods
This study was conducted at two institutions (Hanyang University Seoul Hospital, Soonchunhyang University Seoul Hospital) in Korea from January to December 2017 and conducted in accordance with good clinical practice and the Declaration of Helsinki.
We performed our study after approving IRB from Hanyang University Hospital Institutional Review Board (IRB) (HY 2018-06-024-007). After approval was obtained with indication to have informed consent from IRB, written informed consent was obtained from all the patients. We reviewed the medical records of patients with postoperative anemia who were undergoing elective urologic surgery and patients with anemia due to sepsis. We defined anemic condition as patients with Hb ≤12.0 g/ dL (7.45 mmol/L) for women and Hb ≤13.0 g/dL (8.1 mmol/L) for men, according to the WHO criteria. First, we excluded patients with iron overload or disturbances in utilization of iron (eg, hemochromatosis and hemosiderosis), s-ferritin >800 ng/mL, and diagnosed with inflammatory bowel disease such as Crohn's disease and ulcerative colitis. Also we excluded patients with known hypersensitivity to any excipients in the investigational drug products, history of multiple allergies, decompensated liver cirrhosis and hepatitis, alanine aminotransferase >3 times the normal upper value, acute infection, and rheumatoid arthritis with symptoms or signs of active joint inflammation and pregnant or nursing women. In addition, patients with preoperative anemia and those who received blood transfusions due to urosepsis were excluded.
Iron isomaltoside (IIM) was administrated when postoperative anemia was observed on the first day after urologic elective surgery, while in the group of anemic patients with urosepsis, the administration was done on the day of admission. We measured the change in Hb in 7 days for both groups.
Patients receiving single doses of isomaltoside 1000 over 15 min were divided into three groups: 200 mg, 400 mg, and 600 mg. We decided the dose of IIM according to the degree of Hb change level.
Any concomitant medication or treatment deemed necessary to provide adequate supportive care was allowed throughout the trial, except for erythropoiesis stimulating agents and any iron supplementation other than the investigational drug, as they would have influenced the outcome of the study.
## Objectives and outcomes
The primary efficacy objective of the study was to evaluate the change in Hb level after IV IIM administration in patients with anemia undergoing elective urologic surgery and patients with anemia and urosepsis. Except for patients with severe anemia who required blood transfusion those who underwent urologic elective surgery, IIM was administrated. Then we assessed the safety (adverse events, vital signs, electrocardiogram (ECG), and biochemistry parameters).
## Statistical analyses
We analyzed demographics, change in hemoglobin (Hb) level, and Hb level according to the type of surgery using submit your manuscript | www.dovepress.com
## Dovepress
Drug Design, Development and Therapy 2020:14 the Chi-squared test or Wilcoxon test, as appropriate. All statistical analyses were computed using SPSS (version 22.0 for Windows). All statistical tests were two-tailed, and the significance level was <0.05.
# Results
## Patients
In total, 82 patients were included in this study from January to December 2017 [fig_ref] Table 1: Characteristics of Patients According to Group Notes [/fig_ref]. We classified the patients into two groups: patients with anemia that underwent urologic elective surgery (n=66) and anemic patients with urosepsis (n=16). Patients with urosepsis (70.1 years of age) were older than those undergoing urologic elective surgery (66.3 years of age). The patients were classified into three groups according to the dosage of iron isomaltoside 1000: 200 mg/mL (3.7%), 400 mg/mL (52.4%), and 600 mg/mL (43.9%). Patient demographics and baseline characteristics are summarized in [fig_ref] Table 1: Characteristics of Patients According to Group Notes [/fig_ref]. Overall, more men (63.4%) than women (36.6%) were included in this study.
## Efficacy of iron isomaltoside 1000 (iim)
The primary efficacy analysis was the change in Hb concentration from baseline, following administration of IIM. We observed a significant difference between pretreatment Hb and post-treatment Hb according to the type of surgery after IIM administration (p=0.01) in patients with postoperative anemia. The results of the Hb level change were measured by dividing patients into those who underwent stone surgery (including percutaneous nephrolithotomy) and those who underwent radical surgery and other endoscopic surgery. Seven days after surgery, Hb level had increased to an average of 11.4 g/ dL from 9.5 g/dL in the patients who underwent stone surgery, and also increased to an 11.0 g/dL from 10.1 g/dL in the patients who underwent other surgery. We also found a significant difference between stone surgery (including percutaneous nephrolithotomy) and other types of urologic surgery (p=0.009) [fig_ref] Table 2: Outcomes of Elective Surgery Type [/fig_ref]. The mean preoperative Hb before IIM administration was 8.5 g/dL, and it was 9.9 g/dL after IIM administration (p=0.006) in patients with urosepsis [fig_ref] Table 3: Comparison of Hb Change Before and After Treatment with Iron Isomaltoside 1000 [/fig_ref]. Mean preoperative Hb changed from 10 g/dL to 11 g/dL after administration (p<0.001) in the whole cohort.
The Hb level change differed according to the type of surgery in this cohort [fig_ref] Figure 1: The change of hemoglobin level according to the type of surgery [/fig_ref] , and the pre-treatment Hb level and post-treatment Hb level were different for each surgery type [fig_ref] Figure 2: The differences of hemoglobin level for each surgery type [/fig_ref]. The pre-treatment Hb level and post-treatment Hb level in patients with urosepsis were also different [fig_ref] Figure 3: The pre-treatment Hb level and post-treatment Hb level in patients with urosepsis [/fig_ref].
## Safety
Among the 82 patients available for safety analysis in the present study, no adverse drug reactions were reported to be related to the IIM. The hematological and biochemistry parameters and vital signs were checked at each trial visit, and no cases of hypophosphatemia (defined as a phosphate level <2 mg/dL) were observed.
# Discussion
This study is, to the best of our knowledge, the first cohort study to investigate the effects of intravenous iron
## 5681
administration in place of allogenic blood transfusion in patients with postoperative anemia in the field of urologic surgery. The etiology of anemia is diverse and includes blood loss, decreased RBC production owing to functional iron deficiency, and altered erythropoiesis in critically ill patients. In addition, perioperative anemia, which occurs in 33% to 50% of surgical patients, is one of the major predictive factors for allogenic blood transfusion (ABT) in surgeries with moderate to high perioperative blood loss and it induces postoperative anemia and aggravates existing anemia. [bib_ref] Very-short-term perioperative intravenous iron administration and postoperative outcome in major orthopedic surgery:..., Munoz [/bib_ref] ABT carries increased risks for transmission of infectious diseases, postoperative nosocomial infection and has been clearly demonstrated to be associated with adverse outcomes related to postoperative acute kidney injury, neurological complications and postoperative atrial fibrillation after cardiac surgery. [bib_ref] Red cell transfusion is associated with an increased risk for postoperative atrial..., Koch [/bib_ref] Despite efforts to reduce ABT, transfusion requirements still remain high. In this regard, a discussion on patient blood management (PBM) programs aim at identifying patients at risk for transfusions has been conducted in Europe. [bib_ref] Strategies to reduce the use of blood products: a European perspective, Theusinger [/bib_ref] PBM programs are based on multimodal approach including early preoperative recognition and treatment of anemic patients, surgical efforts to minimize blood loss. From this point of view, preoperative optimization of anemia seems to be a key aspect of PBM.
Iron deficiency is common, either owing to the depletion of whole-body iron stores or because of the restricted availability of iron for erythrogenesis (functional iron deficiency). Thus, iron therapy is commonly used to restore the iron reserve to prevent anemia after bleeding. Iron plays an essential role in erythropoiesis and hemoglobin synthesis. 14 Therefore, here, we tried to investigate the effect of the preoperative administration of intravenous iron isomaltoside 1000 (Monofer ® ) in patients undergoing elective urologic surgery. In our study, a significant elevation in Hb was observed without blood transfusion in endoscopic stone surgery and percutaneous nephrolithotomy (PNL), which can cause postoperative anemia or bleeding. The new IV iron supplement, iron isomaltoside 1000 (IIM), is composed of iron and chemically modified isomalto-oligosaccharides that have a mean molecular weight of 1000 Da and consist predominantly of 3-5 glucose units. In contrast to dextran polysaccharides present in iron dextran, isomaltoside is a linear and unbranched molecule with a theoretically reduced anaphylactogenic potential. [bib_ref] Successful prevention of an anaphylactoid reaction to iron dextran, Altman [/bib_ref] Physiochemically, the strong binding of iron within the IIM formulation enables a controlled and slow release of bioavailable iron to the iron-binding proteins, with only a low risk of free iron toxicity. [bib_ref] A comparative study of the physicochemical properties of iron isomaltoside 1000 (Monofer),..., Jahn [/bib_ref] This allows the rapid administration of high single doses of IIM. Several studies employing IIM have shown it to be well tolerated and to improve iron-related parameters in patients with various conditions, including patients with CKD receiving dialysis, 17 patients with chronic heart failure, patients with inflammatory bowel disease, [bib_ref] A 1-year trial of repeated high-dose intravenous iron isomaltoside 1000 to maintain..., Reinisch [/bib_ref] and patients undergoing cardiac surgery, 20 major orthopedic surgery, [bib_ref] Very-short-term perioperative intravenous iron administration and postoperative outcome in major orthopedic surgery:..., Munoz [/bib_ref] or major abdominal surgery. [bib_ref] The important role for intravenous iron in perioperative patient blood management in..., Froessler [/bib_ref] To date, clinical trials of IIM have demonstrated a good efficacy and a good safety profile, and systemic reviews and meta-analysis of randomized clinical trials comparing intravenous iron to other comparators have indicated that all currently available intravenous iron preparations are safe. [bib_ref] The safety of intravenous iron preparations: systematic review and meta-analysis, Avni [/bib_ref] [bib_ref] Efficacy and safety of ferric carboxymaltose and other formulations in iron-deficient patients:..., Rognoni [/bib_ref] Perioperative anemia is common among patients scheduled to undergo elective surgery and it is also a predictor of blood transfusion. The prevalence of perioperative anemia is reported to range from 5% to 75% of electively scheduled patients. [bib_ref] Detection, evaluation, and management of anemia in the elective surgical patient, Goodnough [/bib_ref] [bib_ref] Prevalence and outcomes of anemia in surgery: a systematic review of the..., Shander [/bib_ref] In our study, the average Hb level of 66 patients who were scheduled for elective surgery was 10.2 g/dL, which is quite low according to the definition provided by the World Health Organization. Van Straten et al 4 reported that a lower preoperative Hb level is an independent predictor of late mortality in patients undergoing coronary artery bypass grafting, whereas anemia is a risk factor for early and late mortality. Iron-deficiency anemia is the most common form of anemia and may be associated with other hematologic deficiencies, particularly among the elderly population; it compromises patients' ability to recover Hb after surgery. [bib_ref] Treatment of iron deficiency anemia in orthopedic surgery with intravenous iron: efficacy..., Theusinger [/bib_ref] Based on previous studies and our experiences, we expect intravenous iron administration to be able to reduce the incidence of postoperative infections associated with blood transfusion, reduce the Another notable result of our study was that intravenous iron administration caused a statistically significant elevation of the Hb level (from 8.5 g/dL to 9.9 g/dL) in patients with urosepsis. Consensus guidelines for the management of sepsis have been published. [bib_ref] Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock, Dellinger [/bib_ref] Early in the course of sepsis, the cornerstone of emergency management of sepsis is early, goaldirected therapy plus administration of lung protective ventilation broad-spectrum antibiotics. [bib_ref] Early goal-directed therapy in the treatment of severe sepsis and septic shock, Rivers [/bib_ref] [bib_ref] The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes..., Ibrahim [/bib_ref] Similar to the management of patients with sepsis, the guideline for patients with urosepsis recommends antibiotic treatment as soon as possible after diagnosis, but only after blood and urine cultures have been obtained (recommendation grade B, evidence level Ic). [bib_ref] Urosepsis-etiology, diagnosis, and treatment, Dreger [/bib_ref] According to the concept of early goal-directed therapy, hemodynamic stabilization promotes the delivery of an adequate oxygen supply to the tissues. Therefore, as soon as the diagnosis of urosepsis is suspected, the intravenous administration of isotonic crystalloid solution should be started, and blood products should be administered to target a hemoglobin level of 7-9 g/dL. Anemia is common in sepsis, and erythropoietin requires between days to weeks to induce red blood cell production; thus, it may not be effective. [bib_ref] Proinflammatory cytokines lowering erythropoietin production, Jelkmann [/bib_ref] To date, the criteria for which blood products should be used to correct anemia with supportive treatment in urosepsis are unclear and lack supporting evidence. In our study, 16 patients with urosepsis showed a significant increase in Hb level (from 8.5 g/dL to 9.9 g/dL) after the administration of IIM. Our study also showed that the degree of elevation of the Hb level was significantly greater in the group with initial Hb ≤10 g/dL compared to that in the group with initial Hb ≥11 g/dL when comparing the effects of intravenous IIM. Based on these findings, IIM may be an alternative to allogenic blood transfusion, with a reduced incidence of adverse effects due to blood transfusion, which may increase mortality.
To date, there are no clinical guidelines to support the use of prophylactic intravenous iron before major elective surgery. Therefore, we searched the literature in MEDLINE (January 1, 1990, through December 31, 2017) that examined intravenous iron therapy in patients scheduled to undergo elective surgery, except for patients with iron-deficiency anemia, inflammatory bowel disease, and chronic kidney disease. We selected and summarized five studies that were well-designed. One paper described treatment with IIM and four papers detailed treatment with iron sucrose [fig_ref] Table 4: Summary of Studies About Intravenous Iron Preparations for Patients Undergoing Elective SurgeryOne... [/fig_ref]. Owing to the differences in the type, timing, and dosage of the intravenous iron supplement used prior to surgery, the results of the Hb level and transfusion requirements were statistically different. When comparing our study with the PROTECT trial, which evaluated the effect of perioperative IV IIM in patients without anemia undergoing elective or subacute coronary artery bypass graft or valve replacement, we found that the Hb level had significantly increased, and fewer patients were anemic in the IV IIM-treated group in both studies. [bib_ref] Intravenous iron isomaltoside 1000 (MonoferVR) reduces postoperative anaemia in preoperatively non-anaemic patients..., Johansson [/bib_ref] This trial was an exploratory and retrospective study with the limitation of a small sample size of the patients undergoing elective urologic surgery. And the limitation of this study is that there was no data on iron-related parameters (transferrin saturation, ferritin, reticulocytes) as a retrospective study to find out the effects of IIM as an alternative to blood transfusion in patients who underwent urologic surgery with postoperative anemia. In this cohort, there was no control group receiving either placebo or comparing result about transfusion requirement. We only compared and reported the treatment before and after at anemic status. Designed as a retrospective study, there is a limitation that each group could be heterogenous. The novelties of this study are the feasibility of Intravenous IIM in patients with anemia after elective urologic surgery and the efficacy of Intravenous IIM in patients with anemia due to urosepsis.
## 5685
Hence, we are planning additional clinical studies employing a large, randomized, controlled, prospective trial to allow further evaluation of the differences in effect according to iron status (serum iron, ferritin, and transferrin saturation index) for the treatment of anemia in clinical settings.
# Conclusion
This study has demonstrated that IIM can be used safely and effectively to prevent anemia after elective urologic surgery and reduce the need for blood transfusion. The reviewed data in patients who underwent urologic elective surgery (n=66) and patients with anemia and urosepsis (n=16) showed adequate efficacy and a good safety profile of IIM. The results also showed that the hemopoietic response was evident in urosepsis. The safety profile of IIM is favorable. Therefore, we expect that the administration of IIM is attractive not only for patients with anemia that are undergoing urologic surgery, but also for patients with anemia due to sepsis.
## Abbreviations
IIM, iron isomaltoside; Hb, hemoglobin; ICU, intensive care unit; ABT, allogenic blood transfusion; PBM, patient blood management; PNL, percutaneous nephrolithotomy; CKD, chronic kidney disease.
[fig] Figure 1: The change of hemoglobin level according to the type of surgery. Drug Design, Development and Therapy 2020:14 submit your manuscript | www.dovepress.com DovePress 5683 length of hospital stay, and facilitate better recovery and outcome. [/fig]
[fig] Figure 3: The pre-treatment Hb level and post-treatment Hb level in patients with urosepsis. [/fig]
[fig] Figure 2: The differences of hemoglobin level for each surgery type. (A) Pre-treatment (B) Post-treatment. submit your manuscript | www.dovepress.com DovePress Drug Design, Development and Therapy 2020:14 [/fig]
[table] Table 1: Characteristics of Patients According to Group Notes: Urologic elective surgery*: Radical surgery (cancer), Stone surgery (endoscopic, PNL), Other endoscopic surgery. Drug Design, Development and Therapy 2020:14 submit your manuscript | www.dovepress.com [/table]
[table] Table 2: Outcomes of Elective Surgery Type [/table]
[table] Table 3: Comparison of Hb Change Before and After Treatment with Iron Isomaltoside 1000 [/table]
[table] Table 4: Summary of Studies About Intravenous Iron Preparations for Patients Undergoing Elective SurgeryOne month after surgery, Hb concentration had increased to an average of 12.6 g/dl vs 11.8 g/dl (p=00012) and significantly more patients were non-anaemic in the IV iron isomaltoside 1000-treated group compared to the placebo group (38.5% vs. 8.0%; p=0.019).Prospective study: 900 mg IV iron sucrose over 10 days starting 4 weeks before surgery Hb increased significantly (p<0.001), the mean maximum increase was 0.2-2.2 g/dl. The maximum increase of hemoglobin was observed 2 weeks after the start of IV iron treatment.Double-blind, randomized, placebo-controlled trial (Group 1: treated with 300mg of IV iron sucrose, group 2: treated with oral ferrous fumarate, group 3: oral and IV placebo)The use of IV or oral iron supplementation proved ineffective in correcting anaemia after cardiopulmonary bypass and did not reduce blood transfusion requirements Prospective, randomized study: divided into two groups, standard treatment or IV iron sucrose (600 mg) Transfusion requirements in patients with intracapsular fracture or baseline Hb level of 12 g/dL or more appear to be reduced by IV iron sucrose therapy, but there was no difference in morbidity, mortality, or length of hospital stay.Drug Design, Development and Therapy 2020:14 submit your manuscript | www.dovepress.com [/table]
|
From the Heart to the Lung: A Case of Drug Toxicity
# Background
Amiodarone, an iodinated benzofuran derivate, is a Vaughan-Williams class III antiarrhythmic drug used to treat ventricular and supraventricular dysrhythmias [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref]. The daily maintenance dose, which is usually the lowest effective dose, varies from 100 to 600 mg [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref]. Despite its excellent anti-arrhythmic effect, this drug is associated with a relatively high incidence of multiple organ toxicity, including thyroid, lungs, heart, eyes, skin, and liver. In about 1% to 5% of cases, lung toxicity occurs, and is the most serious adverse effect, depending on the dose of amiodarone [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref]. Pulmonary toxicity can be acute, subacute, or chronic. Amiodarone has a long half-life and high lipid solubility; it can accumulate in the lung parenchyma, liver, thyroid and adipose tissue. This explains why, even after discontinuation of the drug, adverse effects occur. The most important risk factors for amiodarone-induced pulmonary toxicity are older age, duration of therapy exceeding 2 months, and cumulative dose. There is evidence that exposure to supplemental oxygen, especially in high concentrations, can potentiate amiodarone-induced lung toxicity. The prognosis of amiodarone-induced lung toxicity is favorable [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity, Colby [/bib_ref]. It usually responds to amiodarone discontinuation, with or without steroids. Pulmonary changes can completely resolve, or some fibrosis may persist. Mortality is not easy to assess and varies among studies. More advanced age, the need for hospitalization, and acute respiratory distress syndrome (ARDS) are associated with higher mortality, reaching 50% [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref].
## Case report
We report the case of a 71-year-old woman with a medical history of multinodular goiter in euthyroidism, a former smoker with a 5-pack/year smoking history, with supraventricular and ventricular dysrhythmia, with several episodes of bigeminism and trigimenism, taking amiodarone 200 mg/day for 18 months. She had no other known diseases or environmental exposure to known risk factors.
The patient presented to our Emergency Department with a 2-month history of dry cough, dyspnea on exertion with progressive worsening, and weight loss of 5 kg in this period. She denied orthopnea, night sweats, and fever. The patient did not complain of any other symptoms suggestive of other organ involvement.
At presentation, she was afebrile (36.5ºC), with normal blood pressure (130/75 mmHg) and a normal heart rate (78 bpm). She was eupneic, with peripheral oxygen saturation 97% on ambient air. Cardiopulmonary auscultation revealed an irregular pulse. Bilateral crepitations were worse at bases. The rest of the examination was unremarkable.
The laboratory tests revealed: hemoglobin 12.8 g/dL; white blood cell count 7280 cells/μL; 65.4% neutrophils, C-reactive protein 1.0 mg/dL, platelet count 350 000/μL, LDH 280U/L, brain natriuretic peptide 50 pg/mL, TSH 1.69 μUI/L, free T4 1.36 ng/dL; free T3 1.81 pg/mL. Autoimmune markers were negative (antinuclear antibodies; rheumatoid factor; anti-DNA; anti-Ro and La; anti-centromere; antineutrophil cytoplasmic antibody, and anti-ribonuclear protein).
The chest X-ray [fig_ref] Figure 1: Chest X-ray -lung bases with bilateral interstitial infiltrates [/fig_ref] showed bilateral interstitial infiltrates.
A high-resolution computed tomography (HRCT) lung scan [fig_ref] Figure 2: Axial reconstruction of chest HRTC, lung [/fig_ref] showed extensive areas of bilateral fibrosis and pleural thickening, predominantly basal, associated retractable bronchiectasis and ground-glass opacities, with a scattered and nodular pattern.
Flexible bronchoscopy had no relevant findings. The transbronchial lung biopsy performed revealed mild fibrosis and an inflammatory process of the parenchyma. These findings were suggestive of interstitial lung disease.
Bronchoalveolar lavage was negative for microbiological, mycological, and Mycobacterium smears, and cultures were negative. Malignant cell research was also negative. The cytomorphological study showed 526/uL nucleated cellular elements, with lymphocytosis of 80% and 16% eosinophils. Immunophenotyping showed 64.36% CD3+/CD4+/CD8+ T lymphocytes. Lung function tests showed a decreased diffusing capacity (DLCO) with moderate impairment of the alveolar-capillary transfer of CO, without other alterations. These changes were admitted as a pattern of fibrous interstitial pneumonia associated with Given her good general condition and the absence of hypoxemia, it was decided not to start corticosteroid therapy and to do close surveillance of the patient. She was then discharged to outpatient follow-up.
At a 2-month follow-up visit, the patient had radiological improvement.
At a follow visit 8-month after amiodarone withdrawal the patient was asymptomatic. She presented with radiologic and lung function tests, including DLCO improvement.
At 18-month follow-up, the patient had experienced no relapse.
# Discussion
Amiodarone-induced lung toxicity is one of the most serious effects in patients on this therapy. Pulmonary toxicity can be divided into manifestations associated with the parenchyma or pulmonary interstitium [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref]. Factors such as age, doses higher than 400 mg/day, duration of treatment, the existence of earlier respiratory diseases, and thoracic or non-thoracic surgery seem to influence the appearance of lung injury [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref]. Overall, low-dose amiodarone seems safer. However, lung toxicity can occur with doses lower than 200 mg/day, especially for periods longer than 2 years [bib_ref] Pulmonary toxicity in patients receiving low-dose amiodarone, Ott [/bib_ref]. Our patient was taking amiodarone for less than 2 years and at a low daily dose of 200 mg. Even so, there is evidence that a 2-month period is enough for lung toxicity to develop. In this case, age and treatment duration were risk factors.
Symptoms may be absent or include a nonproductive cough, dyspnea, weight loss, fever, and pleuritic chest pain [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref] [bib_ref] A crazy cause of crazy paving: An unusual case of amiodarone induced..., Mahmoud [/bib_ref]. In this case, the patient had nonspecific symptoms compatible with the findings described in the literature.
Pulmonary involvement can present in various ways, including, interstitial pneumonitis with different degrees of fibrosis, eosinophilic pneumonia, organizing pneumonia, diffuse alveolar hemorrhage, ARDS, pulmonary masses or nodules, and, rarely, pleural disease, and exudative pleural effusions isolated or occur in association with interstitial pneumonitis [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref] [bib_ref] A crazy cause of crazy paving: An unusual case of amiodarone induced..., Mahmoud [/bib_ref] [bib_ref] Eosinophilic pneumonia: A rare manifestation of amiodarone toxicity diagnosed using traditional bronchoscopy, Levee [/bib_ref]. Interstitial pneumonitis is the most common presentation of amiodarone-induced lung toxicity, and appears after 2 or more months of therapy [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref]. Amiodarone-induced pulmonary toxicity can be found unilaterally [bib_ref] A crazy cause of crazy paving: An unusual case of amiodarone induced..., Mahmoud [/bib_ref].
The physiopathology behind amiodarone-induced lung toxicity is not well understood. Different hypotheses have been suggested, including direct toxicity to interstitial and alveolar cells by alteration of intracellular metabolic pathway and an immunologic reaction with lymphocytic infiltration, and stimulation of the renin-angiotensin-aldosterone system that may lead to lung cells apoptosis [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity, Colby [/bib_ref].
Pulmonary function testing in amiodarone-induced pulmonary toxicity often shows a restrictive pattern and a reduction in DLCO, but these findings are nonspecific [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref].
This is a diagnosis of exclusion, and cardiac heart failure and infection need to be excluded in all cases. Idiopathic and secondary interstitial lung diseases are also in the differential diagnosis [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref] [bib_ref] Adverse reactions of Amiodarone, Biancatelli [/bib_ref] [bib_ref] Amiodarone-induced pulmonary toxicity with an excellent response to treatment: A case report, Terzo [/bib_ref]. The clinical, laboratory, and radiologic evaluation are essential to achieve the diagnosis. The diagnosis of amiodarone-induced lung toxicity is difficult to establish because it lacks specific respiratory symptoms.
In most cases, the prognosis is favorable after drug withdrawal. The disease usually responds to amiodarone discontinuation within 1 to 6 months [bib_ref] Amiodarone: Review of pulmonary effects and toxicity, Papiris [/bib_ref]. In our case, in a short period of 2 months, the patient showed clinical, radiological, and functional improvement. The good clinical evolution remained at 8-month reassessment. During the follow-up of the patient, there were no recurrences.
# Conclusions
Although amiodarone toxicity is widely known, this case stands out by its presentation of amiodarone-induced lung toxicity with parenchymal and pleural manifestations. This was a successful case, with an excellent outcome. The patient showed clinical and radiological improvement associated with normal DLCO values. Discontinuation of amiodarone is the primary treatment of all forms of lung toxicity. Not all cases need systemic corticosteroid therapy, as in this case, which was clinically mild, without hypoxia. Steroids are indicated in clinically severe cases. It also highlights that interstitial lung disease associated with amiodarone toxicity may be reversible on discontinuation of the drug. It is important to consider this diagnosis in patients with respiratory symptoms who are receiving amiodarone therapy, even in low drug doses, as in the present case. An early diagnosis and immediate withdrawal of the drug with or without corticosteroids are crucial to obtain a good outcome.
This clinical case highlights the importance of weighing the risks and benefits of using this drug, especially for long periods. Patients taking amiodarone and presenting with increased risk factors for drug toxicity may benefit from close surveillance monitoring.
[fig] Figure 1: Chest X-ray -lung bases with bilateral interstitial infiltrates. [/fig]
[fig] Figure 2: Axial reconstruction of chest HRTC, lung (A-D) and mediastinal (E, F) window: extensive fibrotic areas are observed in both pulmonary fields, predominantly at the base, and pleural thickening, associated retractable bronchiectasis, and with groundglass opacities. toxicity. Give the high suspicion of drug-induced lung toxicity, amiodarone was suspended. [/fig]
[fig] Figure 3: (A-D) Axial reconstruction of chest CT, lung window: evaluation 8 months after drug withdrawal with disappearance of the ground-glass opacities, and resolution of parenchymal and pleural fibrotic changes. [/fig]
|
Scleromyxedema in a 21 year old female patient with acute lymphoblastic leukemia: a case report
Background: Scleromyxedema is a rare, para-neoplastic, chronic, progressive condition of the Lichen myxedematosus (LM) family. The clinical picture consists of generalized confluent papular eruptions with possible systemic manifestations, which may be fatal as it still constitutes a therapeutic dilemma. Histologically, it is characterized by dermal mucin deposition, fibroblast proliferation with fibrosis, with monoclonal gammopathy in the absence of thyroid disease. Some atypical forms of the disease were reported in the literature, but none were reported in acute leukemia. Case presentation: Herein, we report a case of a 21 years old female patient, known case of acute lymphoblastic leukemia (ALL), who developed numerous hyper-pigmented erythematous papules and plaques, mainly over her thighs, lower abdomen, and sub-mammary flexures. Histopathology of skin lesions confirmed the diagnosis of atypical scleromyxedema. Her symptoms significantly improved with the use of high dose intravenous immunoglobulin (IVIG). Conclusions: Despite that scleromyxedema is associated with many hematologic disorders, it is very rarely associated with acute lymphoblastic leukemia, and a high index of suspicion is needed for diagnosis. IVIG remains a reasonable management of such a disabling disease.
# Background
Cutaneous mucinoses are a group of rare dermatological disorders, in which there is an excessive accumulation of mucin in the skin. Their clinical presentation may vary considerably making them similar to many other systemic diseases. Cutaneous mucinoses are divided into 2 main broad categories: the primary mucinoses, in which the mucin deposit is the main histologic feature producing a clinically distinctive lesion, and the secondary mucinoses, where other disorders show mucin deposition as a secondary response. Diagnosis of dermal mucinoses is particularly challenging. Lichen myxedematosus (LM) is an idiopathic form of primary cutaneous mucinosis. LM includes 3 clinico-pathologic subsets, which are summarized in, with their diagnostic criteria.
In some cases, LM may be associated with certain diseases, including multiple myeloma and lymphoma. It also can lead to deleterious systemic consequences, which include: dermato-neuro syndrome, and cardiomyopathy. The treatment of these disorders is difficult and often ineffective. Many therapeutic approaches have been tried. However, until the date of this report, no specific approach proves to be superior to another, despite that intravenous immunoglobulin (IVIG) is becoming more utilized as a treatment.
A search of the literature found very few case reports of cutaneous mucinosis in ALL patients. Herein, we report a case of cutaneous mucinosis in ALL patient, for which IVIG was effective as a treatment.
## Case presentation
We present a case of 21 year old female patient, with unremarkable medical, family, and psychological history, who was diagnosed with ALL. The patient was on the appropriate chemotherapeutic regimen in the hematology ward. There, she developed post chemotherapy pancytopenia with neutropenic sepsis, which was treated with appropriate intravenous antibiotics (tigecycline, colistin, meropenem, and ceftazidime), and blood product transfusion as needed.
During her hospitalization, and due to the low platelet counts, the patient developed a pulmonary hemorrhage that required intubation and prolonged mechanical ventilation in the medical intensive care unit (ICU), during which her cell counts have recovered, but she remained in sepsis in the form of intractable fever with positive sputum and blood cultures for Pseudomonas aeruginosa.
During her ICU stay, patient started to develop multiple, well defined velvety, thickened, darkly pigmented papules over her thighs, external genitalia, lower abdomen, and sub-mammary flexures, which gradually enlarged, merged, and thickened to form hyper-pigmented infiltrated plaque (shown in . These lesions were associated with intractable fever which was not fully explained by a persistent sepsis, as the latter was well controlled with hemodynamic stability and normalization of her inflammatory markers.
Her complete blood count, routine examination and culture of urine, liver function test, renal function test, serum protein electrophoresis, blood sugar and thyroid profile were within the normal limits. Computed Tomography scan of her body during a routine evaluation of her persistent fever and sepsis showed incidental left thalamic ischemic stroke and multiple splenic infarctions. Trans-esophageal echocardiography was non contributive.
Based on our clinical suspicion of scleromyxedema, a skin biopsy was performed and revealed abundant amounts of dermal amorphous mucinous material separating increased thickened collagen bundles, which was confirmed by mucicarmine special stain. In addition, the biopsy showed proliferation of stellate fibroblasts, and superficial and deep perivascular lympho-plasmacytic infiltrates (shown in.
The patient was started on high dose IVIG (2 g/kg) given over 2 days, in monthly cycles for a total of 6 months, and this was well tolerated. After starting the first dose of IVIG, our patient's state considerably ameliorated in the form of improvement of cutaneous lesions (shown in and complete disappearance of her intractable fever.
# Discussion and conclusion
Scleromyxedema is considered one of the rare dermatologic diseases with a chronic, progressive course of unknown etiology. Scleromyxedema is considered one of the paraneoplastic dermatoses. The pathogenesis of scleromyxedema is unknown, but cytokines such as interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta, known to stimulate fibroblast proliferation in the skin, could play a role. Clinical remission following stem cell transplantation suggests that the bone marrow may be a source of these circulating factors.
Some patients have atypical forms with features intermediate between generalized and localized LM. - Generalized papular and sclerodermoid eruption.
- Mucin deposition, fibroblast proliferation, and fibrosis.
- Monoclonal gammopathy.
- Absence of thyroid disease.
- Papular or nodular plaques.
- Mucin deposition with variable fibroblast proliferation.
- Absence of both: monoclonal gammopathy and thyroid disease.
- Features that mix between the generalized and the localized forms.
## Fig. 1 infiltrated plaques on the thigh
Clinically, it is characterized by indurated erythematous, waxy papules, disseminated on the face, chest and limbs, that then coalesce to form generalized plaques causing extensive thickening and hardening of skin. Comparing this with our patient, we can find that she has the typical skin lesions of this disease, but a different distribution pattern notably the lack of involvement of the face and upper trunk. As a para-neoplastic disease, different malignancies were reported in the literature in association with scleromyxedema. These included, but not limited to, monoclonal gammopathy, lymphoma, advanced gastric cancer, and very rarely, acute leukemia. Scleromyxedema can lead to disabling systemic manifestations, these include: dysphagia, myopathy, cardiomyopathy, central nervous system involvement, including: intractable fever, convulsion, and coma.
According to the clinical presentation and histopathological evidence, the diagnosis of atypical scleromyxedema was established in our patient, with systemic manifestations that might be related to her disease.
Literature review revealed no consensus regarding the therapeutic approach. Multiple regimens were used. These included the use of melphalan chemotherapy with autologous bone marrow transplant, and thalidomide with prednisolone. High dose IVIG has recently become a more popular option and has been a first line strategy used in the recently reported cases. For example, high dose IVIG for 6 months was used in a patient who was diagnosed with scleromyxedema and polyneuropathy, with improvement started after the first infusion of IVIG, and complete resolution was achieved after 3 infusions. Another example of the effectiveness of IVIG was reported in 2 cases of scleromyxedema at different age groups, where skin lesions dramatically improved after the first 2 infusions of high dose IVIG (2 g/kg/month). Our case report supports the hypothesis that high dose IVIG could be adopted in treating scleromyxedema.
In conclusion, scleromyxedema is a rare but disabling skin disease. It is very rarely associated with ALL. Atypical forms require a high index of suspicion for diagnosis. A constellation of clinical and pathological features is needed for diagnosis. The management of scleromyxedema is controversial. Based on the improvement of the symptoms noted in our case, we conclude that high dose IVIG could be beneficial in treating this disease in ALL patients, supporting the recently published literature. |
Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020
Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named 'spore crystal association' (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.
# Introduction
Bacillus thuringiensis is a Gram-positive, spore-forming bacterium with one of its most important features being the formation of parasporal crystals. The insecticidal activity of B. thuringiensis lies primarily in its parasporal crystals. B. thuringiensis strains carry one or more different crystal genes which are usually located on large, transmissible plasmids [bib_ref] Bacillus thuringiensis and its pesticidal crystal proteins, Schnepf [/bib_ref]. Crystal protein is typically deposited against the forespore and develops outside the exosporium. Parasporal crystals are then separated from spores after lysis of the mother cell. However, in a few strains, such as in B. thuringiensis subsp. finitimus strains [bib_ref] Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus, Debro [/bib_ref] [bib_ref] Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp...., Wojciechowska [/bib_ref] and B. thuringiensis subsp. oyamensis strain LBIT-113 [bib_ref] Characterization of a Novel Strain of Bacillus thuringiensis, Lopez-Meza [/bib_ref] , the parasporal crystals are located between the exosporium and the spore coat and continue to adhere to the spore after mother cell lysis. This phenotype has been previously described as spore-crystal association (SCA) [bib_ref] Promoters of crystal protein genes do not control crystal formation inside exosporium..., Ji [/bib_ref].
The SCA phenotype was identified a half century ago [bib_ref] Fowler's bacillus and its parasporal body, Hannay [/bib_ref]. SCA strains were originally designated as Bacillus finitimus; ''finitimus'' meaning ''neighboring'' or ''adjacent'' in Latin, and this species was later made a subspecies of B. thuringiensis [bib_ref] The fine structure of Bacillus finitimus and Bacillus thuringiensis spores with special..., Short [/bib_ref]. Debro et al. [bib_ref] Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus, Debro [/bib_ref] described that the SCA phenotype depended on a 98 MDa plasmid, suggesting that the plasmid contained all the genes essential for crystal formation within the exosporium, and that the inclusion that formed within the exosporium contained a major polypeptide of approximately 135 kDa. Two crystal protein encoding genes, cry26Aa1 and cry28Aa1, were identified from B. thuringiensis subsp. finitimus B-1166 VKPM; encoding 131 and 125 kDa proteins respectively [bib_ref] Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp...., Wojciechowska [/bib_ref]. An unusual nontoxic strain of B. thuringiensis subsp. oyamensis was isolated from living larvae of Anopheles pseudopunctipennis [bib_ref] Characterization of a Novel Strain of Bacillus thuringiensis, Lopez-Meza [/bib_ref]. In this strain, the crystal protein was found to be enclosed within the exosporium and composed of two proteins of 88 and 54 kDa. A survey of B. thuringiensis strains isolated from Spanish citrus orchards, conducted by Vidal-Quist et al. [bib_ref] Diversity of Bacillus thuringiensis strains isolated from citrus orchards in Spain and..., Vidal-Quist [/bib_ref] , showed that 25 out of 376 strains produced crystals that adhered to spores. Four morphological types of crystals with four different protein profiles were described using SDS-PAGE.
To date, the gene(s) conferring such localization have not been reported. B. thuringiensis subsp. finitimus strain YBT-020 is a typical strain with the SCA phenotype. In our previous studies, two crystal protein genes, cry26Aa and cry28Aa, were isolated from this strain. When cry26Aa and cry28Aa were transferred into the acrystalliferous B. thuringiensis strain BMB171, the crystals were separated from the spores after mother cell lysis, even when they were transferred into the plasmid-cured strain of YBT-020, that had been cured of all plasmids. These results revealed that the expression of cry26Aa and cry28Aa alone from their own promoters was not sufficient for SCA phenotype [bib_ref] Promoters of crystal protein genes do not control crystal formation inside exosporium..., Ji [/bib_ref].
To isolate the key genes that determining the SCA phenotype in strain YBT-020, the following work was performed: testing which plasmid was required for the SCA phenotype by plasmid conjugation, and then constructing a shuttle bacterial artificial chromosome (BAC) library for complementation testing to enable screening of clones exhibiting the SCA phenotype. We found that two native, large plasmids, pBMB26 and pBMB28, were essential for the formation of SCA phenotype, and two 35 kb fragments located on plasmids pBMB26 and pBMB28 were able to recover the SCA phenotype in an acrystalliferous mutant strain BMB171. Deletion analysis and gene disruption indicated that six genes are indispensable for the SCA phenotype in strain YBT-020.
# Results
Discovery of native plasmids pBMB26 and pBMB28 determining the SCA phenotype Strain YBT-020 harbors two native plasmids, named pBMB26 and pBMB28. In our previous study [bib_ref] The plasmid harboring cry26Aa may contribute to the phenomenon of spore-crystal connection..., Jv [/bib_ref] , a plasmid-curing experiment suggested that plasmid pBMB26, harboring the crystal protein gene cry26Aa, was indispensable for the SCA phenotype in strain YBT-020. To test whether plasmid pBMB26 contains all the genes essential for the SCA phenotype, a plasmid conjugation experiment was performed. The conjugational donor, strain BMBJ1, was generated by inserting a chloramphenicol resistance cassette into gene cry26Aa via homologous recombination. By screening of the acrystalliferous mutant strain BMB171, a spontaneous rifampin resistance mutant was obtained. Plasmid pBMB0617, harboring gene cry26Aa, was transformed into this strain to generate the recipient strain BMB171R1. Two kinds of phenotype were isolated from the transconjugants, with crystals enclosed inside spores [fig_ref] Figure 1: Micrographs of parasporal crystals from transconjugants [/fig_ref] , or crystals separated from spores [fig_ref] Figure 1: Micrographs of parasporal crystals from transconjugants [/fig_ref]. The morphology of fifty randomly selected transconjugants was observed, and the ratio of the two phenotypes was found to be approximately 1:4.
Based on the complete sequences of plasmids pBMB26 and pBMB28 [bib_ref] Complete Genome Sequence of Bacillus thuringiensis Serovar finitimus Strain YBT-020, Zhu [/bib_ref] , PCR primers were designed to detect the existence of both plasmids. PCR verification showed that the strains with separated crystals contained plasmid pBMB26, while the strains with the SCA phenotype contained the two plasmids. This mating experiment suggested that the two plasmids together determined the SCA phenotype.
Characterization of fragments from pBMB26 and pBMB28 recovering the SCA phenotype in BMB171
To locate the critical regions detemining the SCA phenotype, six fragments covering the full length of pBMB26, and four others covering pBMB28, were screened from the shuttle BAC library of strain YBT-020. The plasmid pBMB26-cured mutant BMB1151 of strain YBT-020 [bib_ref] Complete Genome Sequence of Bacillus thuringiensis Serovar finitimus Strain YBT-020, Zhu [/bib_ref] , in which crystals were not formed [fig_ref] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h [/fig_ref] , was used as a host strain to locate the crucial region for the SCA phenotype within plasmid pBMB26. Six BAC clones, covering different regions of pBMB26, were transferred into mutants BMB1151 and BMB171 by electroporation. The SCA phenotype of the transformants was detected by microscopic observation after sporulation. A fragment of 35 kb (pBMB275), carrying cry26Aa, was confirmed to direct the formation of crystals adhering to spores, and to recover the SCA phenotype in the pBMB26-cured mutant BMB1151 of strain YBT-020 [fig_ref] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h [/fig_ref] , but not in acrystalliferous mutant BMB171 [fig_ref] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h [/fig_ref]. This further suggested that both pBMB26 and pBMB28 were indispensable to the SCA phenotype. Following this, we determined the location of the crucial region within plasmid pBMB28 that was essential to the SCA phenotype. As a requirement of resistance screening, the 35 kb fragment (pBMB275) was inserted into another shuttle BAC vector pEMB0603, to give rise to pBMB275A. Using the same methods, four BAC clones, covereing different regions of pBMB28, were transferred into strains BMB171/pBMB275A and BMB171. The SCA phenotype of the transformants was detected after sporulation. A 35 kb fragment (pBMB251) was demonstrated to recover the SCA phenomenon in strain BMB171/pBMB275A [fig_ref] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h [/fig_ref] , but not in strain BMB171 [fig_ref] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h [/fig_ref]. Thus, by large fragment complementation testing, two 35 kb fragments, from plasmids pBMB26 and pBMB28, were identified to determine the SCA phenotype.
Determination of minimal regions essential to the SCA phenotype Firstly, pBMB251A (a fragment of pBMB28), was constructed by inserting the 35 kb fragment from pBMB251 into shuttle BAC vector pEMB0603, and then transferring this into BMB171. The subclones of the 35 kb fragment within pBMB275 (a fragment of pBMB26) were then transferred into strain BMB171/pBMB251A. The results demonstrated that a minimal fragment of 4 kb (pBMB0617), carrying an intact cry26Aa gene was sufficient for the SCA phenotype [fig_ref] Figure 3: The search for minimal DNA fragments essential to the SCA phenotype in... [/fig_ref] , [fig_ref] Table 1: ORFs involved in the spore-crystal association [/fig_ref]. Following this, the subclones of the 35 kb fragment within pBMB251 (a fragment of pBMB28), were transferred into strain BMB171/pBMB275A. A 7 kb fragment (pBMB251B2), was confirmed as the minimal sufficient fragment for the SCA phenotype [fig_ref] Figure 3: The search for minimal DNA fragments essential to the SCA phenotype in... [/fig_ref]. Sequence analysis showed that this 7 kb fragment carried five putative genes [fig_ref] Table 1: ORFs involved in the spore-crystal association [/fig_ref]. Three partial sequences from this fragment could not recover the SCA phenotype in strain BMB171/pBMB275A, and separated crystals were formed after sporulation [fig_ref] Figure 4: Phase-contrast micrographs of different plasmid derivatives of pBMB275 and pBMB251 in strain... [/fig_ref]. The combination of the above described 4 kb and 7 kb fragments in strain BMB171 led to the SCA phenotype [fig_ref] Figure 4: Phase-contrast micrographs of different plasmid derivatives of pBMB275 and pBMB251 in strain... [/fig_ref]. This meant that the two fragments of 4 kb and 7 kb, from pBMB26 and pBMB28 respectively, contained the critical genetic information for the SCA phenotype. (Nucleotide sequences of two fragments have been deposited in GenBank under accession numbers DQ242519 and HQ695909).
DNA sequence analysis of the 7 kb fragment revealed that it contained five ORFs [fig_ref] Table 1: ORFs involved in the spore-crystal association [/fig_ref]. The genes orf1 and orf2 encoded the putative peptides of 300 and 268 amino acid residues with predicted molecular weights of 34,509 daltons and 30,872 daltons respectively. These two proteins showed low similarity with the crystal proteins NT40KD and NT32KD [bib_ref] Cloning and characterization of two novel crystal protein genes from a Bacillus..., Kim [/bib_ref] , exhibiting 30% and 22% sequence identity. Gene orf1, located 101 nucleotides upstream of orf2 in the same orientation, was preceded by potential ribosome-binding sites. No putative promoter was found upstream of orf2. It is probable that the two genes are involved in a single operon. The genes orf3, orf4, and orf5 were similar to the genes encoding the germination complex, and were particularly similar to the spore germination genes pBt086, pBt085, and pBt084, which are located on the plasmid pBtoxis. These had 58%, 54%, and 44% amino acid sequence identity with Orf3, Orf4, and Orf5 [bib_ref] Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis..., Berry [/bib_ref] [bib_ref] Genetic basis for alkaline activation of germination in Bacillus thuringiensis subsp. israelensis, Abdoarrahem [/bib_ref]. This germination complex operon has been demonstrated to be a single operon by transcriptional analysis [bib_ref] Transcriptional analysis of the toxin-coding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis, Stein [/bib_ref]. The three orfs shared sequence overlaps; orf3, for example, was shown to overlap with orf4 by 8 bp, while orf4 overlapped with orf5 by 4 bp. This suggests that the three genes are organized within a single operon.
## Construction and analysis of mutant strains at the critical genes
To confirm that the six genes were involved in determining the SCA phenotype in strain YBT-020, three mutant strains were constructed and characterized. The operon containing orf1 and orf2 was disrupted to create mutant strain BMBJA. We observed that the crystals of this mutant were formed outside the exosporium during sporulation [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref] , and were separated from the spores after sporulation [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref]. In complementation experiments, the plasmid pBMB251B5 carrying orf1 and orf2 (2.9 kb, [fig_ref] Figure 3: The search for minimal DNA fragments essential to the SCA phenotype in... [/fig_ref] , was capable of recovering the SCA phenotype in BMBJA [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref].
To confirm that orf1 and orf2 are necessary to the SCA phenotype, a frameshift was introduced into these genes to generate plasmids pBMB251B5A and pBMB251B5B respectively (see Materials and Methods), These plasmids were transferred into strain BMBJA. No SCA phenotyope was observed in the transformants [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref]. The combination of the two frameshift mutant plasmids in strain BMBJA resulted in the SCA phenotype (data not shown). This indicated that both the products of orf1 and orf2 are indispensable for the SCA phenotype. The operon containing orf3, orf4, and orf5, was disrupted, resulting in mutant strain BMBJB. Microscopic observation revealed that the crystals of this mutant formed inside the exosporium during sporulation [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref] and the SCA phenotype remained after sporulation [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref] , However, the SCA phenotype in the mutant appeared impermanency compared with wild strain YBT-020. After growth for 100 h, the crystals were no longer attached to the spores [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref]. This observation suggested that the products of orf3, orf4, and orf5 were only essential in maintaining the stability of the SCA phenotype, and were not involved in the formation of the SCA phenotype in strain YBT-020. In complementation experiments, the plasmid pBMB251B3 carrying intact orf3, orf4, and orf5 (5.3 kb, [fig_ref] Figure 3: The search for minimal DNA fragments essential to the SCA phenotype in... [/fig_ref] was demonstrated to be capable of restoring the stability of the SCA phenotype in BMBJB [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref].
Strain BMBJ1 was a cry26Aa gene disruption mutant from strain YBT-020. Microscopic observation revealed that BMBJ1 had lost the ability to form crystals [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref]. After the plasmid pBMB0617 harboring the cry26Aa gene was transferred into BMBJ1, the crystals were observed and the SCA phenotype was regained [fig_ref] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains [/fig_ref]. This confirmed that the crystal protein gene, cry26Aa, was essential for the SCA phenotype. Through the characterization of mutant strains, we thus revealed that the six genes were indispensable to the formation of the SCA phenotype in strain YBT-020.
Crystal proteins that are normally deposited outside the exosporium were not able to restore the SCA phenotype in BMBJ1
In a previous study [bib_ref] Promoters of crystal protein genes do not control crystal formation inside exosporium..., Ji [/bib_ref] , we found that the Cry1Ca protein could not be deposited inside the exosporium in strain YBT-020. To determine if the crystal proteins that were normally deposited outside the exosporium were able to replace Cry26Aa and restore the SCA phenotype in BMBJ1, several crystal protein genes, including cry1Ac, cry2Aa, cry5Ba, cry6Aa, cry7Ba, cry51Aa, and cry55Aa, were transferred into mutant strain BMBJ1 to detect the formation of the SCA phenotype. Microscopic observation revealed that only separated crystals were formed (data not shown). This suggested that the Cry26Aa protein was specific to the formation of the SCA phenotype in strain YBT-020.
# Discussion
Aronson [bib_ref] Sporulation and delta-endotoxin synthesis by Bacillus thuringiensis, Aronson [/bib_ref] proposed two possible mechanisms for SCA phenotype formation. In the first, he suggested that the synthesis time of the crystal protein was synchronized with the formation of the exosporium. In a previous study [bib_ref] Promoters of crystal protein genes do not control crystal formation inside exosporium..., Ji [/bib_ref] , we demonstrated that crystal protein genes and their promoters were not able to cause crystal formation inside the exosporium. Thus, Aronson's second hypothesis seems reasonable. This was that the plasmid gene encodes a protein that binds the crystal protein to the inner surface of the exosporium. For determining the critical regions, we used a shuttle BAC library to carry out complementation testing and identified that five genes from pBMB28 and a crystal protein gene cry26Aa from pBMB26 were indispensable to the SCA phenotype in strain YBT-020. By screening shuttle BAC library, we have isolated several novel crystal protein genes [bib_ref] New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis..., Guo [/bib_ref] , and a thuringiensin synthesis gene cluster [bib_ref] Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus..., Liu [/bib_ref]. Our results testify to the convenience of this method in searching for unknown functional genes or gene clusters. This is the first report of the isolation of genes determining the SCA phenotype.
When other types of crystal protein genes were transferred into strain BMBJ1, only separated crystals were observed under phase microscope. This suggested that the formation of the SCA involved recognition of the specific amino acid sequences of the Cry26Aa protein. We also isolated the gene cry28Aa from strain YBT-020 as well, and by screening the library of YBT-020, we isolated a fragment of pBMB28 carrying the cry28Aa gene. The combination of this fragment and plasmid pBMB0617 harboring the cry26Aa gene in strain BMB171 was able to prolong the stability of separated inclusion bodies (unpublished). We speculated that the co-existence of proteins Cry26Aa and Cry28Aa provided a way of preventing the crystal protein from degradation. The genes orf1 and orf2 were found to determine the deposition of crystal proteins inside the exosporium. The genes orf3, orf4 and orf5 were found to be necessary for the stability of the SCA phenotype. This implied that the formation of the SCA phenotype was involved in protein interaction and was closely linked with spore differentiation and development. We have revealed the genes which were indispensable to the SCA phenotype. However, the functions of the proteins encoded by these genes remain unclear and need investigation.
The exosporium is a prominent structure with a paracrystalline basal layer and an external hair-like nap [bib_ref] Ultrastructure of the Exosporium Enveloping Spores of Bacillus Cereus, Gerhardt [/bib_ref]. It is composed of at least 20 proteins and glycoproteins [bib_ref] Structure, assembly, and function of the spore surface layers, Henriques [/bib_ref]. Exosporium assembly is a non-uniform process, and exosporium formation begins with the synthesis of a cap substructure [bib_ref] Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model..., Steichen [/bib_ref]. How do proteins, which are essential to the formation of SCA phenotype, carry out their function during the process of SCA? We propose the following explanatory mechanism to explain it. The Orf1 and Orf2 proteins play a role as bridges between spores and crystals. We infer that these two proteins are located on the spore coat, and interact with crystal proteins and other spore proteins (such as the products of the orf3, orf4, and orf5 operon). They would therefore bind crystal proteins to the inner surfaces of the exosporium cap. The inclusion assembly could then be confined to such a site in order to ensure a location within the exosporium. This needs detailed investigation in future.
Untill now, many strains with SCA phenotype have been isolated, and the crystal proteins enclosed within the exosporium are distinct among subspecies. Four morphological types of the SCA phenotype and four different crystal protein profiles were isolated from citrus orchards in Spain [bib_ref] Diversity of Bacillus thuringiensis strains isolated from citrus orchards in Spain and..., Vidal-Quist [/bib_ref]. Genome sequence analysis of B. thuringiensis strain C15 (another strain with the SCA phenotype maintained in our lab), showed that the genes involved in the formation of the SCA phenotype in YBT-020 could not be found (data not shown). This indicates that the genes controlling the formation of the SCA phenotype differ among subspecies.
# Materials and methods
## Bacterial strains, plasmids, growth conditions and dna manipulations
The bacterial strains and plasmids involved in this study are listed in [fig_ref] Table 2: Bacterial strains and plasmids [/fig_ref]. Conditions and media used for growing and maintaining different strains of E. coli and B. thuringiensis have been described previously [bib_ref] New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis..., Guo [/bib_ref]. Chloromycetin, tetracycline, erythromycin, kanamycin, ampicilin, and rifampin were supplemented at the final concentrations of 5 or 25, 10, 25, 50, 100, 100 mgmL 21 when needed, respectively. Plasmids were extracted from B. thuringiensis following the procedure described by Andrup et al. [bib_ref] Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by..., Andrup [/bib_ref]. All regular DNA manipulations were carried out following standard methods. E. coli-B. thuringiensis shuttle vectors, pEMB0557 and pEMB0603, were used to clone large DNA fragments. The two vectors were constructed from BAC vector pBeloBAC11 by adding erythromycin and kanamycin resistance genes, as the selectable marker in B. thuringiensis, and B. thuringiensisoriginated plasmid replication origins ori60 and ori44 respectively. The 2.3-kb ori60 and 2.25-kb ori44 replication origins were amplified from large plasmids in B. thuringiensis strain YBT-1520 [bib_ref] Determination of plasmid copy number reveals the total plasmid DNA amount is..., Zhong [/bib_ref] , and show 99% and 100% sequence identity with that of the 91 kb and 66 kb plasmids in B. thuringiensis subsp. kurstaki HD-263 [bib_ref] Characterization and comparative sequence analysis of replication origins from three large Bacillus..., Baum [/bib_ref]. B thuringiensis transformation was conducted as described previously [bib_ref] Elaboration of an electroporation protocol for large plasmids and wild-type strains of..., Peng [/bib_ref].
Construction of shuttle BAC library and screening of clones covering the full length of plasmids pBMB26 and pBMB28
Shuttle vector pEMB0557 was used to construct a genomic BAC library of B. thuringiensis strain YBT-020. Construction of library was performed following the method described by Liu et al [bib_ref] Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large..., Liu [/bib_ref] , with slight modifications. Genomic DNA embedded in agarose plugs was partially digested with HindIII and separated by pulse field gel electrophoresis (PFEG). 30-50 kb fragments of genomic DNA were recovered by electroelution (Bio-Rad) and were ligated with HindIII dephosphorylated vector pEMB0557 to generate a genomic library. The fragments which covered the full length of pBMB26 and pBMB28 were screened from library of strain YBT-020.
## Construction of recombinant plasmids
(i). Large fragment complementation testing. Two fragments, which were seclected from the shuttle BAC library and were able to recover the SCA phenotype in BMB171, were named as pBMB275 and pBMB251. And then, they were sub-cloned into pHT304 for sequencing by a primer walking strategy. The two fragments were digested with NotI and inserted into another vector pEMB0603 to generate pBMB275A and pBMB251A.
(ii). Minimizing the regions that determine the SCA phenotype. Deletion derivatives of pBMB275 containing either a BamHI fragment of 16.7 kb or a HindIII fragment of 13.7 kb were designated pBMB290 and pBMB291 respectively. A 6.3 kb NdeI fragment from pBMB291 was first inserted into pDG1514, then digested with BamHI and SalI, and inserted into pHT304 to generate pBMB292. A fragment of 4 kb was amplified from pBMB275 using the pair of primers cry26-F and cry26-R [fig_ref] Table 3: Primers used for PCR [/fig_ref]. This fragment was cloned into pEMB0603 between BamHI and HindIII to generate pBMB0617A. Deletion derivatives of pBMB251, which containing either a 12.5 kb BamHI fragment, a 7 kb ScaI-HindIII fragment, a 5.3 kb PstI-HindIII fragment, or a 1.7 kb ScaI-EcoRI fragment, were designated pBMB251B1, pBMB251B2, pBMB251B3, and pBMB251B4, respectively. A fragment of 2.9 kb was amplified from pBMB251 using the the pair of primers 251B1 and 251B2 [fig_ref] Table 3: Primers used for PCR [/fig_ref] , and cloned into pMD18-T simple vector to generate pEMB251B5. After that, it was digested with ScaI and HindIII and then cloned into pHT304 between SmaI and HindIII, to generate pBMB251B5.
(iii). Construction of frame-shift mutant plasmids. To assess the role of Orf1 and Orf2 protein, two plasmids pBMB251B5A and pBMB251B5B were constructed as follows: Briefly, frame shift mutation was introduced in the pEMB251B5 by AccI and EcoRI cleaving respectively, filling-in of the recessed ends with Klenow enzyme, and then blunt-end self-ligated. This was then digested with ScaI and HindIII and cloned into pHT304 between SmaI and HindIII, to generate pBMB251B5A and pBMB251B5B. The DNA sequencing confirmed the effectiveness of the mutation by introduction of stop codons generating truncated orf1 and orf2.
## Construction of gene disruption mutants of strain ybt-020
To construct a temperature-sensitive shuttle vector pHT304Ts, an EcoRV fragment of 2.1 kb, carrying the replication protein gene from the E.coli-B. thuringiensis shuttle vector pHT304 was replaced by a HpaI fragment containing the temperature-sensitive replication origin from pEG491 [bib_ref] Tn5401, a new class II transposable element from Bacillus thuringiensis, Baum [/bib_ref].
To verify the necessary of the genes for SCA in strain YBT-020, three gene disruption strains were constructed via homologous recombination. The first was the cry26Aa disruption strain BMBJ1. Using primers cry26A and cry26B [fig_ref] Table 3: Primers used for PCR [/fig_ref] , a fragment of 2 kb carrying the partial region of cry26Aa was amplified by PCR, cloned into pMD18-T simple vector, and then digested with EcoRI. A chloramphenicol resistance cassette, derived from pAD123, was amplified with primers CM1 and CM2 [fig_ref] Table 3: Primers used for PCR [/fig_ref] , and cloned into the EcoRI site, yielding pEMBJ1. The HindIII fragment was inserted into pHT304Ts to generate pBMBJ1. To generate the orf1 and orf2 deletion mutant strain BMBJA, a fragment of 3.8 kb was amplified by reverse PCR with primers 251B4 and 251B5 [fig_ref] Table 3: Primers used for PCR [/fig_ref] using pEMB251B5 as template, and then digested with EcoRI. A chloramphenicol resistance gene was amplified and cloned into the EcoRI site to give rise of pEMBJA. The plasmid was digested by ScaI and HindIII and inserted into pHT304Ts between SmaI and HindIII to generate pBMBJA.
To obtain the operon deletion mutant strain for orf3, orf4, and orf5, a PCR fragment of 3.8 kb was amplified with primers 251B6 and 251B7 [fig_ref] Table 3: Primers used for PCR [/fig_ref] using pBMB251 as template, and inserted into pMD18-T simple vector to generate pEMBJB. Using the plasmid as template, primers 251B8 and 251B9 [fig_ref] Table 3: Primers used for PCR [/fig_ref] were used to amplify a 4 kb fragment by reverse PCR. This was then digested with EcoRI. A chloramphenicol resistance gene was amplified and cloned into the EcoRI site, yielding pEMBJB1 which was then digested by BamHI and HindIII and inserted into pHT304Ts to generate pBMBJB.
Mutants were selected following the method described by Fang et al. [bib_ref] Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa..., Fang [/bib_ref]. Briefly, the plasmids were transferred into strain YBT-020, and the transformants were cultivated in LB medium with 2.5 mg/mL chloramphenicol for 8 h. Then the transformants were incubated at 42uC for 4 days to eliminate unintegrated temperaturesensitive plasmids. The expected mutant strains, which were resistant to Chloramphenicol and meanwhile sensitive to erythromycin colonies, were harvested and confirmed by PCR using appropriate primers and sequencing.
## Conjugation experiments
Plasmid conjugation transfers were conducted following the protocols described by Andrup et al. [bib_ref] Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus..., Andrup [/bib_ref] with slight modifications. In short, the overnight cultures of donor and recipient strains were incubated separately at 28uC in LB medium with appropriate antibiotics. Equal quantities of donor and recipient cells (250 ml per OD 600 unit) in logarithmic growth were mixed and shaken in 5 ml prewarmed LB medium at 28uC with moderate shaking (80 rpm). After 8 h, appropriate dilutions were plated onto appropriate selective medium to determine the number of transconjugants Controls of donors and recipients grown separately were also tested.
## Microscopic observation
For phase contrast microscopy, all B. thuringiensis were sporulated at 28uC and 220 rpm in a liquid medium (ICPM medium) containing 0.6% peptone, 0.5% glucose, 0.1% CaCO 3 , 0.05% MgSO 4 , and 0.05% KH 2 PO 4 (pH 7.0) until almost all mother cell lysis. Spores and crystals were collected by centrifugation and washed three times with a solution containing 1 mol of NaCl per liter and then three times with water. The mixture of spores and crystals was then resuspended in water [bib_ref] Effects of the 20-kilodalton helper protein on Cry1Ac production and spore formation..., Shao [/bib_ref]. By phase-contrast microscopy, spores appear as phase-bright, and crystals appear as phase-dark [bib_ref] Parasporal body formation via overexpression of the Cry10Aa toxin of Bacillus thuringiensis..., Hernandez-Soto [/bib_ref]. Each sample was chosen five fields to observe morphology. Transmission electron microscopy was performed following the method described by Bailey-Smith et al. [bib_ref] The ExsA protein of Bacillus cereus is required for assembly of coat..., Bailey-Smith [/bib_ref]. Sections were examined under a FEI Tecnai G 2 20 TWIN transmission electron microscope at an accelerating voltage of 200 kV.
[fig] Figure 1: Micrographs of parasporal crystals from transconjugants. (A) Electron micrograph of a thin section of transconjugant with the SCA phenotype during sporulation. (B) Transconjugants with the SCA phenotype after sporulation; (C) Electron micrograph of a thin section of transconjugant with separated crystal during sporulation. (D) Transconjugants with separated crystal after sporulation. (A) and (C) were Grown for 20 h, Scale bar = 0.5 mm; the arrowheads indicate the exosporium. (B) and (D) were grown for 48 h and observed by phase-contrast microscopy. pc = parasporal crystal. Magnification, 61,000. doi:10.1371/journal.pone.0027164.g001 [/fig]
[fig] Figure 2: Phase-contrast micrographs of recombinant strains after growth for 48 h. (A) BMB1151 (pBMB26-cured mutant of YBT-020). (B) Strain BMB1151 containing the 35 kb fragment of pBMB26. (C) Strain BMB171 containing the 35 kb fragment of pBMB26. (D) Strain BMB171 containing the fragments of pBMB26 and pBMB28. (E) Strain BMB171 containing the 35 kb fragment of pBMB28. Magnification, 61,000. doi:10.1371/journal.pone.0027164.g002 [/fig]
[fig] Figure 3: The search for minimal DNA fragments essential to the SCA phenotype in B. thuringiensis strain BMB171. (A) Determination of the minimal sequence of pBMB275 (a fragment of pBMB26) in strain BMB171 harboring pBMB251A (a fragment of pBMB28). (B) Determination of the minimal sequence of pBMB251 (a fragment of pBMB28) in strain BMB171 harboring pBMB275A (a fragment of pBMB26). The ability of the region to recover SCA is indicated by ''+'', and inability by ''2''. doi:10.1371/journal.pone.0027164.g003 [/fig]
[fig] Figure 4: Phase-contrast micrographs of different plasmid derivatives of pBMB275 and pBMB251 in strain BMB171 after growth for 48 h. (A) Strain BMB171/pBMB251A+pBMB0617. (B) Strain BMB171/pBMB275A+pBMB251B2. (C) Strain BMB171/pBMB275A+pBMB251B3. (D) Strain BMB171/pBMB275A+pBMB251B5. (E) Strain BMB171/pBMB0617A+pBMB251B2. Magnification, 61,000. Data not shown for pBMB251B4. doi:10.1371/journal.pone.0027164.g004 [/fig]
[fig] Figure 5: Micrographs of mutant strains of YBT-020 and their corresponding complementary strains. (A) and (B) Strain BMBJA (genes orf1 and orf2 disrupted). (C) and (D) Complementary strain BMBJA/pBMB251B5 (carrying orf1 and orf2). (E) Complementary strain BMBJA/ pBMB251B5A (carrying a frameshift at the AccI site in orf1). (F) Complementary strain BMBJA/pBMB251B5B (carrying a frameshift at the EcoRI site in orf2). (G), (H) and (I) strain BMBJB (genes orf3, orf4, and orf5 disrupted). (J) Complementary strain BMBJB/pBMB252B3 (containing orf3, orf4, and orf5). (K) Strain BMBJ1 (gene cry26Aa disrupted); (L) Complementary strain BMBJ1/pBMB0617 (carrying the gene cry26Aa). (A), (C) and (G) Electron micrographs of a thin sections during sporulation, arrowheads indicate exosporium. Scale bar = 0.5 mm. (B), (D), (E), (F), (K) and (L) after Growth for 48 h. (H) after growth for 36 h. (I) and (J) after growth for for 36 h, 100 h. Magnification, 61,000. doi:10.1371/journal.pone.0027164.g005 [/fig]
[table] Table 1: ORFs involved in the spore-crystal association (SCA) phenotype.The numbers correspond to the nucleotide coordinates of inserted fragments in pBMB275 or pBMB251. doi:10.1371/journal.pone.0027164.t001 [/table]
[table] Table 2: Bacterial strains and plasmids. Amp r , ampicillin resistance; Cm r , Chloromycetin resistance. Erm r , erythromycin resistance; Kan r , kanamycin resistance; Tet r , Tetracycline resistance. doi:10.1371/journal.pone.0027164.t002 [/table]
[table] Table 3: Primers used for PCR. doi:10.1371/journal.pone.0027164.t003 [/table]
|
Variables associated with general practitioners taking on serious mental disorder patients
Background: As part of community-based initiatives to strengthen integrated care and promote patient recovery, GPs are asked to play a greater part in treating serious mental disorder (SMD) patients. All current healthcare reforms favour the reinforcement of primary care. More information on enhancing the role of GPs in mental health would benefit policymakers, especially as regards SMD patients, where little research has been published as yet. This article assesses variables associated with GPs taking on SMD patients.Methods:The study, encompassing multiple sites, is based on a sample of 398 GPs, representative of the GP population in the Canadian province of Quebec. GPs were asked to answer a 143-item questionnaire on their socio-demographic and clinical practice profiles, patient characteristics, perceived inter-professional relationships and quality of care. Descriptive, bivariate and multivariate analyses were performed.Results: Our data highlighted that GPs currently followed up only a minority of SMD patients on a continuous basis and far fewer for both physical and mental health problems. A linear regression model that accounts for 43% of the variance was generated. The best variables associated positively with GPs taking on SMD patients were: frequency of referrals for joint follow-up with other resources, and involvement in post-hospitalization follow-up. Conversely, lack of expertise in mental health (related in our model to frequency of mental disorder patient transfer due to insufficient mental health training) is associated with a lower incidence of GPs taking on patients.Conclusion:As advocated in current healthcare reforms, our study confirms the need to promote greater GP involvement in integrated care models and enhance their training in mental health -thereby helping to reverse the trend among GPs of transferring SMD patients to specialized care. Patients with stable SMDs ought to have the same care access as the general population.
# Background
In light of efforts to improve healthcare efficiency, enhancing the integration of primary care within the men-tal healthcare system is strongly recommended [bib_ref] General practitioners and mental health staff sharing patient care: working model, Horner [/bib_ref] [bib_ref] Establishing a collaborative service model for primary mental health care, Meadow [/bib_ref]. It has been reported that in countries with more fully developed primary care, the healthcare system is more effective with regard to service accessibility, service continuity and patient outcomes [bib_ref] A new approach to primary care for Australia. Sydney, NSW: Centre for..., Doggett [/bib_ref]. The trend toward extending primary mental health care is related to the communitybased movement in mental health whose objective is to promote patient recovery [bib_ref] Clinical characteristics and service use of persons with mental illness living in..., Anderson [/bib_ref] [bib_ref] Effectiveness of psychiatric rehabilitation approaches for employment of people with severe mental..., Bond [/bib_ref] [bib_ref] Integrating Psychiatric Rehabilitation into Managed Care, Anthony [/bib_ref]. Over the past 40 years, deinstitutionalization has returned individuals with mental disorders to the community. Accordingly, general practitioners (GPs) are increasingly viewed as major partners in the mental healthcare system [bib_ref] General practitioners and mental health staff sharing patient care: working model, Horner [/bib_ref] [bib_ref] Primary care for those with severe and persistent mental illness, Crews [/bib_ref].
In recent years, many primary care models have proliferated, favouring collaboration, care continuity, and bestpractices for the management of patients with chronic and complex problems (e.g., Wagner's Chronic Care Model; the patient-centered medical home approach). In Canada, two such examples in mental health are sharedcare [bib_ref] Better practices in collaborative mental health care: an analysis of the evidence..., Craven [/bib_ref] [bib_ref] Models of Integrated Service Networks and Key Conditions for their Operationalization, Fleury [/bib_ref] and integrated service network models [bib_ref] Models of Integrated Service Networks and Key Conditions for their Operationalization, Fleury [/bib_ref] [bib_ref] Integrated service networks: the Quebec case, Fleury [/bib_ref] , which aim at improving care co-ordination among GPs, psychiatrists and multidisciplinary mental health providers, or within the healthcare system as a whole. These models usually include a broad spectrum of integration strategies and best practices such as clinical guidelines, electronic medical records, case management, capitation and performance incentives for GP remuneration, and patient self-management support [bib_ref] Better practices in collaborative mental health care: an analysis of the evidence..., Craven [/bib_ref] [bib_ref] Integrated service networks: the Quebec case, Fleury [/bib_ref] [bib_ref] Creating Integrated Service Systems for Homeless Persons with Mental Illness: The ACCESS..., Randolph [/bib_ref]. In the province of Quebec, multidisciplinary group practices such as "family medicine groups" involving several GPs working closely together with nurses responsible for patient screening, follow-up, referral and patient registration are other innovations designed to improve service continuity and patient outcomes. It has been shown that these innovative integrated models not only improve care continuity but also more appropriately meet the needs of mental health patients living in the community [bib_ref] Sorohan H: Patients' and health professionals' views on primary care for people..., Lester [/bib_ref] [bib_ref] Care of long-term mentally ill patients by British general practitioners, Burns [/bib_ref].
Compared to specialized care, services provided primarily by GPs for patients with mental disorders are found to be more accessible, less stigmatizing and more comprehensive, since physical problems are managed along with mental disorders [bib_ref] Chronic Illness Management: What Is the Role of Primary Care?, Rothman [/bib_ref]. As the main entry point into the healthcare system [bib_ref] Shared mental health care. The way ahead, Kates [/bib_ref] [bib_ref] Collaboration and referral practices of general practitioners and community mental health workers..., Lockhart [/bib_ref] , GPs play a pivotal role in screening, detecting and treating mental disorders [bib_ref] Challenges faced by general practitioners and allied mental health services in providing..., Bambling [/bib_ref] [bib_ref] Avancement des objectifs des soins de santé mentale axés sur la collaboration, Gagné [/bib_ref]. In the course of a single year, about 80% of the population consults a GP, and between 20 and 40% of visits are related to mental health [bib_ref] Populationbased use of mental health services and patterns of delivery among family..., Watson [/bib_ref]. In Canada, of all patients with a mental disorder seeking help, 45% consult a GP while 25% consult other healthcare practitioners [bib_ref] Shared mental health care. The way ahead, Kates [/bib_ref] [bib_ref] Avancement des objectifs des soins de santé mentale axés sur la collaboration, Gagné [/bib_ref]. About 25% of patients with chronic psychosis see only their GPs. Depression and anxiety are the predominant common problems in mental health patients seen by GPs [bib_ref] Challenges faced by general practitioners and allied mental health services in providing..., Bambling [/bib_ref] [bib_ref] Caring for depressed patients in rural communities: general practitioners' attitudes, needs and..., Wright [/bib_ref] [bib_ref] Primary care physicians' experience with mental health consultation, Kushner [/bib_ref]. In the context of current reforms designed to promote patient recovery and further deinstitutionalization, GPs are increasingly being asked to play a pivotal role with regard to stabilized serious mental disorder (SMD) patients (e.g., schizophrenia). This is a trend not only in Canada, but in most countries.
Although current reforms encourage GPs to manage more SMD patients, few studies have been published as yet on this subject [bib_ref] Sorohan H: Patients' and health professionals' views on primary care for people..., Lester [/bib_ref] [bib_ref] Caring for seriously mentally ill patients, Brown [/bib_ref] [bib_ref] On the front line: survey on shared responsibility. General practitioners and schizophrenia, Stip [/bib_ref]. Most studies involving GPs focus on common mental disorders (e.g. depression and anxiety), examine best practices for treatment and assess outcomes for various types of intervention and programs [bib_ref] Better practices in collaborative mental health care: an analysis of the evidence..., Craven [/bib_ref]. In the other hand, studies on SMD patients examine almost exclusively the mental health network, rarely including GPs' care [bib_ref] Primary care for those with severe and persistent mental illness, Crews [/bib_ref]. To our knowledge, no prior study has investigated variables that promote or hinder the involvement of GPs with SMD patients, which is a prerequisite for the development of optimal integrated care models for these patients. Accordingly, this study is designed to test the association of GPs taking on SMD patients and multiple correlates such as GPs' socio-demographic profile, clinical practice, perceived inter-professional relationships and quality of care, and patient characteristics. Although based on the Quebec/Canada context, the findings from this study should be of wider relevance since primary mental health care in most of the industrialized countries share similar reform objectives (e.g., optimizing GPs' role, accessibly and continuity of care), and organizational and practice features (e.g., United Kingdom, Australia, Ireland).
# Method
## Design and study population
This cross-sectional study was conducted among GPs practising in the province of Quebec, in Canada. Quebec has a population of 7.5 million, and 7,199 full-time GPs [bib_ref] Direction de la planification et de la régionalisation -FMOQ, Savard [/bib_ref]. The study sites represented rural, semi-urban and urban territories (with or without a university-affiliated psychiatric hospital). In each of these sites, participants were selected in a variety of settings, including solo or group practices in private clinics, local community-based service centers (CLSCs), hospitals (acute, psychiatric or long-term), walk-in clinics, family medicine groups, and network clinics. CLSCs are local organizations that provide a broad range of health and psychosocial services, including mental health. Network clinics are similar to family medicine groups, except that patients are not registered with their GPs, and nurses act mainly as liaison agents. The sample list was provided by the Quebec Federation of General Practitioners (FMOQ), the professional union representing Quebec GPs. Recruitment took place from September 2006 to February 2007. Each participant was required to sign a consent form approved by the Douglas Mental Health University Institute research ethics board.
## Data collection process
As no prior questionnaire existed, a self-administered questionnaire including six main domains and 143 items, based on a literature review, was designed by the research team. It was validated by a multidisciplinary group of twenty experts (researchers, GPs and psychiatrists). The RAMQ (Régie de l'assurance maladie du Québec) 2006 data bank -the public register for all GPs' medical actswas also used (e.g., number of GP medical acts, percentage of patients with mental disorders) for the purpose of comparison. The questionnaire was pre-tested with ten physicians not included in the study sample. Its structure reflects our goal to cover every possible aspect of GP practice in mental health, without exceeding a maximum of 30 minutes to complete the questionnaire. No financial incentive was offered to respondents.
The questionnaire covered six main domains: (1) GP socio-demographic and attitudinal profile, (2) patient characteristics, (3) clinical practice features, (4) collaboration between GPs and other medical or psychosocial mental health professionals, (5) GP perception of quality of mental health services, and (6) GP opinions about supportive strategies to be promoted for better care integration. It includes either categorical or continuous items or five-point Likert scales (1 = strongly disagree to 5 = strongly agree).
The questionnaire was sent by mail. Each questionnaire was assigned a tracking number and accompanied by support letters from the Quebec College of Physicians and FMOQ. There were three follow-ups. The first was conducted by mail. In the second, a nurse called the GPs. For the third, GPs were contacted by network medical administrators of the target territories. More information on the questionnaire and the sampling procedure can be found in another publication [bib_ref] Variables Associated with GPs Taking on Patients with Common Mental Disorders, Fleury [/bib_ref].
## Statistical analyses and definition of variables
Univariate, bivariate and multivariate analysis were performed on the questionnaire items linked to the dependent variable. The model was built using linear regression analysis. The proportion of SMD patients taken on by GPs, out of the total number of SMD patients seen, was the dependent variable, which was measured as a continuous variable. The term "taking on" patients goes further than seeing patients during a medical visit (one-time basis), and implies relational continuity and follow-up over time for the same or subsequent condition, including medical tests (physical and/or mental health), medication, side-effect monitoring, psychotherapy or any kind of psychosocial support. It was based on answers given by GPs to the following question: "Among patients seen with SMD in your medical practice weekly, what is the proportion of SMD patients you follow up on a continuous basis (i.e., accepted as your own patients)?" Mental disorders in the study were divided into two broad categories: (1) common mental disorders, which include anxiety, depression, adaptation disorders, personality disorders and substance abuse co-morbid disorders; and (2) SMD, which excludes the latter and for which three examples were pro-vided: schizophrenia, bipolar disorder and delirious disorder.
Independent variables were organized in five sets related to the five first main dimensions of the questionnaire (identified above). Associations yielding a p value of less than 0.10 in bivariate analyses were considered for the multiple regression model. In each of the five variable sets, a partial model was constructed using the backward stepwise method (p ≤ 0.05). The final model was designed using the same technique of elimination by adjusting all the variables from the five sets. It was validated for goodness of fit, proportion of variance explained, and collinearity diagnostics.
# Results
## Sample
The sample comprised 398 GPs, for a response rate of 41%. More information is presented on the sampling procedure in another publication [bib_ref] Variables Associated with GPs Taking on Patients with Common Mental Disorders, Fleury [/bib_ref]. The sample was compared to non-respondent GPs for gender distribution, which yielded a non-significant result (χ 2 = 3.44, df = 1, P = 0.0637). Other important parameters were used to compare our sample with the overall GP population in Quebec [bib_ref] Direction de la planification et de la régionalisation -FMOQ, Savard [/bib_ref] [bib_ref] Douglas Institut Universitaire en santé mentale, Ouadahi [/bib_ref]. Where data were available, tests were carried out comparing the GP population in Quebec and Canada [bib_ref] Institut canadien d'information sur la santé, Pong [/bib_ref]. As shown in [fig_ref] Table 1: Comparison between our GP sample and the Quebec/Canada GP population [/fig_ref] , no significant difference was found.
## Gp profiles
In [fig_ref] Table 2: Proportion of medical consultations related to mental disorders, both common and serious... [/fig_ref] , pertinent information is provided on GP socio-demographic, clinical and inter-professional collaboration profiles regarding the management of SMD patients. The study showed that one quarter of GP consultations concerned mental health problems. Of all the patients visiting GPs for any mental health reasons, approximately one out of ten was found to consult for SMD. Only one third of SMD patients (34% ± 36) were followed up on a continuous basis by GPs. The long-term mental health management of these patients related mostly to medication follow-up (49% ± 39) and supportive therapy (35% ± 37), with GPs seeing them on average six times (± 5) a year. GPs estimated referring a majority of SMD patients (71% ± 3) mainly to emergency rooms and psychiatric services. They primarily referred those patients for advice on medication and diagnostic evaluation. When they instead transferred mental disorder patients (for any types of care and for either a short-term or a long-term period), the main reasons were case severity or complexity.
## Variables associated with gps taking on smd patients
The final multiple linear regression model is shown in [fig_ref] Table 4: Variables independently associated with GPs taking on SMD patients [/fig_ref] , consisting of six variables organized in three sets. The most significantly associated variable was negatively related to taking on SMD patients: frequency of transfer of patients with mental disorders, owing to GPs' insufficient mental health training. The six variables included in the model accounted for 43% of the variance in the dependent variable. The model fit was significant (F = 45.88; p < 0.001).
# Discussion
As found in other studies [bib_ref] Caring for depressed patients in rural communities: general practitioners' attitudes, needs and..., Wright [/bib_ref] [bib_ref] On the front line: survey on shared responsibility. General practitioners and schizophrenia, Stip [/bib_ref] [bib_ref] Douglas Institut Universitaire en santé mentale, Ouadahi [/bib_ref] , our research findings showed that only a minority of SMD patients are managed primarily by GPs. Three sets of variables were associated with GPs taking on SMD patients: (1) their level of expertise toward treating those patients; (2) their inter-professional relationship feature; and (3) their clinical practice profile. Those three sets of variables, especially the first one that contains the most strongly associated variable in the model, should be considered as significant hindering or enabling factors for optimizing primary mental healthcare service planning.
Consistent with our findings, various studies have cast doubt on the ability of GPs to detect and treat more com- plex forms of mental disorders, particularly major depression with suicidal tendencies, schizophrenia, and bipolar disorders [bib_ref] Caring for depressed patients in rural communities: general practitioners' attitudes, needs and..., Wright [/bib_ref] [bib_ref] High prevalence of mental disorders in primary care, Ansseau [/bib_ref] [bib_ref] Increased recognition of depression in primary care. Comparison between primary-care physician and..., Berardi [/bib_ref]. They have also highlighted GP discomfort with such patients. GPs either consider these disorders too specialized for routine primary care, deeming their skills and experience inadequate for effective diagnosis and treatment, or they position themselves as complementary to specialized care, treating essentially physical problems [bib_ref] Sorohan H: Patients' and health professionals' views on primary care for people..., Lester [/bib_ref] [bib_ref] Collaboration and referral practices of general practitioners and community mental health workers..., Lockhart [/bib_ref]. All of these conditions encourage GPs to transfer SMD patients to specialized care (i.e., psychiatric departments of acute-care or psychiatric hospitals, or emergency rooms).
Nevertheless, when GPs practice in integrated primary care models such as in shared-care or a patient-centered medical home approach, they are reportedly more at ease with managing patients with mental disorders [bib_ref] Better practices in collaborative mental health care: an analysis of the evidence..., Craven [/bib_ref]. This is confirmed by our findings: joint follow-up with 4 (± 15) Crisis center 4 (± 13) Among GPs reporting transferring mental disorder patients (i.e., all GP mental disorder consultations, both SMD and common mental disorders), proportion of GPs who transferred patients occasionally and often (for any types of care, and for either a short-term or a long-term period) to other resources for the following reasons [mean %]: severity of the disorder 93.6 case complexity 92.1 lack of support from psychiatrists 62.7 insufficient mental health training 58.9 lack of interest in mental health 17.5 insufficient financial incentives 18.0 other resources and involvement in post-hospital followup were found to be the second and third most important variables associated with GPs taking on these patients. Research showed that when SMD patients are successfully managed and stabilized by psychiatrists, GPs are more comfortable following them in the community with medication and/or supportive therapy, jointly with mental health teams, as required [bib_ref] Attitudes and roles of general practitioners in the treatment of schizophrenia compared..., Carr [/bib_ref]. SMD patients generally need continuous community follow-up of varying intensity over time. They usually have numerous bio-psychosocial needs that require teamwork to avoid relapse and help adapt to a recovery-oriented life [bib_ref] Reliability of the Camberwell Assessment of Need-European Version. EPSILON Study 6. European..., Mccrone [/bib_ref] [bib_ref] Patient's Report of Help Provided by Relatives and Services to Meet Their..., Fleury [/bib_ref].
Along with the need for psychiatric team assistance, GPs who offer medication and supportive therapy follow-up and have a greater volume of SMD patients were also found, in our study, to be more likely to take on SMD patients. This is consistent with previous research [bib_ref] Collaboration and referral practices of general practitioners and community mental health workers..., Lockhart [/bib_ref] [bib_ref] On the front line: survey on shared responsibility. General practitioners and schizophrenia, Stip [/bib_ref] highlighting links between mental health knowledge and training, and the ability of GPs to manage these patients. Conversely, lack of both knowledge and training are major factors resulting in the transfer of patients by GPs to specialized care. A higher volume of SMD patient consultations would be expected to result in greater GP willingness to take them on. This further leads us to hypothesize that some GPs may specialize in the follow-up of these patients, being more able to manage both patient medication follow-up and supportive therapy. When all conditions favour SMD patient management by GPs, our findings also show that GPs apparently offer good continuity of care, seeing these patients on average six times a year. Half the time, SMD patients were followed-up by GPs either solely for their physical problems or for both physical and mental health problems
There are many reasons explaining why only a minority of GPs take on SMD patients. Such patients are deemed to require more care and time, more frequent visits, and be more difficult to treat [bib_ref] How bipolar disorders are managed in family practice, Balanchandra [/bib_ref]. Often, they have concurrent diagnoses (e.g., substance abuse) and interrelated physical or social problems [bib_ref] Comorbid mental and somatic disorders: an epidemiological perspective, Iacovides [/bib_ref] [bib_ref] Social class, family history and type of schizophrenia, Jones [/bib_ref]. As for GPs, their poor collaboration with psychiatry services, their busy schedules and the competing demands of other patients are other impending factors [bib_ref] Better practices in collaborative mental health care: an analysis of the evidence..., Craven [/bib_ref] [bib_ref] Sharing psychiatric care with primary care physicians: the Toronto Doctors Hospital experience, Turner [/bib_ref]. The historical separation between psychiatry and primary care [bib_ref] Primary care for those with severe and persistent mental illness, Crews [/bib_ref] , and GPs' limited training or experience with effective team practice [bib_ref] Chronic Illness Management: What Is the Role of Primary Care?, Rothman [/bib_ref] may also explain their reluctance to take on these patients -especially if they consider hospital psychiatric teams to be more appropriate. But none of this would suggest GPs' removal from the treatment equation of those patients. SMD patients are in great need of adequate physical care and mental health follow-up as they face higher risks of interrelated morbidity. Moreover, as psychiatric teams are usually concentrated more in urban settings, GPs are often the sole available source of care. This is the case in Quebec where almost half of the psychiatrists practice in the Montreal metropolitan area, and where in more remote regions, specialized care is scarce.
# Conclusion
While our model yields various strong and interesting associations, the study has certain limitations. First, it has a cross-sectional design, which does not permit causal inference as in experimental or longitudinal studies. Second, as the data collected are from GP self-reporting, the results must be viewed as an approximation of actual GP practice. Third, our questionnaire was complex, which may have discouraged some GPs from participating. As a result, the response rate was limited, but was not substantially lower than that reported in other surveys involving GPs. Finally, no data was collected on GPs' adequacy in treating mental-disorder patients, which is considered a major issue giving rise to conflicting results in the literatureIn the context of current reforms designed to enhance healthcare efficiency and support SMD patients' integration in the community, our findings sustain the development of integrated care models favouring service coordination, exchange of expertise between bio-psychosocial professionals and healthcare lines of services, and reinforcement of GPs' mental-health training. Our data show that GPs currently follow up only a minority of SMD patients on a continuous basis, and far fewer for both their physical and mental health problems. However, GPs may play a pivotal role in taking on patients with stable SMDs, if they have psychiatrists' and mental health team support. Lack of expertise in mental healthcare was also found to be a strong impediment. Without psychiatrists' and mental health team support or sufficient expertise in mental health, GPs are likely to transfer SMD patients to specialized care.
In the Canadian context of high GP shortages, increasing the development of mental-health integrated care models could nevertheless be a major challenge. Patients with stable SMDs ought to have the same care access as the general population, however, and services that are the least stigmatizing. Group practice models such as family medicine groups with nurses working closely with GPs to assist in patient screening and follow-up could serve as the basis for more refined integrated care models in Quebec's mental healthcare system. Improvements in access to direct communications with psychiatrists or shared-care initiatives, for diagnostic and therapeutic consultations, and to mental health teams, for patient case management of various intensities, should also be considered. For managing chronic and complex illnesses such as SMDs, various studies [bib_ref] Creating Integrated Service Systems for Homeless Persons with Mental Illness: The ACCESS..., Randolph [/bib_ref] [bib_ref] Engaging people with enduring psychotic conditions in primary mental health care and..., Cook [/bib_ref] have indicated the major positive impact of comprehensive and continued multimodal strategies (e.g.
clinical guidelines, electronic medical records, financing incentives, medical education sessions), which should be further encouraged.
[table] Table 1: Comparison between our GP sample and the Quebec/Canada GP population [/table]
[table] Table 2: Proportion of medical consultations related to mental disorders, both common and serious mental disorders [mean %] 25 (± 19) Proportion of serious mental disorders (SMD) patients (e.g. schizophrenia, bipolar and delirious disorder) diagnosed among GP patient consultations related to mental disorders [mean % (SD)] [/table]
[table] Table 3: General practitioner (GP) inter-professional collaboration features (n = 398) [/table]
[table] Table 4: Variables independently associated with GPs taking on SMD patients [/table]
|
The impact of Action Schools! BC on the health of Aboriginal children and youth living in rural and remote communities in British Columbia
Objectives: The aim of the study was to determine the short-term impact of a 7-month whole-school physical activity and healthy eating intervention (Action Schools! BC) over the 2007Á2008 school year for children and youth in 3 remote First Nations villages in northwestern British Columbia. Study design: A pre-experimental pre/post design was conducted with 148 children and youth (77 males, 71 females; age 12.592.2 yrs). Methods: We evaluated changes in obesity (body mass index [wt/ht 2 ] and waist circumference z-scores: zBMI and zWC), aerobic fitness (20-m shuttle run), physical activity (PA; physical activity questionnaire and accelerometry), healthy eating (dietary recall) and cardiovascular risk (CV risk). Results: zBMI remained unchanged while zWC increased from 0.4691.07 to 0.5791.04 (p B0.05). No change was detected in PA or CV risk but aerobic fitness increased by 22% (25.4915.8 to 30.9920.0 laps; pB0.01). There was an increase in the variety of vegetables consumed (1.1091.18 to 1.4591.24; pB0.05) but otherwise no dietary changes were detected. Conclusions: While no changes were seen in PA or overall CV risk, zWC increased, zBMI remained stable and aerobic fitness improved during a 7-month intervention.
W hile 26.3% of Canadian children and youth are overweight or obese, 34.5% of off-reserve Aboriginal young people are considered overweight or obese [bib_ref] Obesity and physical activity among aboriginal Canadians, Katzmarzyk [/bib_ref]. Preliminary results of the 2008 First Nations Regional Longitudinal Health Survey suggest that 56% of children and 43% of youths living in Canadian First Nations communities are either overweight or obese, but obesity levels vary considerably by region. In specific First Nations communities the rate of on-reserve Canadian Aboriginal pediatric obesity and overweight has been reported as varying widely from 38% to as high as 70% [bib_ref] Obesity, adiposity, physical fitness and activity levels in Cree children, Ng [/bib_ref]. Obesity not only tracks from childhood into adulthood (5) but adults who were obese during childhood also have an in-creased risk of developing chronic diseases [bib_ref] A 40-year history of overweight children in Stockholm: life-time overweight, morbidity, and..., Dipietro [/bib_ref]. Aboriginal people in Canada suffer from obesity-related conditions such as diabetes, hypertension and heart disease at 2Á3 times the rates of the general population. Clearly, something must be done to reverse this trend.
The school environment is an attractive context to promote healthy eating (HE), obesity prevention and physical activity (PA) as children from diverse ethnic and socio-economic strata spend 5 or more hours per day in school [bib_ref] Schools, health literacy and public health: possibilities and challenges, Leger [/bib_ref]. Elementary-school-based interventions have successfully increased the amount of PA undertaken during school time (9Á11) and have improved dietary habits (12Á14). Action Schools! BC (AS!BC), as described in detail by , is a whole-school framework for action on PA and HE. Efficacy of the model for improving a host of chronic disease risk factors was established in an urban setting in British Columbia . Although the model included Aboriginal children residing and attending school in urban communities, the initiative did not explicitly target issues and contexts specific to Aboriginal children. In response to concerns about type 2 diabetes and obesity among their children [bib_ref] Prevalence of impaired glucose tolerance and the components of metabolic syndrome in..., Zorzi [/bib_ref] , 3 remote Tsimshian Nation communities collaborated with the AS!BC team to implement and evaluate the AS!BC intervention and measurement tools to understand its impact within the specific context of rural, remote Aboriginal communities.
Therefore, our aim was to determine the short-term impact of the AS!BC intervention in 3 rural, remote northern Aboriginal communities. Specifically, we addressed the question: Is there a significant change in health outcomes (obesity-related body composition, PA levels, aerobic fitness, CV risk and HE) for First Nations children and youth living in rural and remote northern communities after participation in AS!BC for 7 months?
# Materials and methods
Study design and population Our study was part of a larger community-based participatory action research project. The initiative was undertaken in response to an invitation from 3 First Nations communities in northwestern British Columbia based on: (i) their longstanding relationship with a clinician-researcher on our team; and (ii) their concern regarding the elevated risk of diabetes in their communities [bib_ref] Prevalence of impaired glucose tolerance and the components of metabolic syndrome in..., Zorzi [/bib_ref]. As a result of the unique relationship and context, we implemented a pre-experimental pre/ post design to conduct our study.
## Recruitment and participants
We received approval from the University of Victoria Human Research Ethics Board, the University of British Columbia Clinical Research Ethics Board and the Children's and Women's Research Review Committee to conduct the study. We also received approval and support from elected Band Councils, hereditary band elders, hereditary band chiefs, community health directors, school administrators and our target communities to undertake our study in their communities. We obtained written informed consent from parents or caregivers; the children provided verbal and written assent. The process we adopted to acquire consent/assent, to obtain community approval and to travel to very remote locations to undertake this project is described in more detail elsewhere. We invited all children in grades 4Á12 at each of the schools (n 03) in the 3 communities to participate. Of the 170 eligible participants, 163 consented to participate (96%), with 148 measured at baseline and 134 at follow-up. Details of the recruitment and reasons for dropping out are displayed in [fig_ref] Figure 1: Participation numbers and reasons for exclusion from measurement over the 7-month intervention [/fig_ref].
## Action schools! bc intervention
We have described the AS!BC model in detail elsewhere [bib_ref] Lessons learned from Action Schools! BC ÁAn 'active school' model to promote..., Naylor [/bib_ref] and have also described its implementation in these 3 communities [bib_ref] Implementing a whole school physical activity and healthy eating model in rural..., Naylor [/bib_ref]. Briefly, AS!BC is a whole-school model that integrates classroom-based HE education and PA opportunities with recess and after-school activities, and environmental change strategies (e.g., playground equipment) with family and community elements (e.g., school spirit and family events) in order to promote healthy living. A primary feature of the AS!BC model is that it is tailored by each school and teacher according to their context. AS!BC advocates that children should accrue 150 minutes of PA per week during school hours. As well as regular physical education classes (typically 75Á80 minutes/week), teachers in the program incorporated an additional 15 minutes per day of teacherselected, classroom-based PA into their school regimen. For the HE component, teachers strove to incorporate at least one HE activity per month into the ''classroom action zone'' and implemented a fruit and vegetable (FV) and a sugar-sweetened beverage (SSB) campaign into their classroom or school. To facilitate classroom activities, the AS!BC Support Team (who travelled 3 times by seaplane to visit all of the schools) customized the model with teachers and provided an ''Action Bin'' replete with classroom PA and HE equipment and resources that teachers needed to deliver their customized model. The AS!BC Support Team also provided support to teachers to enhance the likelihood of successful implementation and sustain-ability [bib_ref] Implementing a whole school physical activity and healthy eating model in rural..., Naylor [/bib_ref].
## Measurement
We assessed children and youth at baseline (September 2007) and again at 7 months following the school-based intervention (April or May 2008). All measurements were conducted at local schools during regular school hours by the same team of university researchers who travelled by seaplane to each of the communities.
## Status of overweight and obesity
We assessed height, weight and waist circumference (WC) and calculated body mass index (BMI; kg/m 2 ). We assessed height as stretched stature to the nearest 0.1 cm (Seca 214 Portable Stadi-ometer) and weight to the nearest 0.1 kg (Conair digital electronic scale). Height and weight were measured twice and we used the mean for analysis as per Canadian Society of Exercise Physiology protocol. We measured WC to the nearest 0.1 cm at the midpoint between the lowest rib and the iliac crest, at the end of normal expiration. To compare BMI and WC across time periods, we used age-and gender-specific z-scores (zBMI and zWC). zBMI was calculated as per the Centers for Disease Control method [bib_ref] Centers for Disease Control and Prevention 2000 growth charts for the United..., Ogden [/bib_ref] , with overweight defined as ]85 percentile and obese as ]95 th percentile [bib_ref] Expert committee recommendations regarding the prevention, assessment, and treatment of child and..., Barlow [/bib_ref]. zWC was determined from percentile values as per Fernandez et al. [bib_ref] Waist circumference percentiles in nationally representative samples of African-American, European-American, and Mexican-American..., Fernández [/bib_ref].
## Physical activity (pa)
We estimated PA using a modified version of a valid and reliable tool for assessing moderate-to-vigorous PA (MVPA), the physical activity questionnaire for children (PAQ-c) or adolescents (PAQ-a) [bib_ref] Measuring general levels of physical activity: preliminary evidence for the physical activity..., Crocker [/bib_ref]. We made minor changes to the PAQ by replacing 2 activities that were unavailable in the studied communities (ice hockey and ice skating) with 2 common, culturally oriented PA choices (e.g., seaweed gathering and traditional dance). A physical activity summary score (PA score) was calculated [bib_ref] Measuring general levels of physical activity: preliminary evidence for the physical activity..., Crocker [/bib_ref] with values ranging from 1 (low active) to 5 (very active). As well, we measured MVPA with GT1M acceler-ometers (Actigraph, Pensacola, FL) in a subset of participants. With a limited number of accelerometers and a high recruitment rate, participants from 2 of the 3 communities wore accelerometers for 5Á7 days at each measurement period. Accel-erometers were programmed to collect activity counts every 15 seconds, and an age-specific regression equation [bib_ref] Calibration of accelerometer output for children, Freedson [/bib_ref] was used to estimate MVPA. We included all participants with at least 3 monitored days of 10 or more hours/day in our analysis. Our accelerometry protocol is described in detail elsewhere [bib_ref] Habitual moderate-to-vigorous physical activity is inversely associated with insulin resistance in Canadian..., Mitchell [/bib_ref].
## Aerobic fitness
To assess aerobic fitness, participants performed the Leger-Boucher 20-metre shuttle run as per standard protocol [bib_ref] National High Blood Pressure Education Program Working Group on High Blood Pressure..., Leger [/bib_ref] in the school gymnasium; participants ran in groups of no more than 10 students. All participants were verbally encouraged throughout the procedure; the total number of laps completed by each student was recorded by the research assistant.
## Cardiovascular risk (cv risk)
We assessed each child's BMI, WC, blood pressure (BP) and aerobic fitness to determine the percentage of participants with elevated CV risk factors. We measured BP 3 times on the left arm and used the mean of the final 2 measurements for analysis. Risk factors were considered elevated if: (i) BMI was greater than the age-and gender-specific 85th percentile [bib_ref] Centers for Disease Control and Prevention 2000 growth charts for the United..., Ogden [/bib_ref] ; (ii) WC was greater than the age-and gender-specific 90th percentile (24); (iii) BP was greater than the age-, gender-and height-specific 90 th percentile (29); or (iv) aerobic fitness laps were less than the age-and gender-recommended standards.
Healthy eating (HE) HE was assessed using a validated 24-hour dietary recall (31) that a member of the research team administered to each participant individually. The EHSA Food Processor Nutrition and Fitness Software (v. 8.7, Salem, OR) was used to generate macro-nutrient servings and total kilocalories consumed. We used hand counting to tally the number of different fruits and vegetables (FV) consumed as well as to assess the number of FV and sugar-
# Statistical analysis
We used Predictive Analytics SoftWare (PASW; v18.0, Chicago, IL) for statistical analyses. We used independent t-tests to compare descriptive characteristics between included (n0134) and excluded (n 014) participants, and paired t-tests to assess differences in outcomes at baseline and follow-up. We established the significance level a priori at p B0.05. To determine if maturation (growth and age) was related to the aerobic fitness outcome, we conducted Pearson product moment correlations and simple linear regression analyses where the independent variables were entered simultaneously.
# Results
Reasons for participant exclusion are provided in [fig_ref] Figure 1: Participation numbers and reasons for exclusion from measurement over the 7-month intervention [/fig_ref] and baseline participant characteristics are displayed in [fig_ref] Table I: Participant characteristics at baseline [/fig_ref]. Participants ranged from 8.7 to 18.5 years. There were no differences at baseline between participants who were measured in both time periods (n0134) and those measured at baseline only (n 014 drop-outs) with the exception that the dropouts had higher aerobic fitness (32.7 versus 25.4 laps; p B0.05) and were older (14.3 versus 12.3 years; p B0.01).
## Status of overweight and obesity
Over one-half (51%) of participants were overweight or obese at baseline; 20% were overweight and 31% were obese (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref]. Following the intervention, zBMI did not change; however zWC did increase (pB0.05) (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref].
## Physical activity
Participants accumulated significantly less accelerometer MVPA during weekend days compared to weekdays at both baseline ((46.3 minutes; p B0.001) and follow-up ((28.3 minutes: p B0.001). No significant change in daily, weekday or weekend MVPA or in the PA score was detected after the 7-month intervention (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref].
## Aerobic fitness and cv risk
Participants showed a substantial (22%) increase in aerobic fitness laps (pB0.001) (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref]. Age and growth were not significant predictors of the change in aerobic fitness laps00.846, p 00.435). While we observed a significant decrease in the proportion of children and youth at risk for low aerobic fitness (from 57% to 45%; p B0.05), no other changes were seen in the CV risk profile (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref].
## Healthy eating
There was a significant increase in the variety of vegetables consumed at follow-up (1.1091.18 to 1.4591.24; pB0.05); otherwise, patterns of FV consumption, caloric intake and SSB consumption remained unchanged (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref].
# Discussion
We previously reported in a randomized controlled trial that AS!BC implemented over 1 school year significantly enhanced aerobic fitness in mixed-ethnicity children attending urban schools [bib_ref] Action Schools! BC: A school-based physical activity intervention designed to decrease cardiovascular..., Reed [/bib_ref]. Here, we extend our previous work by evaluating the AS!BC model in 3 remote First Nations communities and show a similar cardiovascular benefit in Aboriginal children and youth. Specifically, participants improved their aerobic fitness laps by 22% after 7 months. To place this in context, the magnitude of change we observed in aerobic fitness after less than one year was similar to that seen in other nutrition and PA interventions; while minimal changes in aerobic fitness laps were seen in control groups, intervention groups saw significant increases in laps, with a 27% increase in laps for Crete school children over a similar time period (33), a 21% increase for Swiss children (34), a 20.4% gain for Canadian children living in an urban centre (17) and a similar gain in raw laps for a group of Australian children [bib_ref] A controlled evaluation of a fitness and nutrition intervention program on cardiovascular..., Vandongen [/bib_ref]. Other nutrition and PA interventions*such as the Kahnawake Diabetes Preven- tion Program directed at a Canadian Mohawk community (36) and the Child and Adolescent Trial for Cardiovascular Health study (37)*have reported either no change or a decrement in aerobic fitness following intervention. We acknowledge the clearly established link between overweight/obesity and chronic disease in adults [bib_ref] Impact of overweight on the risk of developing common chronic diseases during..., Field [/bib_ref]. Moreover, an independent, compelling and distinct relationship between aerobic fitness and all-cause mortality has also been established in adults [bib_ref] Changes in physical fitness and all-cause mortality. A prospective study of healthy..., Blair [/bib_ref]. Indeed, in adults, a 1 metabolic equivalent (MET) increase in aerobic fitness decreased the risk of all-cause mortality by approximately 15%. While the 22% improvement in our lap score cannot be directly converted to METs, it does represent an average increased running time of over 40 seconds in a test where each additional 60 seconds of running represents a 1 MET increase in VO 2 max (28). Further, research supports the notion that, for young people, the harmful consequences attributed to overweight and obesity can be counteracted by having high levels of aerobic fitness [bib_ref] Sjö strö m M. Physical fitness in childhood and adolescence: a powerful..., Ortega [/bib_ref]. Thus, we consider the improved aerobic fitness in these First Nations children and youth to be an important finding.
Interventions directed at the primary prevention of diabetes or obesity in First Nations young people have been unsuccessful in their attempts to reduce the prevalence of overweight. The Pathways study showed no significant differences in BMI or percent fat between intervention and control American Indian children after 3 years, with BMI and percent fat increasing similarly in both groups [bib_ref] Pathways: a school-based, randomized controlled trial for the prevention of obesity in..., Caballero [/bib_ref]. The Kahnawake diabetes prevention intervention study reported increased age-and gender-adjusted BMI in both elementary school control and intervention groups.
In other diabetes prevention studies with First Nations young people, mean BMI increased [bib_ref] A pilot school-based healthy eating and physical activity intervention improves diet, food..., Saksvig [/bib_ref] or did not change [bib_ref] A lifestyle intervention improves plasma insulin levels among Native American high school..., Ritenbaugh [/bib_ref] , despite intervention. Thus, we view our outcome*where zBMI remained constant over the intervention period*as positive. While an average zBMI greater than 1 (as seen in our participants) is high, impeding the rise in BMI is an important first step in controlling obesity [bib_ref] Expert committee recommendations regarding the prevention, assessment, and treatment of child and..., Barlow [/bib_ref].
In spite of zBMI not changing, zWC increased, affirming the findings that WC may change independently of BMI [bib_ref] Combined influence of body mass index and waist circumference on coronary artery..., Janssen [/bib_ref]. This may reflect the tendency of Aboriginals to accumulate fat centrally [bib_ref] Central adiposity and associated lifestyle factors in Cree children, Downs [/bib_ref]. Some studies suggested that WC was a better marker of CV risk factors than BMI in children and youth [bib_ref] Waist circumference and waist-to-height ratio are better predictors of cardiovascular disease risk..., Savva [/bib_ref]. Others [bib_ref] Combined influence of body mass index and waist circumference on coronary artery..., Janssen [/bib_ref] demonstrated that young people with a high BMI and high WC were twice as likely to have high triglycerides, high insulin levels and metabolic syndrome when compared to peers who had a normal WC but carried excess body weight. While 47% of participants in the current study had a normal BMI and normal WC, 29% had either a normal BMI and high WC or a high BMI and normal WC; 24% had both a high WC and high BMI. We share these findings with the caveat that we were unable to undertake ethnic-specific comparisons for WC as norms for Aboriginal children are unavailable.
As in previous studies, we observed no change in MVPA measured by accelerometry or questionnaire [bib_ref] Action Schools! BC: A school-based physical activity intervention designed to decrease cardiovascular..., Reed [/bib_ref] [bib_ref] Pathways: a school-based, randomized controlled trial for the prevention of obesity in..., Caballero [/bib_ref]. Even when evaluating accelerometry within and outside of the school day (data not shown), no differences in MVPA were detected. We outlined a number of factors that may have influenced this finding. First, the weather at baseline was considerably warmer (13.491.28C versus 8.993.08C; p B0.001) and drier (43% versus 58% rain days; ns) (47) compared to followup. Since adolescents are more physically active on warmer days and on days with no rain (48), it is not surprising that MVPA remained unchanged at follow-up. Second, enthusiastic teachers began implementing the program before and during baseline measurement in spite of requests to wait until after baseline measurement. Regrettably, this could have elevated baseline MVPA levels, masking potential improvements in PA at follow-up. Third, 3 days of accelerometer monitoring may have been insufficient to capture habitual activity patterns [bib_ref] Standardizing and optimizing the use of accelerometer data for free-living physical activity..., Esliger [/bib_ref]. However, given our limited access to these remote communities and with relatively few participants wearing acceler-ometers for 4 or more days, we accepted a 3-day minimum for our analyses. Given the significant improvement we report for aerobic fitness, the PA measures we adopted may have lacked the sensitivity to capture the change in PA that likely mediated the aerobic fitness benefit.
To achieve a 22% improvement on the aerobic fitness test, frequency, duration and/or intensity of activity must have increased, even though our tools were unable to detect it. From the teachers' activity logs, as reported in , it appears that extra classroom PA was delivered to participants. Classes averaged 75 minutes of weekly physical education plus an additional 65 minutes of PA outside of physical education classes. This classroom PA likely contributed to enhancing aerobic fitness; a similar dose of PA resulted in modest increases in steps for male students during our preliminary trial [bib_ref] An active school model to promote physical activity in elementary schools: Action..., Naylor [/bib_ref].
In terms of overall CV risk, almost three-quarters of participants had at least 1 of 4 CV risk factors both preand post-intervention (73.7% versus 72.9%; p !0.05). It is encouraging that the number of children and youth in the risk category for aerobic fitness decreased at followup, although we observed no improvements in the other risk categories. Comparing CV risk with other studies is difficult as there are many different risk variables, and cut-points for risk are not standardized. Even so, the results of our study are more alarming than results reported elsewhere. , who assessed CV risk in 8Á15-year-old Portugese youth, and Reed et al. [bib_ref] Action Schools! BC: A school-based physical activity intervention designed to decrease cardiovascular..., Reed [/bib_ref] , who assessed 9Á11-year-old Canadian children, both found that approximately 50% of participants presented with at least 1 of 4 CV risk factors. Clearly, the Aboriginal young people in our study are at an unacceptably high health risk. Thus, minimizing cardiovascular and diabetes risk should be a major public health objective for this population.
We observed no change in SSB consumption and only a small improvement in FV intake. This may have been the result of too little time devoted to HE activities. Teacher logs indicated that an average of 15 minutes of HE activities was delivered twice per week over the year, including both the SSB and FV campaigns [bib_ref] Implementing a whole school physical activity and healthy eating model in rural..., Naylor [/bib_ref]. Other successful FV interventions have dedicated 42Á60 or more minutes per week to HE activities (12Á14). While other successful nutrition/SSB interventions involved replacing sugar-sweetened soft drinks with diet (sugarfree) options at school [bib_ref] A lifestyle intervention improves plasma insulin levels among Native American high school..., Ritenbaugh [/bib_ref] or at home [bib_ref] Effects of decreasing sugar-sweetened beverage consumption on body weight in adolescents: a..., Ebbeling [/bib_ref] , or by banning junk food sold or brought to school [bib_ref] Comparison of the dietary intakes of two different groups of children (grades..., Jimenez [/bib_ref] , ours involved classroom education and student tracking. We accept that the clinical relevance of increasing fruit and vegetable variety by just under half a fruit or vegetable each day is not clear. However, this small increase may prove a marker for a more substantial change in health behaviours that may ultimately lead to increased FV consumption and a better micronutrient profile over the students' lifespans. Anecdotal reports highlighted that fruits and vegetables were particularly vulnerable to transportation and storage issues in these remote communities.
One strength of our study was that it represented a ''natural'' experiment that illustrated the potential impact of a focused, comprehensive school health model within the context of remote Aboriginal communities and the challenges faced by teachers and students every day. Another strength was the high consent (96%) and participation (87%) rates that demonstrated the willingness of residents to participate in interventions that aim to promote child health. Furthermore, there was little apparent difference between participants measured at both time periods and those measured only at baseline.
Our findings should be interpreted in light of several limitations related to study design, measurement and the intervention itself. The most significant limitation was our inability to include a comparison group. Our research was based on a long-term partnership with the First Nations communities that we approached and all of these communities wished to be involved in the intervention. These partnerships are key to an ethical approach to working with Aboriginal communities in Canada [bib_ref] Participatory research with native community of Kahnawake creates innovative code of research..., Macauley [/bib_ref]. Thus, we were unable to recruit ''comparator'' communities with which we had no relationship and there was insufficient time to establish new relationships during the relatively short grant-funding time frame. Furthermore, each small community in our study was geographically and socially unique (20) thus it would not have been possible to find a representative comparison community.
Measurement limitations included the use of selfreport to assess PA and HE. While the PAQ is a selfreport measure, it has good internal consistency and validity compared with several other PA measures [bib_ref] Measuring general levels of physical activity: preliminary evidence for the physical activity..., Crocker [/bib_ref]. To overcome issues related to self-reported PA, we also included an objective measure of PA in a subset of participants. However, extreme seasonal differences in weather and early uptake of the intervention by teachers may have influenced our ability to accurately assess change in PA. In addition, many participants failed to comply with accelerometry protocol (a daily wear period of !10 hours was required to be considered a valid day). This resulted in many follow-up files with less than 3 valid days; their data were excluded from analysis. By using a single dietary recall we may have misrepresented usual patterns of food consumption [bib_ref] Between-and within-subject variation in nutrient intake from infancy to old age: estimating..., Nelson [/bib_ref]. Unfortunately, administering multiple dietary recalls was not possible given our travel constraints. In addition, 20% of the 24-hour recalls were carried out on a different day (weekend versus weekday) at baseline compared with follow-up. Finally, our findings related to overweight and obesity may be influenced by our use of zWC measurements that were based on non-Aboriginal population standards; fat distribution differs between non-Aboriginal and Aboriginal children [bib_ref] Estimation of body fat from anthropometry and bioelectrical impedance in Native American..., Lohman [/bib_ref].
Higher lap scores after the 7-month period may, in part, reflect normal growth and development of children. That said, changes in height and weight did not significantly affect the lap score (as per the regression analysis we performed). Further, when we used ageadjusted lap score cut-points to determine CV risk, the CV risk for low cardiovascular fitness had significantly decreased at follow-up (see [fig_ref] Table I: Participant characteristics at baseline [/fig_ref] , confirming that critically low aerobic fitness had improved. Although improvements in the shuttle run may reflect enhanced motivation or effort, based on the magnitude of the increase it is unlikely that these factors alone explain the changes we observed.
Finally, our study may have been limited by the design of the intervention, which was developed for grades K-9. However, given the structure of schools in these remote northern communities, we administered the intervention to all grades. Naylor et al. [bib_ref] Implementing a whole school physical activity and healthy eating model in rural..., Naylor [/bib_ref] reported that some teachers from the higher grades were concerned about the potential for disruptive student behaviour, especially during the PA breaks, as a barrier to implementation of the model in older students.
# Conclusion
In conclusion, it is imperative that effective strategies be devised to improve the health of children and youth residing in remote First Nations communities. In our study, the positive effect of AS!BC on aerobic fitness and the ability of this school-based model to stay the current upward trajectory of age-and gender-adjusted BMI in Aboriginal children and youth holds great promise for effectively enhancing the health of this population.
[fig] 1: Citation: Int J Circumpolar Health 202, 7: 7999 -DOI: 0.3402/ijch.v7i0.7999 [/fig]
[table] Table I: Participant characteristics at baseline [/table]
|
Discrete Modeling of Amoeboid Locomotion and Chemotaxis in Dictyostelium discoideum by Tracking Pseudopodium Growth Direction
Dictyostelium discoideum amoeba is a well-established model organism for studying the crawling locomotion of eukaryotic cells. These amoebae extend pseudopodium -a temporary actin-based protrusion of their body membrane to probe the medium and crawl through it. Experiments show highly-ordered patterns in the growth direction of these pseudopodia, which results in persistence cell motility. Here, we propose a discrete model for studying and investigating the cell locomotion based on the experimental evidences. According to our model, Dictyostelium selects its pseudopodium growth direction based on a second-order Markov chain process, in the absence of external cues. Consequently, compared to a random walk process, our model indicates stronger growth in the meansquare displacement of cells, which is consistent with empirical findings. In the presence of external chemical stimulants, cells tend to align with the gradient of chemoattractant molecules. To quantify this tendency, we define a coupling coefficient between the pseudopodium extension direction and the gradient of an external stimulant, which depends on the local stimulant concentration and its gradient. Additionally, we generalize the model to weak-coupling regime by utilizing perturbation methods.Amoeboid movement is the most common method of locomotion in eukaryotic cells 1 . This structured movement is widely seen in unicellular organisms with amorphous structures, e.g. in leukocyte crawling within interstitial tissues 2 . A model organism for eukaryotic cellular motility research is Dictyostelium discoideum. Dictyostelium is a free-living soil amoeba, feeding on bacteria. When the nutrients are available, Dictyostelium lives as a single-cell amoeba with nearly round spherical shape with average diameter of 10 μm. When the food runs out, as a survival strategy, the cells start to signal by releasing cyclic Adenosine Mono Phosphate (cAMP) in to the environment to attract other cells 3 . The nearby cells respond to this signal both by relaying the signal and moving up the cAMP gradient. This directed movement in response to extracellular chemical stimulants is called Chemotaxis. The chemotaxis process prompt self-accelerating processes results in aggregation 4-6 . The aggregation process leads to form a multicellular organism whose shape evolves in time. Finally, the process forms a structure consists of a stalk and a fruiting body including spores which are capable of long-term survival.In order to probe the medium and move in the environment, the amoeba cells grow pseudopodium, a temporary actin-based protrusion of their membrane. Based on the experiments the cell extends pseudopodia in two types: (1) splitting on certain angles with respect to the existing pseudopodium, (2) growing protrusions occasionally at the rear side of the cell, called de novo 7 . The amoeba is propelled by growing successively splitting pseudopodia in a particular direction, while a random reorientation is generated by extending de novo pseudopodium 8-10 . Before starved cells sense their neighbors and begin to cooperate with them, the locomotion of the cells can be modeled as persistent random walk 9-13 . It is assumed that the mechanism of persistent movement in Dictyostelium discoideum depends on the ratio of splitting and de novo pseudopodia. External chemical stimulants may bias the position and direction of pseudopod extension. For example, in the presence of cAMP concentration the cells tend to align their movement with the stimulant gradient 10 .
1. Pseudopodia are extended perpendicular to the surface curvature at the place where they emerge . Two types of pseudopodia may be formed: frequent splitting of an existing pseudopod, or the occasional extension of a de novo pseudopod at regions devoid of recent pseudopod activity 7 . 3. The angle between two split-split pseudopodia is bimodally distributed with peaks of about 55 degrees to the right or left relative to the previous pseudopod. 4. De novo pseudopodia are extended with equal probability in nearly random directions. 5. A pseudopod can extend to the right (R, positive angle) or to the left (L, negative angle) relative to the previous pseudopod. The alternating RL + LR occur about 3 times more often than the consecutive RR + LL. [bib_ref] Cell polarity: A chemical compass, Bourne [/bib_ref]. The pseudopodia do not bend towards the gradient and still are extended perpendicular to the local cell surface curvature [bib_ref] A Stochastic Model for Chemotaxis Based on the Ordered Extension of Pseudopods, Van Haastert [/bib_ref].
Here, considering the above observations as axioms of the macroscopic dynamics of the cell locomotion, we propose a stochastic model for the movement. We describe the model as a "second order Markov chain" for the direction of movement, meaning that the subsequent direction depends not only on the present direction, as in a standard Markov process, but also on the previous direction. Coupling the centroid's movement to the ordered pseudopod growth process, we show that in the absence of external signaling the model leads to undirected motion. Afterwards, by combining the rules of cell's motility with its inclination towards the gradient of external stimulants concentration, we see that the model result in biased movement. The results matches fairly well with the corresponding experiments. This method might help shed light on the question that what are the functional quantities in collective behavior of cells which undergo chemotaxis during the aggregation process.
# Methods
It is a widely held view that the mechanism of persistent movement in Dictyostelium discoideum likely depends on pseudopodia extension series. We are about to construct a minimal model based on the aforementioned axioms that would be capable of explaining the experimental data. Let us suppose that a pseudopodium can extend only along six equally divided allowed directions with respect to a fixed axis (see [fig_ref] Figure 1: Schematics of the space of states including different possibilities for extending a... [/fig_ref]. Thus, the space of states is equal to
[formula] π = − | ∈ { } S n n ( 1) 3 [/formula]
{1, 2, 3, 4, 5, 6} [bib_ref] Reconstruction of Active Regular Motion in Amoeba Extract: Dynamic Cooperation between Sol..., Nishigami [/bib_ref] In the following, we imitate the cell movement both in homogeneous and inhomogeneous medium by referring to these directions.
## Movement in homogeneous
Medium. Based on the number 5 of aforementioned observation, indicated in the introduction, there are some statistical correlations between the growth direction of two successive pseudopodia. Indeed, the probability of splitting a pseudopodium in the same direction (turning to left or right) as the previous pseudopodium differs from the probability for a step back. In this case, the stochastic variable of pseudopodium growth direction, θ t , is not a Markov chain but a Markovian of second degree. That is because its probability distribution at time t not only depends on its value at t − 1 but also depends on its value at t − 2. However, by applying a trick the stochastic variable can reduce to a Markov chain. One can test that the pair of consequent growing pseudopods X t = (θ t , θ t−1 ) is Markovian. We assume that growing pseudopodia in a row is not allowed. Thus, the range of X t is a discrete set that consists of a 30 possible states of allowed pair angles and the transition probability
[formula] P (0) (X t+1 |X t ) is a 30 × 30 matrix. [/formula]
The transition matrix reads as
[formula] | = | + + + P X X P n m n m ( ) (( , )( , )) (2) t t t t t t (0) 1 (0) 1 1 = | P n m n m (( , ) ( , )),(3)(0) 2 2 1 1 [/formula]
in which the angle of first and second component of each pairs are indicated by n and m (n, m ∈ S), respectively. Note that m t+1 = n t , as both of them address to θ t . We assume that the process is time-homogeneous. Thus the equality in Eq. 3, is straightforward. The zero superscript in P (0) denotes the transition probabilities in the absence of the chemoattractant concentration gradient. Now, after restoring the Markov character, the model can be treated normally by defining the transition probabilities between different states. Let us assume that in every time step there is only one growing pseudopodium. The pseudopodium growth direction in every time step is a stochastic variable, θ t ∈ S. The angle θ t+1 on the next step will be in one of the below forms with respect to θ t (see [fig_ref] Figure 1: Schematics of the space of states including different possibilities for extending a... [/fig_ref] :
- makes a turn of π/3 to the right in the currently active front area of the cell (splitting to the right),
- makes a turn of π/3 to the left in the currently active front area of the cell (splitting to the left),
- makes a turn of +2π/3, −2π/3 or π in the currently inactive rear side of the cell (De novo).
There are four scenarios for proceeding every two subsequent steps, See [fig_ref] Figure 2: Schematics of the model, showing different possibilities for two successive movement [/fig_ref]. Every scenario represents the corresponding entries in the transition matrix P (0) (X t+1 |X t ):
(A) Two subsequent splittings, [fig_ref] Figure 2: Schematics of the model, showing different possibilities for two successive movement [/fig_ref]. where p is the probability of making a de novo pseudopodium in each step and α and β are the probabilities of consecutive and alternative splitting, respectively. (B) Splitting at the side of previous pseudopod at first step and then growing protrusion at the rear side at the second step, [fig_ref] Figure 2: Schematics of the model, showing different possibilities for two successive movement [/fig_ref]. (C) Extending pseudopodium at the rear side (with respect to the position of previous pseudopodium) and then splitting at the side of the current one, [fig_ref] Figure 2: Schematics of the model, showing different possibilities for two successive movement [/fig_ref]. (D) Growing pseudopod on both steps randomly, at least with 2π/3 deviation clockwise or counter-clockwise, with respect to the previous step, [fig_ref] Figure 2: Schematics of the model, showing different possibilities for two successive movement [/fig_ref]. The entire transition matrix is demonstrated in Supporting Information, Appendix A. In general, there are five nonzero entries in each row. Thus, the matrix P (0) is sparse.
[formula] δ δ δ δ δ δ δ δ δ | = − − − + − − − + + − − + − { } P n m n m p (( , ) ( , )) 3 (1 )(1 )(1 ) (1 )(1 )(1 )(5)δ δ δ δ δ δ δ δ δ | = − − − − + − − − + + − − + − { } P n m n m p (( , ) ( , )) 1 2 (1 )(1 )(1 ) (1 )(1 )(1 )(6)δ δ δ δ δ δ δ | = − − − + − − − − + + − { } P n m n m p (( , ) ( , )) 3 (1 )(1 )(1 ) (1 )(1 )(1 ) (7) [/formula]
In the transition matrix P (0) (see Supporting Information, Appendix A for details) all the entries are non-negative and each row adds up to unity. Clearly, the matrix P (0) has the eigenvalue 1, because ∑ = = P 1
[formula] j i j 1 30 ,(0) [/formula]
. This relation in matrix notation can be written as P (0) 1 = 1, where 1 is a column vector whose entries are all 1. P (0) has also a left eigenvector ω 0 corresponding to eigenvalue 1. ω 0 is called a stationary or invariant distribution and can be obtained as
[formula] ω − = . P ( ) 0 (8) 0 (0) [/formula]
where is the unite matrix of size 30. We solve Eq. 8, to find ω 0 as a 30 × 1 vector whose entries are invariant probabilities of every possible pairs of subsequent pseudopodia directions. Then, we rearrange the entries in a 6 × 6 array, in which both horizontal and vertical values are in the space of states. Clearly, in the new arrangement all the diagonal entries are zero, as we assumed that growing pseudopodia in a row is prohibited. ω 0 is a symmetric matrix as it is the invariant distribution of extending pseudopodium along discrete directions in a homogeneous medium. Rearranged form of ω 0 in colored matrix is depicted in [fig_ref] Figure 3: Rearranged invariant distribution of the transition matrix [/fig_ref]. In this figure the blue arrows indicate the change in direction relative to the previous direction. Obviously, the probability of choosing each of six possible directions of S in a homogeneous environment is 1/6, that is why the summation of elements of ω 0 both in a row and in a column gives 1/6. Another notable point in an invariant distribution is disappearing of α and β on longtimes (see [fig_ref] Figure 3: Rearranged invariant distribution of the transition matrix [/fig_ref].
[formula] ω = − − − − − − − − − − − − p [/formula]
As stated before, we assume that the persistence of cell movement is based on the ratio of splitting versus de novo pseudopodia. Thus, it is necessary to couple the cell's centroid movement to the direction of growing pseudopodium. Let us assume that every extended pseudopodium leads to movement of the whole cell body and besides, the cell moves along the same direction of the extended pseudopodium. We define an orthonormal basis with the unit vectors x and ŷ on the surface that the cells are crawling on it. The position of each cell is characterized in Cartesian coordinates by → = +r xx yy. In reality not all of the extended pseudopodia are followed by the cell body displacement. Indeed, only ~60% of these protrusions contribute to cell movement. But the size of proceeded length differs from step to step between ~0.25 μm to ~10 μm, depend on the previous state of the cell [bib_ref] The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of..., Bosgraaf [/bib_ref].
Here for simplicity, we assume that all the extended pseudopodia lead to the whole body displacement but with a fixed step size which is equal to 6 μm. The time resolution of the simulations, i.e. the time steps, are equal to 20 seconds. [fig_ref] Figure 4: The trajectories of 7 cells during 15 min in a homogeneous medium [/fig_ref] illustrates the trajectories of 7 independent tracers in the absence of chemical stimulants concentration gradients. As we expect spreading out of the cells on the surface is isotropic.
Chemotactic Movement. In the absence of chemical gradient, the system is entirely homogenous and isotropic. One of the consequences of this fact is that the corresponding transition matrix P (0) would be invariant . However, in the presence of cAMP concentration gradient the index translation symmetry breaks down. Experiments show that even in such an inhomogeneous medium the pseudopodia do not bend towards the gradient and still are extended perpendicular to the local cell surface curvature [bib_ref] The local cell curvature guides pseudopodia towards chemoattractants, Van Haastert [/bib_ref]. But the gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size [bib_ref] Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation, Bosgraaf [/bib_ref]. This fact suggests that the system still has the parity transformation, i.e. the mirror symmetry with respect to the gradient axis. Let us assume that the state of growing pseudopodia in the presence of external cues are still given by S in the relation (1). It is plausible to assume that the chemotactic movement of the cells are additive to their random movement. Thus the transition matrix between different states in the inhomogeneous medium P, is superposition of transition matrix in the homogeneous medium, P (0) and an additional matrix which corresponds to the cell's attempt to get aligned with the preferred spatial direction, i.e. the gradient of cAMP. Clearly, the additional matrix has to depend on concentration gradient of the chemoattractant. Besides, every entry of this matrix should be sensitive to the angle between the prospective direction of growing pseudopodium and the gradient of cAMP. We opt the inner product between these two vectors as our theoretical counterpart in this case. As the parity symmetry implies, one should discard the probability of protruding along the opposite direction of the current direction at the cell's posterior in the next step. The only restriction on the additional term is that the sum of each row should be zero. Clearly, with all the above characteristics the condition is going to automatically fulfill. Let us assume that ŷ is the direction of the spatial gradient of cAMP and n j represents the probable direction of expanding pseudopodium in the next step, then
[formula] ε = + ⋅P P y n ,(9)ij ij j (0) where ε ε = ∇ C C ( , ) [/formula]
is the function which gives the coupling between cell's overall orientation and the gradient of chemoattractant and depends on both concentration of cAMP and its gradient. We refer it as the coupling coefficient. Dimensional analysis suggests that this function in this linear approximation would be equal to ε γ = ∇
## L c c
, where L is the typical size of a cell (~10 μm) and γ is a numerical constant, called coupling parameter, that cannot be merely determined by the dimensional analysis method. Now one can find the stationary state for the cell's chemotactic movement in this field. Owing to the size of the transition matrix P (0) , finding an analytical solution for the stationary state of the system may be difficult, if possible. Here, applying perturbation methods, we solve the problem for the system in weak-coupling regime (see Supporting Information, Appendix B for details). As every individual probability, P ij , must be between 0 and 1, i.e.
[formula] ε ∀ ≤ + ⋅ ≤î j P y n , 0 1 ij j (0) [/formula]
, the maximum valid value for applying the perturbation method is ε = 0.043. By weak-coupling regime, we refer to the inhomogeneous fields of cAMP concentration for which ε ≤ 0.043. [fig_ref] Figure 5: The tracks of 10 cells during 15 min in an inhomogeneous medium... [/fig_ref] depicts the trajectories of 10 independent tracers in the presence of chemical stimulants concentration gradients, where the coupling coefficient between pseudopodium extension direction and gradient of external stimulant is equal to ε = 0.04. Clearly, there is an upward drift in line with the gradient of stimulant concentration.
# Results
In [fig_ref] Figure 4: The trajectories of 7 cells during 15 min in a homogeneous medium [/fig_ref] we show the trajectories of 7 independent tracers in a homogeneous medium. It is well-known 11 that the relation where N is the number of realizations to be averaged, r n (0) is the initial position of each tracer and r n (t) is the position of each individual in determined time t. Here, N = 10000 and the reference position of each cell is the origin i.e., r n (0) = 0.shows the plot of the function D = 〈r 2 (t)〉/4t with respect to time, which give information of diffusive behavior in a typical 2-dimensional random walk process over time. On long times the function is equal to diffusion coefficient of the tracers. The graph shows a dramatic increase in the first 5 minutes and reaches approximately to a plateau near 80 μm 2 /min within 30 minutes. Inthe mean square displacement of the process in Log-Log scale has been illustrated. The crossover between the ballistic and diffusive regimes is clearly visible on the plot. Fitting the MSD to Eq. 10, yields the values v = 11.64 ± 0.25 μm/min and τ = 1.00 ± 0.35 min for the speed and the persistence time respectively, which is in close agreement with the experiment [bib_ref] The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of..., Bosgraaf [/bib_ref].
[formula] τ τ = − − τ − r t v t ( ) 2 [ (1 exp )](10)∑ = − = − = MSD r r N r t r ( ) 1 ( ( ) (0)) ,(11) [/formula]
In the presence of constant gradient of chemoattractant cAMP, the cells start to co-orient their movements with the gradient direction. Let us assume that the angle between prospective pseudopodium and concentration gradient of cAMP is φ. The population average of this angle 〈cos φ(t)〉 is a proper quantity to measure the cell's biased orientation in comparison with its random orientation in a homogeneous medium. As depicted in, even for a small value of coupling constant, there is a considerable tendency to move forward along the cAMP concentration gradient. Notably, 〈cos φ〉 reaches to a steady amount rather soon even for small values of ε. It is worth to mention that this amount is equal to zero in the homogeneous medium (See [fig_ref] Figure 3: Rearranged invariant distribution of the transition matrix [/fig_ref]. As an important illustration of the general features of chemotactic movement, we consider the Chemotaxis Index quantity,. Chemotaxis Index measures the chemotactic movement of a cell towards the chemical gradient with respect to its entire movement. One can calculate chemotactic index as the time average of 〈cos φ〉. It is apparent from the left-hand plot ofthat chemotaxis index linearly increases with coupling constant for the allowed values of ε. As the range of ε considered here is low, thermal agitation still can affect the cell's movement. To get a feeling of the influence of random thermal agitation one can measure the standard deviation of the chemotaxis index for different values of coupling constant, the right-hand plot of. Evidently, the chemotactic movement get comparable with diffusive propagation of the cells only near ε = 0.04. As a physical analogue, one may think of the low drift velocity of an ion induced by a weak electric field while the ion experiences countless collisions with fixed atoms in a circuit.
# Discussion
In recent years, analyzing the locomotion of various eukaryotic cells have been of interest [bib_ref] A model for a correlated random walk based on the ordered extension..., Van Haastert [/bib_ref] [bib_ref] The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of..., Bosgraaf [/bib_ref] [bib_ref] A Stochastic Model for Chemotaxis Based on the Ordered Extension of Pseudopods, Van Haastert [/bib_ref] [bib_ref] Dicty dynamics': Dictyostelium motility as persistent random motion, Li [/bib_ref] [bib_ref] Persistent Cell Motion in the Absence of External Signals: A Search Strategy..., Li [/bib_ref] [bib_ref] A Stochastic Description of Dictyostelium Chemotaxis, Amselem [/bib_ref] [bib_ref] Dictyostelium discoideum chemotaxis: Threshold for directed motion, Song [/bib_ref] [bib_ref] Quantitative analysis of random ameboid motion, Bödeker [/bib_ref] [bib_ref] Functional Analysis of Spontaneous Cell Movement under Different Physiological Conditions, Takagi [/bib_ref] [bib_ref] Non-Brownian dynamics and strategy of amoeboid cell locomotion, Nishimura [/bib_ref] [bib_ref] Anomalous dynamics of cell migration, Dieterich [/bib_ref]. All of these studies share this outcome that the experimental results deviate from the Ornstein-Uhlenbeck model of persistent random walk. Dieterich et al. [bib_ref] Anomalous dynamics of cell migration, Dieterich [/bib_ref] report superdiffiusive behavior in the movement of the crawler cells. Therefore, they suggest that the fractional Klein-Kramers equation governs the cell's motility. However, Li et al. [bib_ref] Persistent Cell Motion in the Absence of External Signals: A Search Strategy..., Li [/bib_ref] did not observe any apparent intrinsic scale invariance in cell trajectories, which is the essential feature of a typical Lévy walk. There are also theoretical studies which shows that as far as the linear diffusion equation (even anomalous) meets both time-and space-translational invariance, like what the cells experience in homogeneous medium, the variance of movement is an at most linear function of time [bib_ref] Lack of anomalous diffusion in linear translationally-invariant systems determined by only one..., Khorrami [/bib_ref]. Hence, it seems that super-diffusive interpretation of the cell movement is a consequence of short duration of the related experiment and if one be patient enough, can meet the long time diffusive behavior of these cells. Hasstert 8 , Li et al. [bib_ref] Persistent Cell Motion in the Absence of External Signals: A Search Strategy..., Li [/bib_ref] and Cooper et al. [bib_ref] An Excitable Cortex and Memory Model Successfully Predicts New Pseudopod Dynamics, Cooper [/bib_ref] have addressed the same problem by presenting stochastic descriptions that reproduces the so-called zig-zag trajectories of the cells. Haastert 8 proposes a model for the persistent random walk based on the observed angular frequencies of pseudopod extensions. By applying Monte Carlo simulations, he shows that the critical elements of this stochastic process are the ratio of persistent splitting pseudopodia relative to random de novo pseudopodia, the Left/Right alternation, the angle between pseudopodia and the variance of this angle. However, in his simulations, as the direction of the simulated de novo and splitting pseudopodia are random, there is always a limited probability that a simulated pseudopodium in de novo type would be recognized in experiments as splitting pseudopodium and vice versa.
Incorporating the principal elements of Haastert's model 8 , we proposed a discrete model to revisit the question of directed motion of eukaryotic cells. Indeed, discretizing the possible angles along which one pseudopodium can extend, prevents shuffling of the simulated splitting and de novo pseudopodia. Based on our model, the cell selects its pseudopodium growth direction based on a second order Markov chain in the absence of external cues. Making the process second order, which is supported by the experiments 9 , has no effect on the stationary state of the cell's migration. Indeed, the short memory of this process vanishes on the cell's long time diffusive behavior (See [fig_ref] Figure 3: Rearranged invariant distribution of the transition matrix [/fig_ref]. Nevertheless, it can postpone the cross over between ballistic and diffusive regimes (See [fig_ref] Figure 1: Schematics of the space of states including different possibilities for extending a... [/fig_ref]. Thus, to describe the persistent behavior of the cells there is no need to attribute fat tail distributions to spatial steps.
Andrew et al. [bib_ref] Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased..., Andrew [/bib_ref] and Bosgraaf et al. [bib_ref] The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of..., Bosgraaf [/bib_ref] define splitting pseudopods as pseudopods that originate from, or are formed in the domain of, an existing pseudopod. Thus, correct attribution of the membrane protrusions depends on defining the boundary between a pseudopod and the rest of the cell, and the correct experimental detection of this boundary. Based on experimental observation, the detected angle between two split-split pseudopodia has a bimodal distribution with peaks of about 55 degrees to the right or left relative to the previous pseudopod. However, distinction between splitting and de novo pseudopodia at the tails of this distribution is more challenging. Indeed, although cells shape change as they move, their shapes remain relatively smooth and the pseudopods grow perpendicular to the membrane. Therefore, a larger angular difference generally implies a larger spatial distance between the tips of two successive pseudopodia. That is, it is more likely that a fraction of de novo pseudopods have been detected as splitting pseudopods at this region. Hence, the tails of the distribution may be lighter than it is. To build the space of state, we assume that splitting at the side of previous pseudopodium occurs at discrete angles -with approximately π/3 radians turn-in the anterior part of the current active area of cell. Apparently, the main part of simplification has been applied in considering discrete states for extending de novo pseudopodia. However, as this type of pseudopodia rarely occur (growing a splitting pseudopodium is seven times more probable than a de novo pseudopodium 9 ), one may consider this simplification something like a mean-field approximation. The statistical features of the model for the cell's centroid motility are in well agreement with the corresponding measured experimental and also simulated quantities (with considering all the complexities of non-discrete states). This may suggest that the model can describe the locomotion of the wild-type cells with statistically regular shape fairly well. The reported speed and persistence time for a typical wild type cell during 15 minutes are v = 10.4 ± 2.1 μm/min and τ = 3.4 ± 0.5 min, respectively 9 . By applying our discrete model, one obtains v = 11.64 ± 0.25 μm/min and τ = 1.00 ± 0.35 min for these quantities along one hour of simulation. It is seen that within the error bars the measured speed matches well with the experiment. However, the predicted persistence time somehow differs from that of experiment. A possible explanation for this might be that these tracers have to alter their direction at least π/3 in two successive steps. The discrete model provides a framework to develop a suitable computational schemes which is generalizable to chemotactic movement as well.
In the presence of external stimulants, the cell tends to align itself with the gradient of the chemoattractant cAMP [bib_ref] Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased..., Andrew [/bib_ref] [bib_ref] A Stochastic Model for Chemotaxis Based on the Ordered Extension of Pseudopods, Van Haastert [/bib_ref]. This suggests that the tendency and random locomotion of the cell "add up" to propel the cell toward the gradient. In this case both the absolute value of chemoattractant concentration and its gradient incorporate to motivate the cells to crawl through an inhomogeneous field at the same time. In most of the previous studies, these processes has been dealt separately [bib_ref] A Stochastic Model for Chemotaxis Based on the Ordered Extension of Pseudopods, Van Haastert [/bib_ref] [bib_ref] A Stochastic Description of Dictyostelium Chemotaxis, Amselem [/bib_ref] [bib_ref] Dictyostelium discoideum chemotaxis: Threshold for directed motion, Song [/bib_ref]. Haastert 10 extracts the probabilities as well as the gradient induced bias in chemotactic motion from experiments and performs Monte Carlo simulations to quantify Chemotaxis index of a population of individuals with respect to cAMP steepness in the environment. His measured Chemotaxis index is in close agreement with experimental data, however as the model for chemotaxis is firmly based on the ordered extension of pseudopodia in the absence of chemoattractants, the above-mentioned difficulties with Monte Carlo simulation are still relevant. Our model is also built on pseudopod extension-based description which is previously developed by Haastert 10 , but with fewer parameters. We also include the dependency of the chemotactic response to the gradient of cAMP and its local concentration in our model. Previously, this sensitivity has been investigated in some experiments [bib_ref] External and internal constraints on eukaryotic chemotaxis, Fullera [/bib_ref] [bib_ref] Control Parameter Description of Eukaryotic Chemotaxis, Amselem [/bib_ref]. By applying information theory techniques, Fuller et al. [bib_ref] External and internal constraints on eukaryotic chemotaxis, Fullera [/bib_ref] show that for shallow gradients and small local concentrations, the extra cellular fluctuations limit the chemotactic response. In addition, for steep gradients and high local concentrations the observed chemotactic response is lower than what one may predict from a simple ligand-receptor binding process. Inspiring by these observations, we assume that the chemotactic response of the cell to its chemical environment is proportional to the ratio of the steepness ∇C and absolute value of concentration field C (See Eq. 9 and its underneath statement). The parameter with which this proportionality get adjusted is coupling parameter γ. This parameter is a phenomenological variable that have to measure experimentally. Indeed, Eq. 9 suggests a practical procedure to determine coupling coefficient ε values from the transition probabilities quantified from the corresponding experiments for variant pairs of C and ∇C. This is a fundamental issue for future research which requires carrying out more experiments. Investigating the dynamical properties of the cells in weak-coupling regime in a broader sense might pave our way toward gaining a better insight of systems in which responsiveness of the cells to the external field is weak, e.g. in the 'back of the wave' problem.
Here, taking the advantage of perturbation methods, we have studied the cell's migration in weak coupling regime, where ε ≤ 0.043. The approach is not extendable to the strong coupling regime where the coupling coefficient ε is higher. Thus, developing new approaches to study this regime needs more effort and is beyond the aim of this paper.
In summary, we have studied the amoeboid locomotion and chemotaxis in Dictyostelium discoideum by tracking pseudopodium growth direction. We have applied Markov chain of second order to describe the behavior of eukaryotic locomotion in homogenous medium. According to the model, short memory of the second order Markov chain vanishes on the cell's stationary state and hence leads to a smooth transition from persistent random walk to purely diffusive behavior for squared displacement. The chemotactic response of the cell in inhomogeneous medium depends both on the local background concentration and its gradient. Coupling coefficient ε measures this dependency for different possible pairs of them. Utilizing perturbation method, we showed that for the cells which undergo chemotaxis under influence of weak coupling, there is a linear dependence between chemotaxis index and coupling coefficient ε. Our method might help shed light on dealing with the phenomenon of cell aggregation from a theoretical point of view.
[fig] Figure 1: Schematics of the space of states including different possibilities for extending a pseudopodium relative to a given axis, x. Solid arrow shows the cell's decision among all other possible alternatives which are shown in dash arrows. Scientific REPORTs | 7: 12675 | DOI:10.1038/s41598-017-12656-1 [/fig]
[fig] Figure 2: Schematics of the model, showing different possibilities for two successive movement (A) splittingsplitting, (B) splitting-de novo, (C) de novo-splitting, and (D) de novo-de novo. Solid arrow shows the cell's decision among all other possible alternatives which are shown in dash arrows. Scientific REPORTs | 7: 12675 | DOI:10.1038/s41598-017-12656-1 [/fig]
[fig] Figure 3: Rearranged invariant distribution of the transition matrix. The entries are stationary probability for transition between different directions. The blue arrows indicate the change in direction relative to the previous direction, e.g. the (1, 2) element of the diagram indicates the probability of a π/3 change in direction regardless of the current direction. Based on the observations 9 , we set P~1/7. Notably, disappearing of α and β (α + β = 1) on long times remarks that short memory of this Markov chain of second order cannot affect the future of the process on large scales. [/fig]
[fig] Figure 4: The trajectories of 7 cells during 15 min in a homogeneous medium. The black dot indicates the common origin of the trajectories. [/fig]
[fig] Figure 5: The tracks of 10 cells during 15 min in an inhomogeneous medium for which the coupling constant is equal to ε = 0.04. The black dot indicates the common initial point of the stochastic movement of the cells. Scientific REPORTs | 7: 12675 | DOI:10.1038/s41598-017-12656-1 [/fig]
[fig] Figure 6: (a) Diffusion coefficient, D = 〈r 2 (t)〉/4t, plotted as a function of t. For a random walk, diffusion coefficient would give rise to a line with zero slope on long times. (b) Log-log plot of the mean-squared displacement in respect to time. The transition between ballistic and diffusive regimes occurs after around 5 minutes. The number of realizations is N = 10000. [/fig]
[fig] Figure 7: (a) 〈cos φ〉 plotted as a function of t for different values of coupling constants ε, φ is the angle between prospective pseudopodium and concentration gradient of cAMP. (b) Chemotaxis Index (CI) plotted as a function of the coupling coefficient ε. CI is a measure of how much a process does get biased in comparison with random motion. Still, the process includes randomness which can be indicated by the standard deviation of CI.Scientific REPORTs | 7: 12675 | DOI:10.1038/s41598-017-12656-1 [/fig]
|
Arginine68 is an essential residue for the C-terminal cleavage of human Atg8 family proteins
Supplemental Table S1 Gene Bank accession numbers of ESTsBy searching the ESTs database, we listed the representative ESTs which could support the existence of each transcript.SupplementalTable S2 Allele specific primers for each transcript Supplemental Table S3 Primers used in this paper for cloning and mutagenesis SupplementalFigure S1The sequencing results of LC3B-a, GABARAP-a andGABARAPL1-aTo verify the potential alternative spliced transcript, products of RT-PCR were ligated into T-vector. Then these T-vectors were subjected to DNA sequencing. The starting codon and stop codon were annotated with boxes. The alternative splicing positions were annotated with arrow heads.Supplemental Figure S2 Primer specificity test for LC3B and LC3B-a transcriptsLC3B-sense primer was used together with LC3B-antisense primer or LC3B-aantisense primer to amplify 10pg of myc-LC3B or myc-LC3B-a plasmid. The amplification conditions were as follows: 95ºC for 3 min; 35 cycles of 95 ºC for 10 s and 65 ºC for 15 s; final extension at 65 ºC for 1min. Then PCR results were analysed by agarose electrophoresis.Supplemental Figure S3 Alignment of human Atg8 family proteinsProtein sequences of human Atg8 family members and Atg8 from Saccharomyces cerevisiae are aligned. The conserved Glycine in the C-terminus is indicated by arrow and the Arg68 lost in LC3B-a is indicated by arrow head. All the residues are numbered after LC3B. Secondary structures base on Atg8 are shown under the sequences.
## Supplemental
The interaction between LC3B-a and ATG4B is intact as that of LC3B. The interactions between ATG4B with LC3B or LC3B-a were compared by GST pulldown assay. 2 g GST-ATG4B and 5 g LC3B or LC3B-a were subjected to GST pulldown assay in the presence of 1mM PMSF. The proteins binding with beads were then resolved by SDS-PAGE and western blot with anti-GST and anti-LC3B antibodies. Molecular weight is shown on the right.
## Supplemental figure s5 cartoon representation of human atg4b-rat lc3b
complex Human ATG4B-rat LC3B complex (2Z0D) showed the salt bridge between R68 and D171 (yellow dashes in the enlarged box). Primer Names
## Supplemental
Primer Sequence (5' to 3') Note myc-A-sense CCGAATTCGGATGCCCTCAG cloning LC3A into pCMV-myc vector A-his-antisense AAGGTACCTCGAGTTAATGGTGATGGTGATGATGGAAGCCGAAG adding his-tag to the C-terminal of LC3A and cloning LC3A into pCMV-myc vector myc-C-sense CCGAATTCGGATGCCGCCTC cloning LC3C into pCMV-myc vector C-his-antisense GCGGTACCTCGAGTTAATGGTGATGGTGATGATGGAGAGGATTG adding his-tag to the C-terminal of LC3C and cloning LC3C into pCMV-myc vector myc-B-sense CCGAATTCGGATGCCGTCGG cloning LC3B or LC3B-a to pCMV-myc vector B-his-antisense AAGGTACCTTAATGGTGATGGTGATGATGCACTGACAAT adding his-tag to the C-terminal of LC3B or LC3B-a and cloning into pCMV-myc vector myc-G-sense CGAGATCTCAATGAAGTTCG cloning GABARAP or GABARAP-a to pCMV-myc vector G-his-antisense AAGGTACCTCGAGTTAATGGTGATGGTGATGATGCAGACCGTAG adding his-tag to the C-terminal of GABARAP and cloning into pCMV-myc vector myc-L1-sense CCGAATTCGGATGAAGTTCC cloning GABARAPL1 into pCMV-myc vector L1-his-antisense AAGGTACCTTAATGGTGATGGTGATGATGTTTCCCATAG adding his-tag to the C-terminal of GABARAPL1 and cloning into pCMV-myc vector G-a-his-antisense AAGGTACCTCGAGTTAATGGTGATGGTGATGATGTCACCATACCTG adding his-tag to the C-terminal of GABARAP-a and cloning into pCMV-myc vector L1-a-his-antisense AAGGTACCTTAATGGTGATGGTGATGATGTCAGAGCCTTA adding his-tag to the C-terminal of GABARAPL1-a and cloning into pCMV-myc vector
[formula] B-antisense GCGGTACCTTACACTGACAAT cloning LC3B or LC3B-a into pCMV-myc vector GST-4B-sense GTGGATCCATGGACGCA cloning ATG4B into pGEX-4T-1 vector GST-4B-antisense AACTCGAGTCAAAGGGACAG cloning ATG4B into pGEX-4T-1 vector GST-B-sense CGTGGATCCATGCCGTCGG cloning LC3B or LC3B-a into pGEX-4T-1 vector GST-B-antisense CGCTCGAGCACTGACAAT cloning LC3B or LC3B-a into pGEX-4T-1 vector GFP-B-sense TCGAATTCGATGCCGTCGG cloning LC3B or LC3B-a into pEGFP-c1 vector GFP-B-antisense GCGGTACCTTACACTGACAATTTC cloning LC3B or LC3B-a into pEGFP-c1 vector GFP-G-sense TCAGATCTATGAAGTTCG cloning GABARAP into pEGFP-c1 vector GFP-G-antisense GCGGTACCTTACAGACCGTAG cloning GABARAP into pEGFP-c1 vector GFP-G-a-sense [/formula]
ATCTCGAGCTATGAAGTTCGT cloning GABARAP-a into pEGFP-c1 vector GFP-G-a-antisense GCGGTACCTCACCATACCTGGT cloning GABARAP-a into pEGFP-c1 vector GFP-L1-sense TCGAATTCGATGAAGTTCC cloning GABARAPL1 into pEGFP-c1 vector GFP-L1-antisense GCGGTACCTTATTTCCCATAG cloning GABARAPL1 into pEGFP-c1 vector GFP-L1-a-sense ATCTCGAGCTATGAAGTTCC cloning GABARAPL1-a into pEGFP-c1 vector GFP-L1-a-antisense GCGGTACCTCAGAGCCTTAC cloning GABARAPL1-a into pEGFP-c1 vector
## ------m p s e k tf k q r r tf e q r v ed v r l i re q h p t ki p v i i er y k g ek q l p v ld k t k f lv p d h v nm s e l i ki i r r r lq l n a n qa f f l l vn g h s
## Lc3b (1)
## ------m p s e k tf k q r r tf e q r v ed v r l i re q h p t ki p v i i er y k g ek q l p v ld k t k f lv p d h v nm s e l i ki
[formula] I R - LQ L N A N QA F F L L VN G H S LC3B-b (1) M P P P Q KI P S V R PF K Q R K SL A I R Q EE V A G I RA K F P N KI P V V V ER Y P R ET F L P P LD K T K F LV P Q E L TM T Q F L SI I R S MV L R A T EA F Y L L VN N K S LC3C (1) --------M K F VY K E E H PF E K R R SE G E K I RK K Y P D RV P V I V EK A P -KA R I G D LD K K K Y LV P S D L TV G Q F Y FL I R K R IH L R A E DA L F F F VN -N V GABARAP (1) --------M K F VY K E E H PF E K R R SE G E K I RK K Y P D RV P V I V EK A P -KA R I G D LD K K K Y LV P S D L TV G Q F Y FL I R K R IH L R A E DA L F F F VN -N V GABARAP-b (1) --------M K F QY K E D H PF E Y R K KE G E K I RK K Y P D RV P V I V EK A P -KA R V P D LD K R K Y LV P S D L TV G Q F Y FL I R K R IH L R P E DA L F F F VN -N T GABARAPL1 (1) --------M K F QY K E D H PF E Y R K KE G E K I RK K Y P D RV P V I V EK A P -KA R V P D LD K R K Y LV P S D L TV G Q F Y FL I R K R IH L R P E DA L F F F VN -N T GABARAPL1-b (1) --------M K W MF K E D H SL E H R C VE S A K I RA K Y P D RV P V I V EK V S -GS Q I V D ID K R K Y LV P S D I TV A Q F M WI I R K R IQ L P S E KA I F L F VD -K T GABARAPL2 ATG8 LC3A LC3B LC3B-b LC3C GABARAP GABARAP-b GABARAPL1 GABARAPL1-b GABARAPL2 [/formula]
(1) Supplemental |
Ant‐induced alopecia: A new differential diagnosis for nonscarring alopecia
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.AbstractAnt-induced alopecia is a rare condition caused by Pheidole pallidula species. This particular cause of nonscaring alopecia should be considered a new differential diagnosis.K E Y W O R D Salopecia, ants, arthropods, hair, Pheidole pallidula 2 of 5 | GANJEI Et Al.ACKNOWLEDGEMENTPublished with written consent of the patient.CONFLICT OF INTERESTThe authors have no conflict of interest to declare.
# | introduction
Ant-induced alopecia is a rare condition; however, due to the increasing number of affected cases, this cause of nonscaring alopecia should be considered a new differential diagnosis. This article presents a case of ant-induced alopecia.
Hair is a part of the body with a bold cosmetic aspect that affects people's life. [bib_ref] The effect of hair loss on quality of life, Williamson [/bib_ref] Some studies have shown the association of alopecia with psychological disorders such as depression and anxiety; therefore, physicians should consider this condition more than a cosmetic disorder. [bib_ref] Psychological impact of alopecia areata, Marahatta [/bib_ref] Different types of alopecia are categorized into two groups, including nonscaring and scaring alopecia. Various conditions can cause nonscaring alopecia, ranging from infections such as tinea capitis to autoimmune conditions like alopecia areata. [bib_ref] Hair loss in women, Shapiro [/bib_ref] An ant species called Pheidole pallidula (P. pallidula) has also been shown to cause hair loss in some parts of the head in some cases. [bib_ref] Ant-induced alopecia: report of 2 cases and review of the literature, Mortazavi [/bib_ref] This study reports on an Iranian woman with a case of ant-induced alopecia.
## | case presentation
A 45-year-old woman was admitted to Shahrekord University of Medical Sciences' dermatology clinic with a sudden hair loss that had occurred overnight. She had no history of any specific diseases or medication consumption. Moreover, she had taken a trip from Isfahan to Shahrekord the day before and this incident had taken place in her mother-in-law's house.
In the examination, there were multiple nonscarring, nearly round patches of alopecia with a diameter of 1-2 cm in the vertex, which was nonexistent in the other regions, and no signs of scaling, erythema, and inflammation were observed in her skin.
The caliber, fragility, and shape of the hair were normal, and there was no exclamation mark hair. In addition, the alopecic patches were rough to touch and the hair looked cut to the same length about a few millimeters above the hair shaft and parts of a dead ant were found in the patient's hair. The pull test was also negative for this case. There were no erythema, scaling, and crusting in the hair loss areas. The patient had no history of any psychiatric disturbances, and there was no broken hair in different sizes on her head. Based on her history and physical examinations, the patient was diagnosed with ant-induced alopecia. The patient was sent home with reassurance and no treatments. She was advised to bring with her a sample of the ants, if possible, to be sent to the entomology laboratory.
## | outcome and follow-up
Three weeks after her discharge from the clinic, she came for her follow-up with her mother-in-law, and her alopecic regions seemed to have been cured and she had brought some of the responsible ants with her . The ants were sent to the entomology laboratory and were then identified as the P. pallidula sp. Her mother-in-law had not experienced the same incident ever in her life.
# | discussion
There are many reasons for hair loss in women, thus necessitating thorough history-taking and physical examinations. [bib_ref] Hair loss in women, Shapiro [/bib_ref] In general, the causes of hair loss can be categorized into two groups, such as scaring and nonscaring alopecia. [bib_ref] Feature characterization of scarring and non-scarring types of alopecia by multiphoton microscopy, Lin [/bib_ref] Physical examination, history-taking, and some other para-clinical tests can help physicians differentiate between the causes of this health condition, including infectious (including tinea capitis) and psychiatric (including trichotillomania) causes, autoimmune reasons (such as alopecia areata), and other metabolic diseases or malnutrition. Moreover, healthcare providers should know the pathophysiology of this condition in different age-groups, since having a thorough understanding of the various causes of hair loss is crucial for its management. [bib_ref] Hair loss in women, Shapiro [/bib_ref] [bib_ref] Hair loss, Imhof [/bib_ref] Besides these frequent causes, there are some other rare causes that could induce alopecia, such as arthropod bite reactions. [bib_ref] Tick bite alopecia: a report and review, Lynch [/bib_ref] Ant-induced hair loss was first described by Radmanesh et al. [bib_ref] Alopecia induced by ants, Radmanesh [/bib_ref] in 1999, who reported three cases of ant-induced F I G U R E 1 Short hairs that has been cut a few millimeters above the surface of the skin F I G U R E 2 Ants brought by the patient alopecia in Ahwaz, Iran. Afterward, some additional cases were also reported, as summarized in . All the cases of ant-induced alopecia were due to the genus P. pallidula . P. pallidula (barber ants) has been reported to cause such types of alopecia, mainly in Iran, but there have been two reported cases in Turkey and Italy as well, one of whom had taken a trip to Iran. It thus seems that this species is becoming more and more dominant in some regions, such as across the Mediterranean countries and in tropical and subtropical regions. [bib_ref] Identification of a component of the trail pheromone of the ant Pheidole..., Ali [/bib_ref] [bib_ref] Field study on foraging by the polymorphic ant species, Pheidole pallidula, Detrain [/bib_ref] Several hypotheses can explain the development of this condition, including the following: First, similarities between the pheromones in P. pallidula and some chemicals in the sebaceous glands and skin of some humans 10 ; second, the infection of P. pallidula with some parasites causes neural shock and seizure-like behaviors in ants, which cause the ants to attack the hair. In a similar study, Lefebvre showed that parasitic infection causes behavioral changes in P. dentateand results in specific gene mutations in some ants of the P. pallidula sp. that may cause the ants to attack the hair. In addition, studies on the behavior of P. pallidula ants have shown that their behavioral changes following environmental changes are based on gene expression switch [bib_ref] Molecular basis for changes in behavioral state in ant social behaviors, Lucas [/bib_ref] [bib_ref] The relation between caste ratios and division of labor in the ant..., Wilson [/bib_ref] [bib_ref] Scavenging byPheidole pallidulaa key for understanding decision-making systems in ants, Detrain [/bib_ref] ; however, further studies are needed to identify the exact cause or causes of this behavior in ants.
In this type of hair loss, the patient complains of sudden hair loss with no history of other diseases and is concerned about having their hair cut short on a certain area of their scalp, usually forming an irregular patch with no scaling or crusting. The diagnosis of this type of alopecia is mainly performed by ruling out the other conditions. [bib_ref] Ant-induced alopecia: report of 2 cases and review of the literature, Mortazavi [/bib_ref] Our patient had no erythema, scaling, and crusting related to tinea capitis on her scalp lesions. There were also no signs of alopecia areata and trichotillomania in her; therefore, they were ruled out from the diagnosis. The patient's mother-in-law had not experienced the same condition even though she had had greater exposure to these ants, making the etiology of this phenomenon more interesting. One possible reason may be the difference in skin greasiness, as our case had greasy scalps while her mother-in-law did not. Studies have shown that this species is lipophilic and more inclined to attack greasy scalps. 5
# | conclusion
Ant-induced alopecia is a rare condition that needs to be differentiated from other causes of hair loss; however, further studies are needed to elucidate the exact mechanism by which ants attack the hair. |
Case report and systematic literature review of a novel etiology of sinistral portal hypertension presenting with UGI bleeding
Introduction: A novel case is reported of upper gastrointestinal (UGI) bleeding from sinistral portal hypertension, caused by a left gastric artery (LGA) pseudoaneurysm (PA) compressing the splenic vein (SV) that was successfully treated with PA embolization.Case report: A 41-year-old man with previous medical history of recurrent, alcoholic pancreatitis presented with several episodes of hematemesis and abdominal pain for 48 hours. Physical examination revealed a soft abdomen, with no abdominal bruit, no pulsatile abdominal mass, and no stigmata of chronic liver disease. The hemoglobin declined acutely from 12.3 to 9.3 g/dL. Biochemical parameters of liver function and routine coagulation profile were entirely within normal limits. Abdominal CT revealed a 5cm-wide peripancreatic mass compressing the stomach and constricting the SV. Esophagogastroduodenoscopy showed blood oozing from portal hypertensive gastropathy, small nonbleeding gastric cardial and fundal varices, gastric compression from the extrinsic mass, and no esophageal varices. MRCP and angiography showed that the mass was vascular, arose from the LGA, compressed the mid SV without SV thrombosis, and caused sinistral portal hypertension. At angiography, the PA was angioembolized and occluded. The patient has been asymptomatic with no further bleeding and a stable hemoglobin level during 8 weeks of follow-up.Discussion: Literature review of the 14 reported cases of LGA PA revealed that this report of acute UGI bleeding from sinistral portal hypertension from a LGA PA constricting the SV is novel; one previously reported patient had severe anemia without acute UGI bleeding associated with sinistral portal hypertension from a LGA PA.Conclusion: A patient presented with UGI bleeding from sinistral portal hypertension from a LGA PA compressing the SV that was treated by angiographic obliteration of the PA which relieved the SV compression and arrested the UGI bleeding. Primary therapy for this syndrome should be addressed to obliterate the PA and not the secondarily constricted SV. Abbreviations: CT = computerized tomography, EGD = esophagogastroduodenoscopy, GI = gastrointestinal, LGA = left gastric artery, MRCP = magnetic resonance cholangio-pancreatography, PA = pseudoaneurysm, SRH = stigmata of recent hemorrhage, SV = splenic vein, SVT = splenic vein thrombosis, UGI = upper gastrointestinal.Previously reported:GI bleeding from gastric varices or portal hypertensive gastropathy can occur from sinistral (left-sided) portal hypertension, typically secondary to splenic vein injury. Reported etiologies include splenic vein thrombosis, embolism, or compression by various enlarged, adjacent lesions.What is new here:A novel case is reported of UGI bleeding from sinistral portal hypertension from a left gastric artery pseudoaneurysm compressing the splenic vein. In this case the UGI bleeding at EGD was from portal hypertensive gastropathy rather than gastric varices, as determined by SRH. Angiographic embolization of the left gastric artery pseudoaneurysm relieved the splenic vein compression, reversed the sinistral portal hypertension, and arrested the UGI bleeding.
# Introduction
Although numerous etiologies of gross gastrointestinal (GI) bleeding from sinistral portal hypertension have been reported ), systematic literature review revealed no case of gross GI bleeding from sinistral (left-sided) portal hypertension from splenic vein (SV) compression by a left gastric artery (LGA) pseudoaneurysm (PA). A patient is reported who presented with this novel syndrome from this PA. This syndrome is important to diagnose because therapy for this syndrome should be directed at the PA to eliminate SV compression and reverse the sinistral portal hypertension rather than the secondarily affected SV; this case illustrates this principle by the novel report of angioembolization of this PA to achieve hemostasis.
# Methods
The literature was systematically reviewed using PubMed with the following medical subject headings (MeSH) or keywords: {"left gastric artery"} and {pseudoaneurysm} OR {"sinistral" or "left"} and {"portal hypertension"} and [{"gastrointestinal bleeding"} or {"gastrointestinal hemorrhage"}]. Two authors independently reviewed the literature and decided by consensus which articles to incorporate in this study. Two articles written in French were professionally translated into English. includes only cases of bleeding from PAs of the LGA or its branches and excludes PAs of other arteries arising from the celiac trunk. [bib_ref] Pseudocysts and pseudoaneurysms: surgical strategy, Bender [/bib_ref] [bib_ref] Bleeding pseudocysts and pseudoaneurysms in chronic pancreatitis, Hamel [/bib_ref] Two cases of bleeding from "gastric arteries" in a study of 104 arterial complications of pancreatitis were excluded because the specific bleeding "gastric" artery and case details were not reported. [bib_ref] Arterial complications of pancreatitis: diagnostic and therapeutic aspects in 104 cases, Boudghène [/bib_ref] This case report received exemption/approval by the IRB at William Beaumont Hospital, Royal Oak, on July 21, 2016. year-old man with a history of alcoholism for 10 years and sober for the last 3 years, recurrent alcoholic pancreatitis, and no known liver disease, presented with several episodes of hematemesis and abdominal pain for 2 days. Esophagogastroduodenoscopy (EGD), performed 3 years earlier for abdominal pain, had revealed no esophageal varices, gastric varices, portal hypertensive gastropathy, or other GI lesions. Physical examination revealed a blood pressure = 100/60 mm Hg, pulse = 60 beats/min, no jaundice, no stigmata of chronic liver disease, a soft abdomen with mild epigastric tenderness but no rebound tenderness, no abdominal bruit, and no pulsatile abdominal mass. Rectal examination revealed gross melena. Laboratory tests revealed hemoglobin = 12.5 g/dL, platelets = 301,000/mL, INR (international normalized ratio) = 1.0, blood urea nitrogen = 20 mg/dL, and creatinine = 1.1 mg/dL. Serum aspartate aminotransferase = 21 IU/L, alanine aminotransferase = 16 IU/L, total bilirubin = 0.6 mg/dL, alkaline phosphatase = 64 IU/L, albumin = 4.4 gm/dL, and lipase = 32 U/dL. The hemoglobin declined acutely to 9.3 g/dL. Abdomino-pelvic computerized tomography (CT), with intravenous contrast, revealed a 5-cm wide, irregular, pancreatic/peripancreatic mass, compressing both the lesser curvature of the stomach and the SV, a normal portal vein, and normal liver parenchyma. The SV compression was pathophysiologically significant as indicated by proximal SV dilatation. EGD revealed in the proximal stomach a fine, reticular, pale-white, polygonal, mucosal, network in a snakeskin pattern, and characteristic of portal hypertensive gastropathy that was actively oozing; extensive coffee-ground, blood clots in the stomach; small gastric cardial and fundal varices without stigmata of recent hemorrhage (SRH); and no esophageal varices [fig_ref] Figure 2: Endoscopic retroflexion at esophagogastroduodenoscopy shows a prominent, pale white, reticular, network surrounding... [/fig_ref]. The extrinsic mass produced a large, round bulge extending into the lumen of the proximal gastric body along the lesser curvature [fig_ref] Figure 2: Endoscopic retroflexion at esophagogastroduodenoscopy shows a prominent, pale white, reticular, network surrounding... [/fig_ref]. Magnetic resonance cholangio-pancreatography (MRCP) revealed a 5-cm wide, enhancing, vascular mass likely arising from the LGA and located between the gastric lesser curvature and distal pancreatic body; compressing the stomach; compressing the middle SV; and resulting in large collateral veins draining the SV into the superior mesenteric vein. Abdominal ultrasound with Doppler studies demonstrated large, turbulent arterial flow into this vascular mass, suggesting a large PA [fig_ref] Figure 4: Abdominal ultrasound with Doppler studies reveals a strong arterial waveform in tracing... [/fig_ref]. Visceral arteriogram showed a 5.3 Â 2.2-cm-wide PA supplied by an LGA branch [fig_ref] Figure 5: Arteriogram during arterial phase after selective injection of contrast into left gastric... [/fig_ref] , which was embolized and occluded with microcoils [fig_ref] Figure 5: Arteriogram during arterial phase after selective injection of contrast into left gastric... [/fig_ref]. Eight weeks later, the patient had a stable hemoglobin level with no further GI bleeding. Abdomino-pelvic CT angiography demonstrated the PA had markedly decreased in diameter, contained numerous microcoils, and had no blood flow.
## Case report
# Discussion
Comprehensive literature review revealed 14 cases of LGA PA, including the currently reported case [fig_ref] Table 2: Fourteen reported cases of left gastric artery pseudoaneurysm including the current case [/fig_ref] [bib_ref] An uncommon cause of melena -left gastric artery pseudoaneurysm, Yang [/bib_ref] [bib_ref] Left gastric artery aneurysm, Jani [/bib_ref] [bib_ref] Left gastric pseudoaneurysmal hemorrhage: a rare endoscopic detection, Marilley [/bib_ref] [bib_ref] EUS diagnosis of a left gastric artery pseudoaneurysm and aneurysmogastric fistula seen..., Falodia [/bib_ref] [bib_ref] Hemosuccus pancreaticus: an uncommon cause of gastrointestinal hemorrhage. A case report, Kapoor [/bib_ref] [bib_ref] Experience of surgical management of pseudo-aneurysms of branches of the coeliac axis..., Negi [/bib_ref] [bib_ref] A rare left gastric artery pseudoaneurysm presenting as a pancreatic mass, Polansky [/bib_ref] [bib_ref] Traumatic false aneurysm of the left gastric artery, Allorto [/bib_ref] [bib_ref] 45-year-old man with left-upperquadrant abdominal pain, Johnson [/bib_ref] [bib_ref] Left gastric artery pseudoaneurysm following traumatic pancreatic transection, Mathew [/bib_ref] [bib_ref] Gastrointestinal case of the day. Pseudoaneurysm of the left gastric artery as..., Smith [/bib_ref]. Twelve patients were male and 1 was female (sex not reported in 1 patient). Twelve patients were adults and 1 was an infant, with a mean age of 46.9 ± 20.4 years old (age not reported in 1 patient). PA etiologies included: recurrent/chronic pancreatitis -9, blunt abdominal trauma -2, gastric ulcer penetrating into LGA -1, recent laparoscopic cholecystectomy -1, and alcoholic cirrhosis -1. Nine patients had alcoholic pancreatitis and 1 had alcoholic cirrhosis. Pancreatitis is the most common cause of PAs of this branch or other branches of the celiac axis. [bib_ref] An uncommon cause of melena -left gastric artery pseudoaneurysm, Yang [/bib_ref] [bib_ref] Splenic artery pseudoaneurysm presenting as massive hematemesis: a diagnostic dilemma, Varshney [/bib_ref] [bib_ref] Splenic artery pseudoaneurysm causing brisk upper-GI bleed from gastric ulcer, Haivas [/bib_ref] Pancreatitis causes leakage of pancreatic enzymes that injure the intima and media, the 2 innermost vascular layers, which then merge with pancreatic pseudocysts to create a false cavity communicating with the arterial lumen. [bib_ref] EUS diagnosis of a left gastric artery pseudoaneurysm and aneurysmogastric fistula seen..., Falodia [/bib_ref] [bib_ref] Pseudoaneurysm of the gastroduodenal artery secondary to chronic pancreatitis, Volpi [/bib_ref] Clinical presentation in the 14 patients included gross upper gastrointestinal (UGI) bleeding -9, severe anemia without Hakim et al.96: [bib_ref] Acinar cell carcinoma of the pancreas: a rare cause of left-sided portal..., Sheen-Chen [/bib_ref] Medicine gross GI bleeding -2, intrapancreatic (retroperitoneal) hemorrhage -1, intrahepatic (intraperitoneal) hemorrhage -1, and abdominal pain -2 [fig_ref] Table 2: Fourteen reported cases of left gastric artery pseudoaneurysm including the current case [/fig_ref] ]; 1 patient had 2 presentations). Etiologies of gross UGI bleeding in the 9 patients included: PA penetrating through gastric wall into gastric lumen -5, peptic ulcer eroding into PA in gastric wall -2, hemosucuss pancreaticus from PA eroding into pancreatic duct -1, and gastric oozing from portal hypertensive gastropathy with sinistral portal hypertension -1. PAs commonly present with upper or lower GI bleeding due to the underlying vascular injury. [bib_ref] Left gastric pseudoaneurysmal hemorrhage: a rare endoscopic detection, Marilley [/bib_ref] Abdominal CT findings of enhancement of a pancreatic pseudocyst or of an adjacent vascular structure are highly suspicious for PAs. However, endoscopic ultrasound (EUS) is superior to CT for diagnosing small PAs. [bib_ref] EUS diagnosis of a left gastric artery pseudoaneurysm and aneurysmogastric fistula seen..., Falodia [/bib_ref] [bib_ref] A case of chronic pancreatitis in which endoscopic ultrasonography was effective in..., Fukatsu [/bib_ref] [bib_ref] Gastroduodenal artery pseudoaneurysm associated with hemosuccus pancreaticus and obstructive jaundice, Bohl [/bib_ref] PAs usually appear on transabdominal ultrasonography as anechoic, fre-quently pulsatile lesions, and often contain a cyst within a larger anechoic mass. Color and pulse Doppler sonography further enhance test sensitivity and specificity by demonstrating turbulent blood flow ("to and fro" sign) within the PA. Angiography is the gold standard diagnostic test: it identifies the affected artery, determines local vascular anatomy, and permits interventional therapy. [bib_ref] Gastroduodenal artery pseudoaneurysm associated with hemosuccus pancreaticus and obstructive jaundice, Bohl [/bib_ref] This case represents the first case of LGA PA diagnosed by MRCP. Abdominal CT with intravenous contrast showed a pancreatic/peripancreatic mass, initially suspicious for pancreatic adenocarcinoma, but MRCP demonstrated that the mass was vascular, and likely originated from the LGA.
Only 2 of the 14 patients had sinistral portal hypertension. In sinistral portal hypertension, defined as partial portal hypertension without cirrhosis from venous obstruction proximal to the Literature review of reported etiologies of sinistral portal hypertension presenting with gross GI bleeding * .
## Underlying pathology causing sinistral portal hypertension mechanism
Bleeding manifestation from sinistral portal hypertension Reference no
## Benign pancreatic lesions
Acute pancreatitis Splenic vein thrombosis Isolated gastric varices [bib_ref] Repeated pancreatitis-induced splenic vein thrombosis leads to intractable gastric variceal bleeding: a..., Tang [/bib_ref] Pancreatic pseudocyst Splenic vein thrombosis Isolated gastric varices [bib_ref] Acute portal vein thrombosis due to chronic relapsing pancreatitis: a fistula between..., Kikuchi [/bib_ref] [bib_ref] Atypical gastric bleeding in a 55-yearold man, beyond the scope of treatment?, Bird [/bib_ref] Pancreatic pseudotumor (focal pancreatitis in the tail) Splenic vein compression Gastroesophageal varices [bib_ref] Left-sided portal hypertension from pancreatic pseudotumor, Harnar [/bib_ref] Pancreatic cancers Pancreatic adenocarcinoma Splenic vein thrombosis or compression Isolated gastric varices [bib_ref] A rare case of serous cystadenoma of the pancreas presenting with left-sided..., Iwasaki [/bib_ref] [bib_ref] Pancreatic adenocarcinoma presenting as sinistral portal hypertension: an unusual presentation of pancreatic..., Chang [/bib_ref] Acinar cell pancreatic cancer Splenic vein thrombosis Isolated gastric varices [bib_ref] Functioning acinar cell pancreatic carcinoma: diagnosis on mangafodipir trisodium (Mn-DPDP)-enhanced MRI, Sahani [/bib_ref] Neuroendocrine pancreatic tumor Splenic vein thrombosis Gastric varices [bib_ref] Neuroendocrine tumor of the pancreas in a patient with pernicious anemia, Caggiano [/bib_ref] Islet cell carcinoma Splenic vein infiltration Gastric varices [bib_ref] Nonfunctioning islet cell tumor presenting with ascites and portal hypertension, Dalvi [/bib_ref] [bib_ref] A case of splenic vein occlusion caused by the intravenous tumor thrombus..., Watase [/bib_ref] Splenic vein compression Isolated gastric varices [bib_ref] Islet cell carcinoma of the pancreas presenting as bleeding from isolated gastric..., Metz [/bib_ref] [bib_ref] Left-sided portal hypertension from malignant islet cell tumour of the pancreas: review..., Chellappa [/bib_ref] Acinar cell pancreatic cancer Splenic vein compression and thrombosis Gastric varices [bib_ref] Acinar cell carcinoma of the pancreas: a rare cause of left-sided portal..., Sheen-Chen [/bib_ref] Other pancreatic tumors MCN of pancreas Splenic vein compression Isolated gastric varices [bib_ref] Pancreatic mass leading to left-sided portal hypertension, causing bleeding from isolated gastric..., Thrainsdottir [/bib_ref] Solid pseudopapillary neoplasm of pancreas Splenic vein compression and occlusion Isolated gastric varices [bib_ref] A case of solid pseudopapillary neoplasm of the pancreas presenting with left-sided..., Nakamura [/bib_ref] Serous cystadenoma of pancreas Compression of splenic vein Isolated gastric varices [bib_ref] Left-sided portal hypertension caused by serous cystadenoma of the pancreas: report of..., Ito [/bib_ref] Tumors of spleen or other organs Hodgkin lymphoma Splenic vein infiltration Portal hypertensive gastropathy [bib_ref] Hodgkin's lymphoma of colon: an unusual cause of isolated splenic vein obstruction, Seenu [/bib_ref] Splenic lymphoma Splenic vein occlusion Isolated gastric varices [bib_ref] Isolated gastric variceal bleeding caused by splenic lymphoma-associated splenic vein occlusion, Chen [/bib_ref] Postsurgical changes Iatrogenic, from segmental splenic vein resection 8 years earlier
Splenic vein compression Isolated gastric varices [bib_ref] Isolated gastric varices resulting from iatrogenic splenic vein occlusion: report of a..., Tsuchida [/bib_ref] Liver transplantation Ligation of portal tributaries of porto-systemic shunts, proximal splenic vein thrombosis Variceal bleeding [bib_ref] Extrahepatic portal hypertension following liver transplantation: a rare but challenging problem, Malassagne [/bib_ref] [bib_ref] Recurrent variceal bleeding after liver transplantation -persistent left-sided portal hypertension, Stevenson [/bib_ref] Vascular lesions/disorders Isolated splenic vein thrombosis Splenic vein thrombosis Isolated gastric varices [bib_ref] Isolated gastric varices due to spontaneous splenic vein thrombosis, Goldberg [/bib_ref] Splenic artery pseudoaneurysms/aneurysm (s) Splenic vein compression and thrombosis Isolated gastric varices [bib_ref] Multiple giant splenic artery aneurysms causing sinistral (left-sided) portal hypertension, Beksac [/bib_ref] Left [bib_ref] Splenic vein thrombosis with oesophageal varices: a late complication of umbilical vein..., Vos [/bib_ref] Wandering spleen Splenic vein occlusion, torsion of splenic vein Isolated gastric varices [bib_ref] Acute gastric hemorrhage secondary to wandering spleen, Angerås [/bib_ref] [bib_ref] Two unusual cases of wandering spleen requiring splenectomy, Meliko Glu [/bib_ref] Perirenal abscess Isolated splenic vein thrombosis Isolated gastric varices [bib_ref] Isolated splenic vein thrombosis: an unusual cause and review of the literature, Köklü [/bib_ref] Pregnancy Splenic vein compression/stenosis Isolated gastric varices [bib_ref] Gastric variceal bleeding from segmentary idiopathic splenic vein stenosis with left-sided portal..., Addario [/bib_ref] Splenic hydatid cysts Splenic vein compression Isolated gastric varices [bib_ref] Splenic hydatid cyst as a cause of sinistral portal hypertension and isolated..., Kantarçeken [/bib_ref] Gastrectomy Isolated splenic vein thrombosis Isolated gastric varices [bib_ref] An unusual case of portasystemic encephalopathy caused by splenic vein occlusion following..., Honda [/bib_ref] Retroperitoneal fibrosis Isolated splenic vein thrombosis Gastroesophageal varices [bib_ref] Retroperitoneal fibrosis causing localized portal hypertension, Lavender [/bib_ref] Myeloproliferative disorders Splenic vein thrombosis or hepatoportal sclerosis Isolated gastric varices or gastroesophageal varices [bib_ref] Portal hypertension in the myeloproliferative syndrome and the reticuloses, Shaldon [/bib_ref] Tuberculous lymphadenitis Splenic vein compression Isolated gastric varices [bib_ref] Splenic vein occlusion secondary to tuberculous lymphadenitis at the splenic hilum: report..., Takeuchi [/bib_ref] GI = gastrointestinal, MCN = mucinous cystic neoplasm. * Generally only 1 case cited per etiology. portal vein (typically occurring at the SV), patients commonly bleed from gastric varices or uncommonly from portal hypertensive gastropathy, but do not bleed from esophageal varices because the portal hypertension only affects the stomach. One prior patient had sinistral portal hypertension from LGA PA compression of the SV; but this patient presented with anemia (hemoglobin = 7.1 g/dL) without gross bleeding and had gastric varices identified by EGD which presumably caused the anemia. [bib_ref] A rare left gastric artery pseudoaneurysm presenting as a pancreatic mass, Polansky [/bib_ref] The currently reported patient is the first presenting with gross bleeding from sinistral portal hypertension from SV compression by an LGA PA. This patient had hematemesis from portal hypertensive gastropathy (based on endoscopic SRH), without liver disease, from sinistral portal hypertension. Among the 9 patients with acute UGI bleeding, angioembolization achieved hemostasis in 5 of 7 cases, surgery was successful in 2 of 3 cases, and dual mode intraoperative angiography and surgery was successful in 1 case. In the current case, hemostasis was achieved by angioembolizing the LGA to relieve SV compression and reverse the sinistral portal hypertension. Angioembolization provides definitive therapy in about 78% of all bleeding visceral PAs, and provides partially successful therapy to stabilize a patient before definitive surgical therapy in many of the remaining cases. [bib_ref] Gastroduodenal artery pseudoaneurysm associated with hemosuccus pancreaticus and obstructive jaundice, Bohl [/bib_ref] Rebleeding is the most common complication of angioembolization. [bib_ref] Pseudoaneurysm of the gastroduodenal artery secondary to chronic pancreatitis, Volpi [/bib_ref] Alternative therapies include endovascular stents or vascular surgery which is reserved for patients who are appropriate surgical candidates, and who have either failed angioembolization or have other surgical indications, such as pancreatic pseudocyst, pancreatic abscess, or gastric outlet obstruction. [bib_ref] An uncommon cause of melena -left gastric artery pseudoaneurysm, Yang [/bib_ref] [bib_ref] Gastroduodenal artery pseudoaneurysm associated with hemosuccus pancreaticus and obstructive jaundice, Bohl [/bib_ref] It is hoped that prompt angioembolization may reduce the previously reported mortality of bleeding from visceral PAs of 60%. [bib_ref] EUS diagnosis of a left gastric artery pseudoaneurysm and aneurysmogastric fistula seen..., Falodia [/bib_ref] Splenic vein thrombosis (SVT) also causes GI bleeding associated with pancreatitis from formation of gastric varices due to sinistral portal hypertension. [bib_ref] Repeated pancreatitis-induced splenic vein thrombosis leads to intractable gastric variceal bleeding: a..., Tang [/bib_ref] SVT should be suspected in patients with pancreatic disease, especially pancreatitis; splenomegaly without generalized portal hypertension or cirrhosis; and isolated gastric varices. [bib_ref] Splenic vein thrombosis and gastrointestinal bleeding in chronic pancreatitis, Weber [/bib_ref] Notably, this work illustrates different therapies depending upon the etiology: sinistral portal hypertension caused by LGA PA compressing the SV should be treated by obliterating the PA, whereas sinistral portal hypertension caused by SVT should be treated by splenic artery embolization, SV recanalization, or splenectomy. [bib_ref] Repeated pancreatitis-induced splenic vein thrombosis leads to intractable gastric variceal bleeding: a..., Tang [/bib_ref] The pathophysiology of UGI bleeding for sinistral portal hypertension from LGA is illustrated in .
Study limitations include: this represents only 1 case report, the methodology is retrospective, bleeding from leakage from the PA into the gastric lumen or from the small gastric varices cannot be excluded as possible etiologies of the UGI bleeding, and bleeding from portal hypertensive gastropathy, rather than gastric varices, is based primarily on endoscopic SRH. Bleeding from sinistral portal hypertension is usually from gastric varices, but in this reported case was apparently from portal hypertensive gastropathy, as demonstrated by endoscopic SRH. The observed gastric varices were small and lacked SRH, and the bleeding was mild, more consistent with portal hypertensive gastropathy than gastric varices. [bib_ref] Portal hypertensive gastropathy: a systematic review of the pathophysiology, clinical presentation, natural..., Gjeorgjievski [/bib_ref] A prospective clinical series is unlikely because this syndrome is rare.
In conclusion, novel findings in this case include: gross gastric bleeding from sinistral portal hypertension secondary to an LGA PA compressing the SV, and hemostasis achieved by occluding the PA to relieve SV compression. [bib_ref] An uncommon cause of melena -left gastric artery pseudoaneurysm, Yang [/bib_ref] Abdominal CT: contrast leaking into oval structure between stomach and pancreatic tail. EGD: Large extrinsic mass and ulceration of posterior lesser curvature of stomach.
Bleeding from LGA PA reaching to gastric mucosa with overlying gastric mucosal ulcer.
## Angiographic microcoil embolization and occlusion
No further bleeding during hospitalization [bib_ref] Left gastric artery aneurysm, Jani [/bib_ref] Abdominal CT: 4-cm enhancing mass between gastric fundus and pancreatic tail. EUS: 3.7 Â 2.5 cm "cystic" lesion with large pulsating artery at margin of lesion (suspected ruptured PA [bib_ref] Gastrointestinal case of the day. Pseudoaneurysm of the left gastric artery as..., Smith [/bib_ref] Abdominal CT with IV contrast: central enhancement of lesion.
[fig] Figure 1: (A) Axial view of abdominal CT, with IV contrast, shows a 5.2 cm pancreatic/peripancreatic mass (labeled * ) compressing the stomach (which has a radiopaque penumbra due to orally ingested contrast) along the lesser curve, and shows that this mass causes a kink (arrow) that partially obstructs the splenic vein and causes sinistral portal hypertension. (B) Coronal view of abdominal CT, with IV contrast, shows a 5.22 cm pancreatic/peripancreatic mass extending superiorly and compressing the stomach, with a radiopaque penumbra due to orally ingested contrast along the lesser curve, and extending inferiorly to the pancreas. (C) CTA 8 weeks after angiographic embolization of left gastric artery demonstrates the pseudoaneurysm has markedly decreased in size, contains metallic coils (producing a metal streak artifact), and has no blood flow. Occlusion of the pseudoaneurysm reverses the splenic vein compression and relieves the sinistral portal hypertension responsible for bleeding from portal hypertensive gastropathy. CT = computerized tomography, CTA = computerized tomographic angiography, IV = intravenous. [/fig]
[fig] Figure 2: Endoscopic retroflexion at esophagogastroduodenoscopy shows a prominent, pale white, reticular, network surrounding individual pink polygons, a characteristic finding of portal hypertensive gastropathy, in the proximal gastric body. This mucosa bulges into the gastric lumen due to extrinsic compression by the pseudoaneurysm. This lesion is actively oozing, as evidenced by stigmata of recent hemorrhage: an intensely erythematous, oozing, lesion on the dependent side of the bulge (arrow) within the area of portal gastropathy. The proximal stomach along the greater curvature is dark because of the presence of dark blood clots within the lumen. [/fig]
[fig] Figure 4: Abdominal ultrasound with Doppler studies reveals a strong arterial waveform in tracing on bottom of screen, indicating a vascular, arterial mass. Blue and red Doppler signals within the mass demonstrate turbulent (to-and-fro) blood flow. [/fig]
[fig] Figure 3: (A, B) Abdominal MRCP shows a 5-cm-wide mass compressing the gastric mid-body, along the lesser curve. The mass is radiolucent before IV contrast administration (A), but markedly enhances after contrast administration (B), a temporal pattern consistent with a vascular mass. The stomach has a moderately radiopaque penumbra from ingested oral contrast. IV = intravenous, MRCP = magnetic resonance cholangio-pancreatography. [/fig]
[fig] Figure 5: Arteriogram during arterial phase after selective injection of contrast into left gastric artery shows pseudoaneurysm filled with injected contrast (A, before embolization); and shows no blood flow (no contrast) within pseudoaneurysm after angiographic coil embolization (B, note coils in pseudoaneurysm and left gastric artery). [/fig]
[table] Table 2: Fourteen reported cases of left gastric artery pseudoaneurysm including the current case: clinical presentation, diagnostic evaluation, pathophysiology, therapy, and outcome. [/table]
|
The Role of Prognostic and Predictive Biomarkers for Assessing Cardiovascular Risk in Chronic Kidney Disease Patients
Chronic kidney disease (CKD) is currently defined as the presence of proteinuria and/or an eGFR < 60 mL/min/1:73m 2 on the basis of the renal diagnosis. The global dimension of CKD is relevant, since its prevalence and incidence have doubled in the past three decades worldwide. A major complication that occurs in CKD patients is the development of cardiovascular (CV) disease, being the incidence rate of fatal/nonfatal CV events similar to the rate of ESKD in CKD. Moreover, CKD is a multifactorial disease where multiple mechanisms contribute to the individual prognosis. The correct development of novel biomarkers of CV risk may help clinicians to ameliorate the management of CKD patients. Biomarkers of CV risk in CKD patients are classifiable as prognostic, which help to improve CV risk prediction regardless of treatment, and predictive, which allow the selection of individuals who are likely to respond to a specific treatment. Several prognostic (cystatin C, cardiac troponins, markers of inflammation, and fibrosis) and predictive (genes, metalloproteinases, and complex classifiers) biomarkers have been developed. Despite previous biomarkers providing information on the pathophysiological mechanisms of CV risk in CKD beyond proteinuria and eGFR, only a minority have been adopted in clinical use. This mainly depends on heterogeneous results and lack of validation of biomarkers. The purpose of this review is to present an update on the already assessed biomarkers of CV risk in CKD and examine the strategies for a correct development of biomarkers in clinical practice. Development of both predictive and prognostic biomarkers is an important task for nephrologists. Predictive biomarkers are useful for designing novel clinical trials (enrichment design) and for better understanding of the variability in response to the current available treatments for CV risk. Prognostic biomarkers could help to improve risk stratification and anticipate diagnosis of CV disease, such as heart failure and coronary heart disease.
# Introduction
According to the latest classification, edited by the Kidney Disease: Improving Global Outcomes Work Group (KDIGO) in 2012, chronic kidney disease (CKD) is defined as the presence of a reduced kidney function (i.e., an estimated glomerular filtration rate ðeGFRÞ < 60 mL/min/1:73 m 2 ) and/or albuminuria, a strong marker of kidney damage. The cause of CKD was also included in the KDIGO classification, since different causes are associated with disparate outcomes and need specific treatments [bib_ref] Independent role of underlying kidney disease on renal prognosis of patients with..., De Nicola [/bib_ref]. An important aspect that has drawn attention to this topic, in the past decades, is the global impact of CKD. The 2017 Global Burden of Disease study has shown that the number of deaths attributable to CKD increased by 33.7% over the 2007-2017 period and that this trend was higher than that of mortality due to neoplasms (+25.4%) and cardiovascular diseases (+21.1%) and close to that of diabetes mellitus (+34.7%). These general epidemiologic evidences are even more impressive when considering that from 1990 to 2016 the incidence and prevalence substantially doubled worldwide, rising by 88.76% and 86.95%, respectively [bib_ref] Analysis of the Global Burden of Disease study highlights the global, regional,..., Xie [/bib_ref]. The main reasons that have been considered to explain the increase in CKD burden are population growth and aging together with the decrease in age-standardized mortality and morbidity rates in most regions. Furthermore, the tide of type 2 diabetes in high-income countries has also driven the increasing trend of CKD and was confirmed as a leading cause of CKD and the more severe clinical condition of end-stage kidney disease (ESKD) [bib_ref] Analysis of the Global Burden of Disease study highlights the global, regional,..., Xie [/bib_ref] [bib_ref] Cardiorenal prognosis by residual proteinuria level in diabetic chronic kidney disease: pooled..., Minutolo [/bib_ref]. The result of the global dimension, when translated into clinical practice, is that a growing number of patients are exposed to both severe cardiovascular (CV) and renal risks [bib_ref] Unraveling cardiovascular risk in renal patients: a new take on old tale, Provenzano [/bib_ref] [bib_ref] Epidemiology of cardiovascular risk in chronic kidney disease patients: the real silent..., Provenzano [/bib_ref]. In the attempt to improve the management of CKD patients as well as to optimize the individual treatment, a large number of studies have been carried out in the past decades. Indeed, observational analyses have provided clinicians with important evidence on the predictors of poor prognosis in CKD patients, thus improving their risk stratification [bib_ref] Reclassification of chronic kidney disease patients for end-stage renal disease risk by..., Provenzano [/bib_ref] [bib_ref] On behalf of Study group Peritoneal Dialysis of Italian Society of Nephrology, Borrelli ; V. La [/bib_ref]. In addition, a large number of intervention studies testing the effect of antihypertensive drugs, diuretics, albuminuria-lowering agents, sodium-glucose cotransporter 2 inhibitors (SGLT-2i), and endothelin receptor antagonists on CV risk reduction in CKD patients have been conducted [bib_ref] Cardiovascular outcomes in high-risk hypertensive patients stratified by baseline glomerular filtration rate, Rahman [/bib_ref] [bib_ref] Effects of a fixed combination of perindopril and indapamide in patients with..., Heerspink [/bib_ref] [bib_ref] Canagliflozin and renal outcomes in type 2 diabetes and nephropathy, Perkovic [/bib_ref] [bib_ref] Empagliflozin and progression of kidney disease in type 2 diabetes, Wanner [/bib_ref]. However, despite the relevant protective effects that these drugs exert against CV events, they also showed a large variability in individual response, thus determining that a considerable proportion of patients do not respond to the scheduled treatment and remain at very high risk of developing CV events [bib_ref] Variability in response to albuminuria-lowering drugs: true or random?, Petrykiv [/bib_ref]. To overcome individual response variability and to reduce the residual CV risk in CKD patients, several strategies have been adopted in clinical research. The first consists in designing new clinical trials that allow to understand what patient is likely to respond to a specific treatment (ClinicalTrials.gov identifier: NCT03504566), whereas the second is focusing attention on the identification, validation, and implementation of novel CV risk biomarkers that may improve risk stratification of CKD patients and identify aspects of renal disease that are not detected by albuminuria or eGFR such as inflammation, tubular damage, and fibrosis. In general, the term biomarker refers to a defined characteristic that can be measured accurately and reproducibly and evaluated as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. They have been investigated and also used for several diseases or pathologic conditions, including the renal ones [bib_ref] Protein biomarkers in renal transplantation, Chakraborty [/bib_ref] [bib_ref] The ischemic/nephrotoxic acute kidney injury and the use of renal biomarkers in..., Andreucci [/bib_ref] [bib_ref] Blood biomarkers of kidney transplant rejection, an endless search?, Jacquemont [/bib_ref]. The aim of this review is to summarize the strong association between CKD and CV disease and to examine the role of novel biomarkers of CV risk in CKD, dealing with biomarkers' function, clinical application, and future perspectives.
## Cardiovascular disease in ckd patients
The association between CV abnormalities and CKD is an old concept. Indeed, the first scientist who described the interconnection between heart failure and the degree of renal fibrosis was Richard Bright in 1836, in a fascinating manuscript that is still available in PubMed [bib_ref] Cases and observations, illustrative of renal disease accompanied with the secretion of..., Bright [/bib_ref]. Many studies have since confirmed this association, and explanations have been sought in terms of epidemiology, pathophysiology, and clinical perspective. From observational analyses emerged that either low eGFR or increased proteinuria, which are considered the two main kidney measures, is associated with the onset of CV complications, such as CV mortality, heart failure (HF), coronary heart disease (CAD), and stroke [bib_ref] Cardiorenal prognosis by residual proteinuria level in diabetic chronic kidney disease: pooled..., Minutolo [/bib_ref] [bib_ref] Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a..., Matsushita [/bib_ref] [bib_ref] Lower estimated glomerular filtration rate and higher albuminuria are associated with mortality..., Astor [/bib_ref] [bib_ref] Estimated glomerular filtration rate and albuminuria as predictors of outcomes in patients..., Clase [/bib_ref]. Although results of previous studies are controversial, a recent individual-level meta-analysis of the CKD Prognosis Consortium provided strong evidence by analyzing uniformly more than 600,000 CKD patients [bib_ref] Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a..., Matsushita [/bib_ref]. In that meta-analysis, both eGFR and proteinuria (measured as albumin-to-creatinine ratio) predicted CV endpoints even after accounting for traditional risk factors (i.e., blood pressure, serum cholesterol levels, smoking habit, age, and gender). Interestingly, the contribution of either eGFR or proteinuria to the CV risk prediction was equal, or even greater, than any traditional CV risk factor. Moreover, for eGFR, a cut-off point of 60 mL/min/1.73m 2 has been identified as the level below which the CV risk starts to increase, while there is no specific threshold for proteinuria. This means that an increase in proteinuria, even within the normal range, confers CV risk.
These data suggest that eGFR and proteinuria should be considered before estimating the CV risk in patients with CKD, especially if considering that the already available risk scores, such as the Framingham or the Atherosclerotic Cardiovascular Disease (ASCVD), failed in predicting CV risk in CKD [bib_ref] Unraveling cardiovascular risk in renal patients: a new take on old tale, Provenzano [/bib_ref] [bib_ref] Epidemiology of cardiovascular risk in chronic kidney disease patients: the real silent..., Provenzano [/bib_ref] [bib_ref] Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a..., Matsushita [/bib_ref]. The linkage between CV disease and CKD measures has also been recently extended to the peripheral vascular disease (PVD). It has been demonstrated that even slight increases in proteinuria, as well as moderate reductions in eGFR, were found significant predictors of PVD (i.e., peripheral artery disease and leg amputation) beyond traditional CV risk factors [bib_ref] Measures of chronic kidney disease and risk of incident peripheral artery disease:..., Matsushita [/bib_ref]. Taken together, CV events are prevalent in CKD patients and are also responsible for most of the unfavorable outcomes. In the Kidney Early Evaluation Program (KEEP), which enrolled subjects at high risk of developing CKD, the overall prevalence of CV disease (CVD) was 22.1% and rose to 30-50% in CKD populations of MASTERPLAN (Netherlands), Chronic Renal Impairment in Birmingham, United Kingdom (CRIB), African Americans Study (AASK), and CKD Multicohort [bib_ref] Reclassification of chronic kidney disease patients for end-stage renal disease risk by..., Provenzano [/bib_ref] [bib_ref] Cardiovascular disease in chronic kidney disease: data from the Kidney Early Evaluation..., Mccullough [/bib_ref] [bib_ref] Multi factorial approach and superior treatment efficacy in renal patients with the..., Van Zuilen [/bib_ref] [bib_ref] Epidemiological evaluation of known and suspected cardiovascular risk factors in chronic renal..., Landray [/bib_ref] [bib_ref] Cardiovascular outcomes in the African American study of kidney disease and hypertension..., Norris [/bib_ref]. Once CKD is established, up to 50% of patients are reported to die of cardiovascular causes over time [bib_ref] Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention, Gansevoort [/bib_ref]. Indeed, in the CKD populations of Kaiser Permanente Northwest, a healthcare service of the United States of America (USA), as well as among CKD diabetic and nondiabetic patients in the USA Medicare system, patients have died or developed CV disease 2
BioMed Research International with a higher rate than ESKD and the two-year survival probability in patients with previous CV disease was modified by the presence/absence of CKD [fig_ref] Figure 2: Two-year survival [/fig_ref] [bib_ref] Chronic kidney disease and the risk for cardiovascular disease, renal replacement, and..., Foley [/bib_ref] [bib_ref] Longitudinal follow-up and outcomes among a population with chronic kidney disease in..., Keith [/bib_ref]. In the Italian CKD Multicohort, which included CKD patients under stable nephrology care, the incidence rates of ESKD and CV events before ESKD were similar (5.26 vs. 4.52 per 100/pts/year), thus confirming that the CV risk remains a major complication for these patients [bib_ref] Cardiorenal prognosis by residual proteinuria level in diabetic chronic kidney disease: pooled..., Minutolo [/bib_ref].
Hence, the presence of kidney-specific mechanisms contributes to the raised CV risk beyond traditional risk factors and individual comorbidities. It has been shown that in CKD the expression of endothelial nitric oxide synthase is downregulated [bib_ref] Chronic kidney disease, Schiffrin [/bib_ref]. This mechanism has been hypothesized as the main cause of endothelial dysfunction in CKD patients in association with the increased levels of asymmetric dimethylarginine (ADMA). Indeed, ADMA acts by Figure 1: Adjusted associations between eGFR, proteinuria, and risk for cardiovascular (CV) fatal and nonfatal events (i.e., myocardial infarction, congestive heart failure, stroke, revascularization, peripheral vascular disease, nontraumatic amputation, or CV death). Solid line represents hazard ratio (HR), whereas dashed lines represent the 95% confidence intervals. HR is adjusted for the main predictors of CV events (age, gender, type 2 diabetes, history of cardiovascular disease, body mass index, hemoglobin, smoking habit, systolic blood pressure, serum phosphorus, and use of RAAS inhibitors). Knots were located at the 25 th , 50 th , and 75 th percentiles for both proteinuria and eGFR. Rug plots at the top of the x-axis represent the distribution of observations. Data source: CKD Multicohort, a pooled analysis of 3,957 patients referred to Italian nephrology clinics [bib_ref] Reclassification of chronic kidney disease patients for end-stage renal disease risk by..., Provenzano [/bib_ref].
inhibiting generation of nitric oxide and increasing systemic vascular resistance and blood pressure [bib_ref] Asymmetric dimethylarginine: a cardiovascular risk factor and a uremic toxin coming of..., Kielstein [/bib_ref]. The endothelial stretch and the increase in ADMA lead to an impairment in coronary vascular resistance and left ventricular hypertrophy [bib_ref] Left ventricular hypertrophy, cardiac remodeling and asymmetric dimethylarginine (ADMA) in hemodialysis patients, Zoccali [/bib_ref]. Moreover, although arterial hypertension is present in a large number of CKD patients, it seems that the renal anemia and the increased vascular stiffness mainly contribute to the onset of left ventricular hypertrophy in combination with the endothelial dysfunction [bib_ref] Stiffness of capacitive and conduit arteries, Pannier [/bib_ref]. CKD also causes dyslipidemia. In the presence of impaired kidney function, an excessive oxidation of low-density lipoprotein (LDL) cholesterol has been observed together with a defective highdensity lipoprotein (HDL) function. The lipid profile becomes, thus, atherogenic [bib_ref] Lipid disorders in uremia and dialysis, Bakris [/bib_ref] [bib_ref] Prognostic role of LDL cholesterol in non-dialysis chronic kidney disease: multicenter prospective..., De Nicola [/bib_ref]. Accordingly, an observational analysis of 1,162 subjects who died between 1988 and 2005 in a suburban community adjacent to Fukuoka City, in southern Japan, showed that the entity of coronary artery stenosis was raised from state I-II to stage V of CKD and that the vascular stenosis was attributable to a worsening atherosclerosis in advanced stages of CKD [bib_ref] Association of kidney function with coronary atherosclerosis and calcification in autopsy samples..., Nakano [/bib_ref]. CKD is also associated with the presence of systemic inflammation which is, in turn, a trigger for CV damage. The increased oxidative stress and accumulation of toxins, normally excreted in the case of normal kidney function, favor the onset of an imbalance of inflammatory factors. In CKD patients, levels of IL-6 and matrix metalloproteinases (MMPs) have also been found to have increased [bib_ref] Statins, inflammation and kidney disease, Krane [/bib_ref] [bib_ref] The association of matrix metalloproteinases with chronic kidney disease and peripheral vascular..., Provenzano [/bib_ref]. Interestingly, it has been shown that MMPs play an important role in expanding inflammatory response and in the inflammation and rupture of atherosclerotic plaques [bib_ref] The association of matrix metalloproteinases with chronic kidney disease and peripheral vascular..., Provenzano [/bib_ref]. Another key factor of endothelial dysfunction is proteinuria (or albuminuria). A group of researchers from the Steno Memorial Hospital, in Denmark, described for the first time in 1989 that in diabetic patients with proteinuria the presence of proteinuria was strongly associated with raised levels of von Willebrand factor and transcapillary escape rate of fibrinogen, thus testifying that proteinuria is most likely a marker of systemic vascular damage [bib_ref] Albuminuria reflects widespread vascular damage, Deckert [/bib_ref]. Further evidence has confirmed that proteinuria exerts prominent toxic effects on all parts of the nephrons including the renal tubules, thus feeding a vicious circle that moves from kidney to systemic damage [bib_ref] Albuminuria is an appropriate therapeutic target in patients with CKD: the pro..., Lambersheerspink [/bib_ref] [bib_ref] Ruolo della proteinuria nella ricerca clinica: per ogni vecchia risposta, una nuova..., Provenzano [/bib_ref]. Patients with impaired kidney function present a deficiency in vitamin D, because of the weakened function of the 1α-hydroxylase, a renal enzyme which converts the vitamin D precursor to the active hormone. Many studies suggested that vitamin D deficiency is associated with CV risk since the vitamin D pathway directly works in modifying cardiac function [bib_ref] Vitamin D therapy in chronic kidney disease and end stage renal disease, Melamed [/bib_ref] [bib_ref] Prognosis and determinants of serum PTH changes over time in 1-5 CKD..., Borrelli [/bib_ref]. Other factors have been considered as CV risk factors in the early phase of CKD, such as hyperphosphataemia, parathyroid hormone (PTH), and leptin which worsen atherosclerosis, vascular calcifications, and cardiorenal prognosis [bib_ref] Prognosis and determinants of serum PTH changes over time in 1-5 CKD..., Borrelli [/bib_ref] [bib_ref] Calcification and cardiovascular health, Ketteler [/bib_ref] [bib_ref] Epidemiology of low-proteinuric chronic kidney disease in renal clinics, De Nicola [/bib_ref].
## Rationale to incorporate novel biomarkers of cv risk in ckd patients
Owing to the great burden of CV events in patients with CKD, much effort has been initiated to improve prognosis of these patients. One strategy, which we previously mentioned, is to test novel drugs that would probably represent the best possible treatment in the near future. In this context, SGLT-2i have been shown to reduce the rate of CV events in patients with CKD and diabetes [bib_ref] Canagliflozin and renal outcomes in type 2 diabetes and nephropathy, Perkovic [/bib_ref] [bib_ref] Empagliflozin and progression of kidney disease in type 2 diabetes, Wanner [/bib_ref] and the results were so promising to the point that new trials have been started testing the effect of SGLT-2i in patients with nondiabetic CKD (ClinicalTrials.gov identifier: NCT03036150). One major concern of these trials is that they answer the question whether one treatment is able to reduce on average the CV risk compared with the standard treatment (control group) without considering the individual response to treatments. Indeed, a variability in response has already been described for drugs intervening in the renin-angiotensin-aldosterone system (RAAS), but also with respect to SGLT-2i, thus meaning that a consistent proportion of patients continue to remain at increased risk if the response to treatment is suboptimal. Another strategy that has been considered is to evaluate, develop, and implement novel biomarkers of CV risk. Biomarkers may improve the management of CKD patients in several ways. Although the increase in proteinuria and (1) Novel biomarkers that are able to anticipate the diagnosis of kidney damage (at early stage of the disease) would be extremely useful in clinical practice since they help in adopting timely strategies to prevent the progression of kidney disease and CV risk
(2) Novel biomarkers can reveal aspects of kidney disease that are not directly captured by eGFR or proteinuria, for example, by informing about the degree of fibrosis, renal inflammation, or tubular damage (3) The combination of the novel biomarker measurement and renal biopsy could be useful in the case whether eGFR and proteinuria are noninformative, such as in nonproteinuric CKD [bib_ref] Epidemiology of low-proteinuric chronic kidney disease in renal clinics, De Nicola [/bib_ref] (4) Novel biomarkers must be studied in those fields of research where therapeutic strategies are not yet adequately improved. For example, it has been shown that, among CV diseases, CKD patients are more likely at risk of developing HF than CAD, probably due to left ventricular hypertrophy and the impaired preload that are commonly observed in advanced CKD [bib_ref] Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a..., Matsushita [/bib_ref]. It is thus remarkable that, since proteinuria and eGFR may be suboptimal in predicting HF, the role of novel biomarkers in anticipating the clinical diagnosis in order to plan proper therapeutic strategies would be determinant (5) Novel biomarkers could also reveal more information on pathophysiological mechanisms of kidney and CV damage (6) The assessment of clinical utility of biomarkers in large cohorts with proper follow-up is essential in order to understand whether a specific biomarker can be transportable to clinical practice, since it would help to improve monitoring the disease trend over time (prognostic biomarker) or predicting the individual response to a treatment or intervention (predictive biomarker)
## Principally investigated cv biomarkers in ckd
Biomarkers have been differently classified in previous available studies. As far as we know, at least three classification systems exist [bib_ref] Cardiovascular risk prediction in CKD, Ballew [/bib_ref] [bib_ref] Cardiorenal syndrome: classification, pathophysiology, diagnosis, and treatment strategies: a scientific statement from..., Rangaswami [/bib_ref] [bib_ref] Multimarker panels in diabetic kidney disease: the way to improved clinical trial..., Perco [/bib_ref]. The first considers the anatomic origin or the mechanisms of damage and thus identifies kidney and cardiac markers [bib_ref] Cardiorenal syndrome: classification, pathophysiology, diagnosis, and treatment strategies: a scientific statement from..., Rangaswami [/bib_ref]. A second classification encompasses filtration markers, namely, biomarkers that give a better estimation of GFR as compared to creatinine eGFR and nontraditional biomarkers that were derived from imaging techniques (i.e., coronary artery calcium score) or laboratory measurements [bib_ref] Cardiovascular risk prediction in CKD, Ballew [/bib_ref]. A third classification is based on the clinical "intended use" of the biomarker and distinguishes prog-nostic and predictive biomarkers [bib_ref] Multimarker panels in diabetic kidney disease: the way to improved clinical trial..., Perco [/bib_ref] [bib_ref] Use of archived specimens in evaluation of prognostic and predictive biomarkers, Simon [/bib_ref]. A prognostic biomarker is used to identify the likelihood of the patient to develop a clinical outcome regardless of treatment. Indeed, it can be evaluated in untreated patients or patients who undergo heterogeneous treatments that often happen under the standard of care. Such a measure may improve the physician's ability to identify patients with a poor prognosis. On the other hand, predictive biomarkers are used to determine whether the patient is likely to benefit from a specific treatment. In this context, the clinical benefit is interpretable as either a good response to a drug that can be used if the biomarker is positive or, alternatively, a resistance to the same drug that can save a patient from drug toxicity or pointless side effects. Since we are interested in the clinical utility of the biomarkers, we adopt and follow the latter classification.
## Prognostic biomarkers.
In patients with already established CKD, many biomarkers have been shown to improve prediction of CV events. The use of cystatin C to estimate GFR (eGFR cys ) was able to refine risk stratification of CKD patients as compared to creatinine-based GFR (eGFR crea ). eGFR cys affords estimates of kidney function levels that are slightly different from those estimated by eGFR crea . A meta-analysis of the CKD Prognosis Consortium showed that the reclassification of patients according to eGFR cys versus eGFR crea is accurate in the sense that patients with lower and higher eGFR cys than eGFR crea levels were, respectively, at higher and lower risk for all endpoints, including CV events [bib_ref] Cystatin C versus creatinine in determining risk based on kidney function, Shlipak [/bib_ref]. β 2 -Microglobulin is another filtration marker that was found to improve prediction of CV events to an extent similar to cystatin C [bib_ref] Serum β-trace protein and β2-microglobulin as predictors of ESRD, mortality, and cardiovascular..., Foster [/bib_ref]. Strong pieces of evidence toward the utility of cardiac troponins (high-sensitivity cardiac troponin (hs-cTnT)) and natriuretic peptides (N-terminal pro-B-type natriuretic peptide (NT-proBNP)) have been recently published [bib_ref] Association of serial measures of cardiac troponin T using a sensitive assay..., De Filippi [/bib_ref] [bib_ref] Association of troponin T detected with a highly sensitive assay and cardiac..., De Lemos [/bib_ref]. Blood levels of hs-cTnT and NT-proBNP are routinely used for diagnosing CAD and HF, respectively, and reflect subclinical abnormalities in the heart. Interestingly, in CKD patients, both hs-cTnT and NT-proBNP are more consistently associated with the development of HF than CAD over time. More importantly, this association is true even after accounting for the kidney function level which per se alters the serum concentrations of the two biomarkers [bib_ref] Cardiac and kidney markers for cardiovascular prediction in individuals with chronic kidney..., Matsushita [/bib_ref]. The importance of such evidence is enormous when considering that HF is the most represented CV disease in CKD patients and for whom the two kidney measures of CV risk, proteinuria and eGFR, show a suboptimal prediction. The clinical implication is also relevant as these novel biomarkers could be used in the future to identify CKD patients at increased CV risk who could be prescribed with preventive treatments (e.g., statins and/or aspirin therapy). In the context of HF, two further biomarkers are of particular interest: soluble suppressor of tumorigenicity (sST2) and galectin-3. sST2 is a protein produced by the endothelial cells lining the left ventriculus in response to mechanical strain. It has shown to have an incremental value to NT-proBNP to predict deaths and hospitalizations due to HF, irrespective of kidney function [bib_ref] 2017 ACC/A-HA/HFSA focused update of the 2013 ACCF/AHA guideline for the management..., Yancy [/bib_ref]. Galectin-3 is a member of the β-galactoside-binding lectin family that interacts with laminin, synexin, and other extracellular matrix proteins. In observational analyses which included patients with HF, serum galectin-3 levels were independent predictors of hospitalizations due to HF and CV mortality, regardless of kidney measures (proteinuria and eGFR) [bib_ref] Galectin-3: a novel blood test for the evaluation and management of patients..., Mccullough [/bib_ref] [bib_ref] Prognostic value of changes in galectin-3 levels over time in patients with..., Van Der Velde [/bib_ref]. Markers of inflammation or tissue remodeling have also sparked interest in assessing CV risk in CKD patients. Among these, levels of MMPs have been considered as possible biomarkers. Serum levels of MMP-2, MMP-8, and MMP-9 have been found increased in CKD patients and diabetic patients, being correlated, respectively, with serum phosphate (MMP-2), fibroblast growth factor-23 (FGF-23), and the degree of proteinuria (MMP-8 and MMP-9), two relevant predictors of oxidative stress and CV risk [bib_ref] Fibroblast growth factor 23 and matrix-metalloproteinases in patients with chronic kidney disease:..., Peiskerová [/bib_ref] [bib_ref] Matrix metalloproteinases in subjects with type 1 diabetes, Gharagozlian [/bib_ref] [bib_ref] Urinary matrix metalloproteinase-8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in..., Lauhio [/bib_ref]. Moreover, MMP-2 has been directly correlated with vascular calcification, atherosclerotic plaque rupture, and carotid intima-media thickness (cIMT), thus playing an important role in atherogenesis [bib_ref] Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis..., Pawlak [/bib_ref]. Higher serum levels of MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) are involved in the pathogenesis of left ventricular hypertrophy by cleaving intracellular myosin filaments [bib_ref] Relations of plasma total TIMP-1 levels to cardiovascular risk factors and echocardiographic..., Sundstrom [/bib_ref] [bib_ref] Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors..., Rouet-Benzineb [/bib_ref]. Several MMPs, such as MMP-2, MMP-3, and MMP-9, are also implicated in the pathogenesis of vascular aneurysm and their levels after surgical interventions for lower extremity bypass were an independent predictor of CV death [bib_ref] The association of matrix metalloproteinases with chronic kidney disease and peripheral vascular..., Provenzano [/bib_ref]. All these mechanisms of damage are made even worse by the presence of an inflammatory milieu in patients with CKD and by the raised serum concentration of MMPs due to the reduction of GFR. The assessment of measures of CV disease process has been also evaluated as biomarkers of CV risk in CKD. Among these, the coronary artery calcium (CAC) score has been used. CAC score is computed using either an electron beam or multidetector cardiac computed tomography (CT). Afterward, a semiautomated tool called Agatston score is used to create a risk score based on the degree of plaque densities and their areas in all coronary arteries [bib_ref] Subclinical atherosclerosis measures for cardiovascular prediction in CKD, Matsushita [/bib_ref]. CAC score has shown to be a reliable predictor of atherosclerotic CV disease among the general population and in patients with moderate and advanced CKD beyond traditional risk factors and with a discrimination ability that is greater than of other filtration markers such as cystatin C [bib_ref] Cardiovascular risk prediction in CKD, Ballew [/bib_ref] [bib_ref] Coronary artery calcification and risk of cardiovascular disease and death among patients..., Chen [/bib_ref].
## Predictive biomarkers.
One fascinating and advantageous aim of the biomarkers is to identify individuals who will likely respond to a drug of interest. These biomarkers are commonly defined "predictive" biomarkers. The baseline level of a predictive biomarker could also change over time (dynamic predictive biomarker) as a treatment-induced effect, so it can be used for monitoring the course of the disease and its treatment efficacy [bib_ref] Multimarker panels in diabetic kidney disease: the way to improved clinical trial..., Perco [/bib_ref]. Predictive biomarkers can be genes, proteins, metabolites, or others. The most used predictive biomarker in nephrology is the presence of proteinuria. Several clinical trials have shown in the past three decades that the drug-induced reduction in proteinuria is associated with a protection from CV risk over time both in diabetic and nondiabetic CKD patients . Treatments tested in these trials were disparate and included antihypertensive, diuretics, and oral hypoglycemic agents. However, the common pieces of evidence derived from these studies were that (1) the magnitude of treatment effect, i.e., risk reduction for fatal and nonfatal CV events, was greater in patients with proteinuric CKD phenotype as compared to those without CKD and (2) the extent of CV risk reduction after interventions was strictly correlated with the reduction in proteinuria [bib_ref] Chronic kidney disease, cardiovascular events, and the effects of perindoprilbased blood pressure..., Perkovic [/bib_ref]. Two post hoc analyses of clinical trials enrolling CKD patients, the Reduction in Endpoints in Noninsulin-dependent diabetes mellitus with the Angiotensin II Antagonist Losartan (RENAAL) and the Irbesartan Diabetic Nephropathy Trial (IDNT) study, have shown that the greatest protective effect was found in patients with the larger reduction of proteinuria after 6 months from randomization visit that corresponds to the start-of-treatment visit [bib_ref] Individual long-term albuminuria exposure during angiotensin receptor blocker therapy is the optimal..., Kröpelin [/bib_ref]. There is now a general agreement, confirmed by KDIGO guidelines, that proteinuria should be measured in CKD patients to monitor the progression of the disease. However, although further studies are needed to establish how often it should be measured and what the correct threshold that confers a strong protection against CV disease, it is reasonably accepted based on previous trials that a 30% reduction of proteinuria after 6 months is a sufficient target [bib_ref] Albuminuria is an appropriate therapeutic target in patients with CKD: the pro..., Lambersheerspink [/bib_ref] [bib_ref] Ruolo della proteinuria nella ricerca clinica: per ogni vecchia risposta, una nuova..., Provenzano [/bib_ref]. Presently, in clinical research in nephrology, additional predictive biomarkers that are able to predict the response to nephroprotective treatments beyond proteinuria would allow to better control the CV risk and refine the treatment decision toward "the right drugs for the right patient" perspective. There is interesting evidence that MMPs could play a predictive, other than prognostic, role in CKD patients [bib_ref] The association of matrix metalloproteinases with chronic kidney disease and peripheral vascular..., Provenzano [/bib_ref]. In fact, a reduction in serum concentration of MMPs in response to the antibiotic doxycycline and the nonselective inhibitors of MMPs Batimastat and Marimastat has been associated with a reduction of detrimental vascular tissue remodeling and to a significant reduction of proteinuria in patients with CKD [bib_ref] The association of matrix metalloproteinases with chronic kidney disease and peripheral vascular..., Provenzano [/bib_ref] [bib_ref] Matrix metalloproteinases in vascular disease -a potential therapeutic target?, Lim [/bib_ref]. Even more importantly, the novel SGLT-2i medications, which have been widely demonstrated to reduce the cardiovascular risk in CKD patients in several clinical trials, may exert part of their CV and renal risk reduction effect through a mechanism that is independent from the level of proteinuria and is possibly based on the activation of an endogenous inhibitor of MMPs, the reversion-inducing cysteine-rich protein with kazal motifs (RECK) [bib_ref] Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK..., Das [/bib_ref]. This is important for improving clinical trial design in CKD, since novel drugs may be also tested in nonproteinuric subjects, which represent a nonnegligible part of the CKD cohort [bib_ref] Epidemiology of low-proteinuric chronic kidney disease in renal clinics, De Nicola [/bib_ref]. A growing body of evidence is emerging around the role of renal resistive index (RI) as a dynamic biomarker of CV risk. It is well known that impaired RI levels reflect both kidney and systemic vascular damage [bib_ref] Renal resistive index as a novel biomarker for cardiovascular and kidney risk..., Aroor [/bib_ref] [bib_ref] Renal resistive index in chronic kidney disease patients: possible determinants and risk..., Provenzano [/bib_ref]. Moreover, RI also predicts CV events in high-risk patients regardless of eGFR and proteinuria [bib_ref] Response to renal resistive index and cardiovascular and renal outcomes in essential..., Doi [/bib_ref]. Interestingly, recent studies showed that RI can change over time and in response to treatments. Solini and colleagues have demonstrated that the SGLT-2i dapagliflozin improves endothelial function, vascular damage, and RI in type 2 diabetic patients [bib_ref] Dapagliflozin acutely improves endothelial dysfunction, reduces aortic stiffness and renal resistive index..., Solini [/bib_ref]. A similar effect is determined by the RAAS inhibitors [bib_ref] Changes in renal resistive index and urinary albumin excretion in hypertensive patients..., Leoncini [/bib_ref]. Hence, novel studies should assess whether the dynamic changes in RI and its trajectory over time could influence prognosis. An insertion/deletion polymorphism of the angiotensin-converting enzyme gene was able to predict the response to losartan in type 2 diabetic patients enrolled in [bib_ref] Unraveling cardiovascular risk in renal patients: a new take on old tale, Provenzano [/bib_ref] BioMed Research International the RENAAL study trial [bib_ref] ACE gene polymorphism and losartan treatment in type 2 diabetic patients with..., Parving [/bib_ref]. This evidence was also replicated in nondiabetic patients, thus testifying that intrarenal RAAS activity has a role in CV risk prediction as well as in response to treatment prediction [bib_ref] ACE inhibitors to prevent end-stage renal disease: when to start and why..., Ruggenenti [/bib_ref]. Among complex biomarkers, a panel of 185 serum metabolites, including amino acids, energy/sugar lysophosphatidylcholines, phosphatidylcholines, and sphingomyelins, was analyzed to select a subset of metabolites, which predicts accurately the response to the angiotensin receptor blocker (ARB) therapy in diabetic patients. That prediction ability was also independent from main confounding covariates such as age, gender, eGFR, and proteinuria [bib_ref] Serum metabolites predict response to angiotensin II receptor blockers in patients with..., Pena [/bib_ref]. Similarly, another classifier has been developed from the PREVEND study, using plasma proteomic profiles which have been shown to predict the change in albuminuria stage and to improve the prediction ability of standard risk factors like albuminuria, eGFR, and RAAS inhibitor use [bib_ref] Plasma proteomics classifiers improve risk prediction for renal disease in patients with..., Pena [/bib_ref]. A summary of the principal prognostic and predictive biomarkers of cardiovascular risk in chronic kidney disease patients is provided in [fig_ref] Table 1: Summary of the principal prognostic and predictive biomarkers of cardiovascular risk in... [/fig_ref].
## Strategies for implementing novel biomarkers of cv risk in the ckd setting
CV disease is a major complication of CKD patients. Despite the introduction of novel treatments and a stricter monitoring of patients, the frequency of CV fatal and nonfatal events remains disproportionately high [bib_ref] Impact of BMI on cardiovascular events, renal function, and coronary artery calcification, Russo [/bib_ref] [bib_ref] Parathyroid hormone may be an early predictor of low serum hemoglobin concentration..., Russo [/bib_ref]. Moreover, the risk of CV events among these patients equals or even overcomes the competing risk of CKD progression or ESKD [bib_ref] Cardiorenal prognosis by residual proteinuria level in diabetic chronic kidney disease: pooled..., Minutolo [/bib_ref]. The correct detection, assessment, and implementation of novel biomarkers may certainly support the aim of improving CV risk management in the CKD setting. As we previously discussed, several biomarkers have been demonstrated to play a prognostic or predictive role but just a few biomarkers have made it from the discovery phase to clinical use. With the exception of cystatin C, whose adoption allowed a refinement in the estimation of GFR and CV risk prediction, the risk markers widely used currently in CKD patients are eGFR and proteinuria. Although they convey a great part of information for individual prognosis and treatment decision as well, several concerns have been recently raised. Yoshio Hall and Jonathan Himmelfarb, in a recent Editorial, reported in the Clinical Journal of the American Society of Nephrology, defined the eGFR/proteinuria-based classification a "reductionist" approach, since it does not consider that CKD could manifest through a myriad of clinical and histological phenotypes and that each renal diagnosis deserves a proper comprehensive investigation [bib_ref] The CKD classification system in the precision medicine era, Hall [/bib_ref]. The major limitations to the development of previous biomarkers are represented by the small sample sizes, the heterogeneous results from a specific biomarker assessment, and the lack of result validation [bib_ref] Strategies to improve monitoring disease progression, assessing cardiovascular risk, and defining prognostic..., Pena [/bib_ref]. The framework for the development of a prognostic biomarker includes a series of steps [bib_ref] Key concepts and limitations of statistical methods for evaluating biomarkers of kidney..., Parikh [/bib_ref]. Briefly, to determine if a biomarker improves the clinical prediction on top of already available variables included in risk prediction models, it is recommended to report model calibration, meaning that the event rates predicted by the model correspond to those rates observed in a clinical setting; the significant association of the biomarker with a clinical outcome that should be independent from other main confounders (the effect size of the biomarker with the outcome after multiple adjustments and the p value should be considered); discrimination, a measure according which a model has a good performance if it classifies at high-risk patients who develop the outcome of interest and at low risk those who do not. Although sensitivity and specificity are the proper measures for a precise threshold of the biomarker, a summary measure that depicts sensitivity and specificity for all possible thresholds is the Receiver Operating Characteristic (ROC) curve. It is thus suggested to present the ROC derived from the model together with the Area Under the Curve (AUC) that in these cases is also called c-statistic [bib_ref] Statistical methods for cohort studies of CKD: prediction modeling, Roy [/bib_ref]. If the model with the biomarker c-statistic is significantly higher than the model without the biomarker, it could be clinically useful; reclassification measures, such as net reclassification improvement (NRI) and integrated discrimination index (IDI). Indeed, if the prediction model with the standard covariates (e.g., a model with eGFR and proteinuria in nephrology) accounts for most of prognostic information, it is hard to find a significant improvement of c-statistic, following the statement "it is hard to improve an already good thing." For this reason, measures of reclassification could give useful information on the frequency (%) of patients that are reclassified in the true risk category (lower or higher) with the addition of the new biomarker as compared to the traditional model [bib_ref] Extensions of net reclassification improvement calculations to measure usefulness of new biomarkers, Pencina [/bib_ref]. During all these phases, it is important to keep in mind the intended use of the biomarker (e.g., what kind of outcome it may predict) and the clinical setting (CKD, general population, and high-risk population), since different clinical settings may give disparate results and the variables that influence the effect size of the biomarker. To this aim, it is useful to run subgroup analyses (e.g., by age, gender, race, eGFR, or proteinuria categories). After computing and depicting the measures of performance, a crucial step forward is to validate biomarker performance. Indeed, if biomarker performance is measured on the same cohort from which it was developed, this performance is likely overestimated. Two strategies to assess a correct validation and avoid overfitting are the internal and external validation [bib_ref] Statistical methods for cohort studies of CKD: prediction modeling, Roy [/bib_ref]. The internal validation consists in splitting the cohort in multiple samples so that it is possible to develop and validate the biomarker in different samples of the same cohort. Alternatively, cross-validation and bootstrap-based methods can be used [bib_ref] Internal validation of predictive models, Steyerberg [/bib_ref]. External validation allows one to transport and apply the model to different populations. The biomarker performance may be poor in other populations because the baseline characteristics (frequency of diabetes, CV disease, and degree of kidney impairment) are often different, thus varying the baseline risk of the new population. However, strategies to recalibrate and adapt the performance measures to the new population are applicable [bib_ref] Multinational assessment of accuracy of equations for predicting risk of kidney failure, Tangri [/bib_ref]. Hence, external validation is considered the most effective way to validate a biomarker. Predictive biomarker performance should be assessed following the same scheme used for the prognostic biomarkers. However, predictive biomarkers are also useful in research to select patients for new trials testing drugs for CV protection. A strategy that follows this concept is the adaptive enrichment design [bib_ref] Atrasentan and renal events in patients with type 2 diabetes and chronic..., Heerspink [/bib_ref]. This design consists in enrolling patients who respond to a drug 7 BioMed Research International Although a treatment-induced reduction of eGFR is considered a surrogate endpoint of ESKD, the predictive role of eGFR change for CV risk is still controversial [bib_ref] Subclinical atherosclerosis measures for cardiovascular prediction in CKD, Matsushita [/bib_ref].
Proteinuria Presence of an abnormal quantity of proteins in urine; it is considered the principal marker of kidney damage.
The increase in proteinuria is strongly associated with the onset of fatal and nonfatal CV events [bib_ref] Independent role of underlying kidney disease on renal prognosis of patients with..., De Nicola [/bib_ref] [bib_ref] Cardiorenal prognosis by residual proteinuria level in diabetic chronic kidney disease: pooled..., Minutolo [/bib_ref] [bib_ref] Reclassification of chronic kidney disease patients for end-stage renal disease risk by..., Provenzano [/bib_ref] [bib_ref] Estimated glomerular filtration rate and albuminuria for prediction of cardiovascular outcomes: a..., Matsushita [/bib_ref] [bib_ref] Lower estimated glomerular filtration rate and higher albuminuria are associated with mortality..., Astor [/bib_ref] In clinical trials, patients who develop a significant reduction in proteinuria during the first months after treatment were protected against CV events over time [bib_ref] Effects of a fixed combination of perindopril and indapamide in patients with..., Heerspink [/bib_ref] [bib_ref] Canagliflozin and renal outcomes in type 2 diabetes and nephropathy, Perkovic [/bib_ref] [bib_ref] Empagliflozin and progression of kidney disease in type 2 diabetes, Wanner [/bib_ref] [bib_ref] Variability in response to albuminuria-lowering drugs: true or random?, Petrykiv [/bib_ref] [bib_ref] Chronic kidney disease, cardiovascular events, and the effects of perindoprilbased blood pressure..., Perkovic [/bib_ref] [bib_ref] Individual long-term albuminuria exposure during angiotensin receptor blocker therapy is the optimal..., Kröpelin [/bib_ref] ]. Raised RI levels above have been shown to predict CV events in hypertensive and CKD patients [bib_ref] Renal resistive index in chronic kidney disease patients: possible determinants and risk..., Provenzano [/bib_ref] [bib_ref] Response to renal resistive index and cardiovascular and renal outcomes in essential..., Doi [/bib_ref] Medications as RAAS inhibitors and SGLT-2i reduce RI levels over time and improve vascular damage [bib_ref] Dapagliflozin acutely improves endothelial dysfunction, reduces aortic stiffness and renal resistive index..., Solini [/bib_ref] [bib_ref] Changes in renal resistive index and urinary albumin excretion in hypertensive patients..., Leoncini [/bib_ref].
## Biomed research international
ACE ID/DD Insertion (I)/deletion (D) polymorphism of the angiotensinconverting enzyme (ACE) gene influences the circulating and renal activity of RAAS.
The D allele patients showed a poor CV prognosis in the RENAAL trial [bib_ref] ACE gene polymorphism and losartan treatment in type 2 diabetic patients with..., Parving [/bib_ref] Patients with DD genotype, despite being at high risk of CV events, showed the better response to losartan in the RENAAL study [bib_ref] ACE gene polymorphism and losartan treatment in type 2 diabetic patients with..., Parving [/bib_ref].
Classifiers A classifier is the combination of the informative markers which is able to classify patients according to their risk of developing an outcome or likelihood of response to a treatment.
-A panel of 185 metabolites and a proteomicbased classifier have shown to predict the proteinuric response to RAAS inhibitors [bib_ref] Serum metabolites predict response to angiotensin II receptor blockers in patients with..., Pena [/bib_ref] [bib_ref] Plasma proteomics classifiers improve risk prediction for renal disease in patients with..., Pena [/bib_ref]. 9 BioMed Research International rather than randomize all the population irrespective of a response/no response. Advantages from this strategy are several. Firstly, patients under study would avoid a long period of ineffective therapy if they were nonresponders. Secondly, since all the patients are treated with the study drug before randomization (the run-in period), the treatment effect is estimated in a proper fashion. Finally, such a design is close to clinical practice since clinicians are used to continuing a treatment only if patients respond to that treatment. Predictive biomarkers could be also used to better understand the phenomenon of variability in response to treatment. The crossover studies and even the single-patient trials, the socalled n-of-1, may help to answer this important question. Indeed, in these study designs, patients are randomized to 2 or more sequences of different drugs interspersed with a washout period. With such a design, by measuring a panel of biomarkers before starting each treatment, it is possible to assess what are the characteristics of a patient who responds to the first treatment and does not respond to the second treatment or vice versa. This could also lead in the future to dose a biomarker before selecting the correct treatment as well. One example of such a crossover study is the ROTATE trial (Clin-icalTrials.gov identifier: NCT03504566); the results of which are eagerly expected in 2021.
# Conclusions
Owing to the global dimension of CKD and the high prevalence of CV disease in this setting, great effort is currently ongoing with the aim of reducing CV residual risk. One important strategy that can be pursued to this aim is to develop reliable prognostic and predictive biomarkers. In fact, eGFR and proteinuria, despite their great importance, have shown suboptimal performance in predicting several CV outcomes in CKD patients such CAD and heart failure [bib_ref] Contribution of predictive and prognostic biomarkers to clinical research on chronic kidney..., Provenzano [/bib_ref]. Predicting the response to treatments is another important scope of clinical research since it allows to individualize therapies, to improve the clinical trial design, and to better comprehend the variability in the response to different treatments. The implementation of novel biomarkers of CV risk from the discovery to clinical practice should follow a rigorous methodology so that it would be possible to improve the management of patients by clinicians.
## Data availability
The underlying data supporting the results of our study can be asked from the corresponding author.
[fig] Figure 2: Two-year survival (%) of patients with cardiovascular disease (CVD) by chronic kidney disease (CKD) status. Columns in dark gray depict patients without CKD whereas columns in light gray depict patients with CKD. AF: atrial fibrillation; AMI: acute myocardial infarction; CAD: coronary artery disease; CVA/TIA: cerebrovascular accident/transient ischemic attack; HF: heart failure; PAD: peripheral arterial disease; SCA/VA: sudden cardiac arrest and ventricular arrhythmias. Data source: United States Medicare Population [of eGFR define CKD, their presence is often the marker of an already established and possibly irreversible kidney damage. In this context, novel biomarkers would be desirable for several reasons: [/fig]
[table] Table 1: Summary of the principal prognostic and predictive biomarkers of cardiovascular risk in chronic kidney disease patients.Cystatin C Protein produced by all nucleated cells mainly used as marker of kidney functionCystatin C improves the estimation of eGFR and risk prediction of CV events; it also allows to reclassify patients into more accurate CV risk Microglobulin Component of MHC class I molecules and expressed on all nucleated cells in humans Improves risk prediction in CKD patients beyond traditional risk factors[53] hs-cTnT Regulatory protein that is integral to cardiac and skeletal muscle contraction Improves the risk prediction of CV events, particularly heart failure regardless of the level of kidney function[54][55][56] NT-proBNP Prohormone with a 76-amino acid N-terminal inactive protein Improves the risk prediction of CV events, particularly heart failure regardless of the level of kidney function[54][55][56] It has been used as predictive biomarker in the SONAR trial during the run-in phase, in order to exclude patients with sodium retention after treatment with atrasentan[83].sST2 Member of the IL-1 receptor family, which is produced by cardiomyocytes and cardiac fibroblastsIt is delivered in response to mechanical stress conditions and showed incremental prediction ability (over NT-proBNP) for HF-related death and hospitalizations[58] Galectin-3 30 kDa protein that contains a carbohydrate-recognition-binding domain that enables the linkage of β-galactosidesIn patients with already established CV disease, galectin-3 is an independent predictor of hospitalizations and death due to CV causes[59,60] MMPs Six families of zinc-containing endopeptidases that are involved in regulating tissue development and homeostasis Serum MMP-2, MMP-8, MMP-9, and TIMP-1 are associated with atherogenesis, the severity of kidney damage, and the onset of left ventricular hypertrophy and peripheral vascular disease[61][62][63][64][65][66] MMP levels are modified by selective and nonselective drugs. Changes in MMP levels have been associated with a reduction of CV risk[72,73].CAC CAC is a score measured at cardiac TC based on the entity of calcium depositions on artery plaques.Improves risk prediction in CKD patientsbeyond traditional risk factors[48,68] eGFR crea is an estimation of the kidney function level based on serum creatinine, age, gender, and race.A reduction of eGFR is a potent predictor of CV endpoints, regardless of age, gender, and other risk factors[1,2,5,8,22,23] [/table]
|
Is tumour size a contraindication to laparoscopic adrenalectomy? Case report
A b s t r a c tThe authors present a case report of a patient with a large, hormonally silent tumour of the right adrenal gland. Due to the patient's numerous strains, the necessity of two gynaecological operations, and treatment of broken bones, adrenalectomy was contraindicated for 2 years. After that time, the size of the tumour reached 18 cm × 12 cm. The patient was selected for laparoscopic adrenalectomy, which was successful. The size of the tumour and performed abdominal surgery did not constitute substantial obstacles, and the less invasive procedure was additionally justified by computed tomography and magnetic resonance imaging results, which demonstrated a benign lesion.
# Introduction
The size of an adrenal tumour is one of the features which affect the performance of laparoscopic adrenalectomy. It is assumed that the diameter of a tumour should not exceed 8 cm [bib_ref] Adrenalectomy in the era of laparoscopy, Staren [/bib_ref] [bib_ref] Outcomes after laparoscopic adrenalectomy, Gupta [/bib_ref]. However, growing experience has allowed even tumours of 9 cm to be removed successfully by means of laparoscopy, according to some authors [bib_ref] Laparoscopic adrenalectomy in elderly patients, Otto [/bib_ref]. Of decisive importance is the character of the tumour. In the case of benign, hormonally silent tumours, their size does not constitute a contraindication to laparoscopic procedures. This is a case report of laparoscopic adrenalectomy for an 18 cm × 12 cm tumour.
## Case report
A 62-year-old woman, who has stayed in departments of internal medicine many times, with metabolic syndrome (obesity, hypertension, hypercholesterolaemia), advanced osteoporosis (after bone fractures) and with other burdensome factors, had a large tumour (11 cm × 10 cm) of the right adrenal gland diagnosed 2 years before. The tumour was assessed as hormonally silent. Some characteristic features in the computed tomography (CT) scan suggested the diagnosis of a myelolipoma. The patient was qualified for surgery, but in the meantime she suffered two bone fractures of the upper extremity. She also underwent two gynaecological operations, one due to a ruptured ovarian cyst and later hysterectomy. The above factors caused a 2-year delay in the tumour treatment. Computed tomography scan performed next revealed an increase of the tumour size up to 18 cm × 12 cm. The patient was selected for the surgical procedure. Due to obesity and other internal features, open conversion of laparoscopic adrenalectomy was taken into consideration. The procedure was performed by 3 surgeons. The patient was placed in the left lateral position and 4 trocars were inserted along the right costal arch through the transperitoneal route: one, 12 mm, for the optics; two, 10 mm, for dissection tools; and one, 5 mm, for the liver retractor. A few adhesions that developed after gynaecological operations were observed intraoperatively. They were lysed. Then the dissection of the tumour was started. The course of surgery was surprisingly satisfactory. The view of the surgical field was assessed as satisfactory and certainly better than in the open approach. Finally, after clipping and cutting the right adrenal vein, the adrenal gland was completely dissected. Haemostasis was achieved by a harmonic scalpel and electrocoagulation. The specimen was retrieved via a small, 6-7 cm laparotomy site connecting the 2 closest incisions for the middle trocars. The extreme ports were used to push the specimen into the created mini-laparotomy site. Being pulled from the outside and pushed from the peritoneal cavity, the specimen was extracted. The patient was released on the 5 th postoperational day in a good condition. Histological examination confirmed the CT scan results. The tumour was myelolipoma with ordinary glandular tissue flattened over the surface of the tumour.
# Discussion
Simultaneously with the process of learning, as in the case of any method, indications for laparoscopy have been extended to treat different afflictions, including adrenal tumours. Currently, the majority of authors believe that patients with adrenal tumours whose diameter does not exceed 8 cm should be selected for laparoscopy [bib_ref] Adrenalectomy in the era of laparoscopy, Staren [/bib_ref] [bib_ref] Outcomes after laparoscopic adrenalectomy, Gupta [/bib_ref]. There are, however, clinical studies assessing the usefulness of laparoscopy in treatment of large adrenal tumours, with diameter significantly exceeding 8 cm. Parnaby et al. [bib_ref] The role of laparoscopic adrenalectomy for adrenal tumours of 6 cm or..., Parnaby [/bib_ref] compared the results of treatment of adrenal tumours, the size of the mass being below and above 6 cm, and obtained similar results in the compared parameters. In this study, the review of the literature presented confirmed the consistency of our results with the results of other authors. According to the consistent opinion of these authors, in the series of patients with tumours larger than 6 cm, open conversion of laparoscopic adrenalectomy occurred much more often due to massive adhesions or infiltration of adjacent structures. Among 39 patients with large adrenal tumour undergoing laparoscopic adrenalectomy, open conversion took place in 5 cases. In the series of patients with tumours smaller than 6 cm, open conversion was required in one case only. Ramacciato et al. [bib_ref] Is laparoscopic adrenalectomy safe and effective for adrenal masses larger than 7..., Ramacciato [/bib_ref] assessed the results of laparoscopic surgery on adrenal tumours with diameter larger than 7 cm in a series of 18 patients, regardless of the primary pathology. In this group, conversion was necessary in 3 cases (16%), and each time it was due to bleeding from the damaged tumour or surrounding organs. Neoplasms, both primary and secondary, were confirmed in 4 cases, i.e. just above 20% of patients. The percentage is much lower than in the earlier reports assessing the probability of both primary and secondary neoplasms in large adrenal tumours with diameter exceeding 6 cm [bib_ref] The incidentally discovered adrenal mass, Copeland [/bib_ref] [bib_ref] Hormonal evaluation of the patient with an incidentally discovered adrenal mass, Ross [/bib_ref]. This study also concludes that in the case of adrenal neoplasms operated on laparoscopically, the oncological radicality and survival rate are comparable in relation to the open technique. Brix et al. also obtained similar results in adrenocortical carcinoma laparoscopic treatment in comparison to the open procedure. The series consisted of patients with grade 1 and 2 tumours according to WHO which did not exceed 10 cm in diameter [bib_ref] Laparoscopic versus open adrenalectomy for adrenocortical carcinoma: surgical and oncologic outcome in..., Brix [/bib_ref]. Gaujoux et al. made an interesting conclusion in their analyses of 462 laparoscopic adrenalectomies. They stated that being over 70, ASA class 3 or higher, obesity (BMI > 35 kg/m 2 ), abdominal or retroperitoneal surgery, or tu mours > 6 cm should not constitute a contraindication for laparoscopic surgery [bib_ref] Risk factors for conversion and complications after unilateral laparoscopic adrenalectomy, Gaujoux [/bib_ref]. Our experience, based on 180 laparoscopic adrenalectomies performed in our centre, proves that abdominal surgery does not have to constitute a contraindication to intraperitoneal laparoscopic adrenalectomy. It may be slightly prolonged due to the necessity of prior lysis of adhesions. Taking into account the literature analysed and our own experience, it may be assumed that the only absolute contraindications to laparoscopic adrenalectomy are a large (> 10 cm), malignant lesion or one infiltrating adjacent organs, and also regional lymph node or distant metastases. We believe that qualification of large adrenal masses for laparoscopic surgery should be based on thorough imaging diagnostics performed in order to assess their charac ter and possible infiltration of surrounding organs. On the other hand, the laparoscopic procedure itself for large lesions, especially dissection of the tumour surface and other anatomically significant structures, is in our opinion more comfortable, and the view of the surgical field is better than in the open technique.
The only issue is the extraction of the specimen through the mini-laparotomy site, which in times of aesthetic concerns may be a problem for some patients. For those patients, single incision laparo-scopic surgery, which is successfully employed in some centres, may be an appropriate solution.
Patryk Fiszer, Sadegh Toutounchi, Ryszard Pogorzelski, Ewa Krajewska, Bartosz Sutkowski, Piotr Gierej, Maciej Skórski |
Large Electrocaloric Effect in Relaxor Ferroelectric and Antiferroelectric Lanthanum Doped Lead Zirconate Titanate Ceramics
SEM images:Figure S1shows the scanning electron microscopy images of the two samples. Figure S1 SEM micrographs of Pb 0.89 La 0.11 (Zr 0.7 Ti 0.3 ) 0.9725 O 3 (a) and Pb 0.93 La 0.07 (Zr 0.82 Ti 0.18 ) 0.9825 O 3 (b) ceramics XRD Patterns: Figure S2 shows the XRD patterns of two samples, right three panels show the (110), (200) and (222) peaks of two samples.Figure S2 XRD patterns of the PLZT ceramics and extended XRD patterns of (1 1 0), (2 0 0) and(2 2 2) peaks Dielectric Proprties:Figure S3exhibits the permitticities as a function of temperature and Polarization properties: shows the polarization as a function of temperature and external electric field for two samples.
## (b) ceramics
Pyroelectric properties: shows the pyroelectric coefficient (dP/dT) as a function of temperature and external electric field for two samples.
## Relaxor ferroelectric properties:
In general, the relaxation behavior of ferroelectric can be determined by the modified Curie-Weiss law S1
[formula] 1 − 1 = ( − ) ′(S1) [/formula]
where and are the maximum dielectric constant and the corresponding temperature, and T the dielectric constant and corresponding temperature above , ′ the Curie-like constant.
is the critical exponent and associated with the type of ferroelectric. When = 1 and 2, the material is corresponding to an ideal normal ferroelectric and to an ideal relaxor ferroelectric, respectively. The relaxation behavior of the ferroelectric is gradually increasing with when is between 1 and 2. can be worked out by fitting the logarithmic plots of the reciprocal permittivity ( 1 − 1 ) measured at the same frequency as a function of temperature ( −
where b and c are assumed to be temperature-independent phenomenological coefficients. For the parameter a a linear temperature dependence based on the Curie-Weiss law , The Landau-Ginzburg-Devonshire (LGD) phenomenological theory has also been used to explain the phase transition and dielectric properties of the antiferroelectric PZT system . For the antiferroelectric with orthorhombic symmetry, the polarization is along the [110] direction.
[formula] ( ) = ( − 0 ),(S3) [/formula]
The Gibbs free energy of antiferroelectrics orthorhombic (A O ) phase for the PZT system under zero stress conditions can be described as follows S4
(O) = 2 1 3 2 + (2 11 + 12 ) 3 4 + 2( 111 + 112 ) 3 6 ,
where, p i , σ 1 , σ ij , and σ ijk (i, j, k=1, 2, 3) denote the polarization components along the coordinate axis, and antiferroelectric dielectric stiffness at a constant stress, respectively.
It should be noted that the above relations are merely suitable for antiferroelectric single domains . Based on them, the single-domain properties of PLZT can be determined and the intrinsic contributions to the properties understood. Hence, by neglecting extrinsic contributions (e.g. domain wall and defect motions), the theories can be used to further understand the properties of polycrystalline materials .
For the antiferroelectric ceramics, the grains distribute randomly, which leads to disordered orientation of domains. When an electric field is applied on the polycrystalline ferroelectric ceramic, the distortions of at least some of the crystallites, initially randomly distribute, orient along the allowable direction along the poling electric field. Some literatures have reported the polarization of ferroelectric ceramics and crystals with the same composition at the same poling condition . The relationship between upper limits ̅ of the polarization of the ceramic and P of the antiferroelectric/ferroelectric single-domain is as follows : tetragonal ceramic ̅ =0.831 P, rhombohedral ceramic ̅ =0.866 P, and orthorhombic ceramic ̅ =0.912 P. All of the coefficients of the Gibbs free energy function were independent of temperature, except for the antiferroelectric and ferroelectric dielectric stiffness coefficients σ 1 and α 1 , which were given as linear temperature dependences based on the Curie-Weiss law . For the antiferroelectric orthorhombic phase, let σ 1 be β(T-T C ). Further, β, 2σ 11 +σ 12 , and σ 111 +σ 112 in the equation (S6) can be found from the first partial derivative stability conditions:
[formula] 3 = ( 3 ) = 4β ( − ) 3 + 4 (2 11 + 12 ) 3 3 + 12 ( 111 + 112 ) 3 5 ,(S7) [/formula]
where 3 and 3 are the electric field and the polarization components of a single-domain material along the coordinate axis. The electric field strengths, 5, 6 and 7 MV/m and their corresponding polarizations were selected respectively and substituted into Equation (S7) to procure the coefficient β. Then the reversible adiabatic changes in entropy (ΔS) and temperature (ΔT) can be obtained by using the relations as mentioned in Equations (S4) and , and the polarization 3 as well.
The parameters ( ) used for the calculation of electrocaloric effect are listed in the [fig_ref] Table S1: exchanges the heat on a much longer time scale to the surrounding... [/fig_ref].
[formula] ( ) = ℎ + − ,(S8) [/formula]
where ℎ is the surrounding temperature and t the heat transfer time. More details about the test procedure and data analysis can be found in Ref. During this test, an electric field of 3 MV/m was applied to the sample for 15 seconds to obtain temperature equilibrium first, then the electric field was released immediately. Meanwhile, the ECE signal appears as shown in [fig_ref] Figure S6: Representative measured values for Pb 0 [/fig_ref]. The red curves are the fitted curves using equation . is obtained by extrapolating the fitting toward the time of the fall of the step-like pulse. is measured in the temperature range from 303 K to 423 K at successive increments of 10 K in the temperature range of 303 K to 423 K. In the direct measurement of , one concern is the Joule heating in the samples, which will cause the enhancement of temperature when the field is applied. But in this test, the base line temperature T in [fig_ref] Figure S6: Representative measured values for Pb 0 [/fig_ref] is constant except while withdrawing the electric field, which indicates that the observed temperature change is due to ECE.
[fig] XRD: Patterns:Figure S2shows the patterns of two samples, right three panels show the (110), (200) and (222) peaks of two samples. [/fig]
[fig] Figure: S2 XRD patterns of the PLZT ceramics and extended XRD patterns of (Dielectric Proprties: S3exhibits the permitticities as a function of temperature and frequency, and also the log-log plot of inverse of permittivity ( [/fig]
[table] Table S1: exchanges the heat on a much longer time scale to the surrounding bath ( ℎ ). So the relaxation of the temperature of the whole system can be described asS9 [/table]
|
Monitoring the Presence of 13 Active Compounds in Surface Water Collected from Rural Areas in Northwestern Spain
Drug residues are considered environmental contaminants, and their occurrence has recently become a matter of concern. Analytical methods and monitoring systems are therefore required to control the continuous input of these drug residues into the environment. This article presents a suitable HPLC-ESI-MS/MS method for the simultaneous extraction, detection and quantification of residues of 13 drugs (antimicrobials, glucocorticosteroids, anti-inflammatories, anti-hypertensives, anti-cancer drugs and triphenylmethane dyes) in surface water. A monitoring study with 549 water samples was carried out in northwestern Spain to detect the presence of drug residues over two sampling periods during 2010, 2011 and 2012. Samples were collected from rural areas with and without farming activity and from urban areas. The 13 analytes were detected, and 18% of the samples collected showed positive results for the presence of at least one analyte. More collection sites were located in rural areas than in urban areas. However, more positive samples with higher concentrations and a larger number of analytes were detected in samples collected from sites located after the discharge of a WWTP. Results indicated that the OPEN ACCESS Int. J. Environ. Res. Public Health 2014, 11 5252 WWTPs seems to act as a concentration point. Positive samples were also detected at a site located near a drinking water treatment plant.
# Introduction
Around the world, drugs consumption in both human and veterinary medicine has been increasing year after year. The consumption of pharmaceuticals in the EU is substantial, with approximately 3,000 different active substances such as analgesics and anti-inflammatory drugs, contraceptives, antibiotics, beta-blockers, lipid regulators and neuroactive compounds being commonly used in human medicine [bib_ref] Ecotoxicology of human pharmaceuticals, Fent [/bib_ref]. These compounds enter the environment through many routes, including manufacturing, formulation, distribution, use and disposal. The use of pharmaceuticals by individuals is the main route. After ingestion, metabolites or drugs that are not completely metabolised in the body are expelled in faeces or urine and reach drains. Wastewater treatment plants (WWTPs) have no specific technologies to eliminate drugs entirely from the waste stream, so active compounds (drugs and metabolites) enter the aquatic environment straightforward from the effluents are discharged daily into rivers, making these residues pseudo-persistent pollutants. Pharmaceuticals widely used in the treatment of animals such as the antimicrobials, sulfonamides and quinolones can also be employed in the treatment of human infections. These drugs could be released directly to the environment through the extensive livestock-raising operations and can accumulate in manure pits or livestock waste that may be used to fertilise agricultural lands [bib_ref] Veterinary antibiotics in the aquatic and terrestrial environment, Kemper [/bib_ref]. The result of these practices is that any residues of drug administered reach via runoff, rivers, lakes and seas and via filtration, groundwater, aquifers and wells. The direct contamination of aquatic environments by aquaculture can lead to the exposure of aquatic and sediment dwelling organisms to the contaminants. The triphenylmethane dyes, malachite green (MG) and brilliant green (BG), originally used as dyeing agents in the textile and paper industries, are also observed as contaminants of the aquatic environment. The dyes were introduced illegally in 1933 as ecto-parasiticides, fungicides and antiseptics in aquaculture because of their broad fungicidal and antiparasitical activity, and the dyes are effective against Gram-positive microorganisms [bib_ref] Determination of residues of malachite green in aquatic animals, Bergwerff [/bib_ref]. However, MG and BG are toxic to multiple organs in mammals and have negative effects on the immune and reproductive systems as well as genotoxic and carcinogenic properties [bib_ref] The potential for human exposure, direct and indirect, to the suspected carcinogenic..., Oplatowska [/bib_ref] [bib_ref] Toxicological effects of malachite green, Srivastava [/bib_ref] , so their use in aquaculture is forbidden.
The reason for the development of interest in the research is that reports on the occurrence of pharmaceuticals in the aquatic environment show that the pharmaceuticals are ubiquitous. The continued input of these compounds into the environment, even at low concentrations, potentially serves to sustain chronic exposure for aquatic organisms. Toxic effects in non-target organisms have previously been reported by Jung et al., Madureira et al. [bib_ref] Environmental levels of ultraviolet light potentiate the toxicity of sulfonamide antibiotics in..., Jung [/bib_ref] [bib_ref] Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus..., Kim [/bib_ref] [bib_ref] Residual pharmaceutically active compounds (PhACs) in aquatic environment-Status, toxicity and kinetics, a..., Li [/bib_ref] [bib_ref] The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal)-Experimental..., Madureira [/bib_ref]. Sulfonamides, propanolol, trimethoprim and diclofenac, inter alia, have been demonstrated to exert toxicity on plants, soil organisms, freshwater crustaceans, fish and zebrafish.
Pharmaceutical pollutants are being discharged daily and continuously, albeit their detection in the aquatic environment can be complicated because these compounds are typically present at levels of ng·L −1 and pg·L −1. Detection of pharmaceuticals is influenced by the different properties of the multiple classes of drugs. Specific and suitable methods therefore need to be developed to identify and quantify the drugs in the aquatic environment. The analytical methods employed for the analysis of drugs are also varied, and the sample preparation procedure generally involves the use of large sample volumes, multiple extractions, sophisticated sample clean up, and/or derivatisation prior to analysis. Following extraction, depending on the nature of the target compound, pharmaceuticals are identified and quantified using a variety of analytical instruments. Analytical techniques including high performance liquid chromatography (HPLC) and gas chromatography (GC) allow the analysis of multiple analytes in the same extract when coupled to a mass spectrometer (MS), the combination of these techniques is commonly employed in environmental analyses [bib_ref] Fate of b-blocker human pharmaceuticals in surface water: Comparison of measured and..., Alder [/bib_ref] [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref] [bib_ref] Analysis of 70 Environmental Protection Agency priority pharmaceuticals in water by EPA..., Ferrer [/bib_ref] [bib_ref] Antibiotics in the Hong Kong metropolitan area: Ubiquitous distribution and fate in..., Minh [/bib_ref] [bib_ref] Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of..., Tamtam [/bib_ref] [bib_ref] Source, occurrence and fate of antibiotics in the Italian aquatic environment, Zuccato [/bib_ref] [bib_ref] Application of strategic sample composition to the screening of anti-inflammatory drugs in..., Carpinteiro [/bib_ref]. However, detectors other than MS such as ultraviolet (UV), fluorescence (FL), chemiluminescence (CL) and other techniques such as capillary electrophoresis (CE) are also employed [bib_ref] Recent advances in the analysis of antibiotics by CE and CEC, Castro-Puyana [/bib_ref] [bib_ref] Oxidized multi-walled carbon nanotubes for the dispersive solid-phase extraction of quinolone antibiotics..., Herrera-Herrera [/bib_ref] [bib_ref] Application of liquid-liquid-liquid microextraction and high-performance liquid-chromatography for the determination of sulfonamides..., Lin [/bib_ref] [bib_ref] Rapid and sensitive determination of aminoglycoside antibiotics in water samples using a..., Serrano [/bib_ref] [bib_ref] Analysis of sulfonamides in environmental water samples based on magnetic mixed hemimicelles..., Sun [/bib_ref].
In the Mediterranean area of Spain, pharmaceuticals such as antimicrobials and anti-inflammatories and other compounds such as drugs of abuse have been reported several times in the 21st century [bib_ref] Highly sensitive simultaneous determination of sulfonamide antibiotics and one metabolite in environmental..., Díaz-Cruz [/bib_ref] [bib_ref] Determination of 19 sulfonamides in environmental water samples by automated on-line solid-phase..., García-Galán [/bib_ref] [bib_ref] Ultraperformance liquid chromatography-tandem mass spectrometry analysis of stimulatory drugs of abuse in..., Huerta-Fontela [/bib_ref] [bib_ref] Screening of antibiotics in surface and wastewater samples by ultra-high-pressure liquid chromatography..., Ibáñez [/bib_ref] [bib_ref] Analysis and occurrence of pharmaceuticals, estrogens, progestogens and polar pesticides in sewage..., Kuster [/bib_ref] [bib_ref] Simultaneous determination of macrolides, sulfonamides, and other pharmaceuticals in water samples by..., Pedrouzo [/bib_ref]. The presence of pharmaceuticals has also been reported in Sevilla, Madrid and Galicia [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref] [bib_ref] Detection of pharmaceutically active compounds in the rivers and tap water of..., Valcárcel [/bib_ref] [bib_ref] Determination of drugs of abuse in water by solid-phase extraction, derivatisation and..., González-Mariño [/bib_ref] surface waters. However, none of the publications cited, to the author's knowledge, have previously investigated the presence of antimicrobials, anti-hypertensives, anti-cancer drugs, corticosteroids, anti-inflammatories and triphenylmethane dyes in river water samples, simultaneously. The geographical area investigated in this work contains the most important river in the Galician area, running through areas with high population density as well as areas dedicated to agriculture and livestock, thus indicating the importance of this community in the production of food from animal and vegetable origins. Because there are trout farms and paper industries in the sampling area, the possibility of finding residues of MG and BG in the aquatic environment must be considered.
The aim of this work is to present a method using HPLC-MS/MS for the simultaneous identification and quantification of 13 target compounds (seven antimicrobials, one anti-hypertensive, one anticancer drug, one synthetic corticosteroid, one non steroidal anti-inflammatory and two triphenylmethane dyes) in surface water. In particular, diclofenac, sulfamethoxazole, trimethoprim and oxolinic acid were included in the study because they are drugs commonly used in human medicine and their presence in environmental waters has been reported by different authors [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref] [bib_ref] Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of..., Tamtam [/bib_ref] [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref].
The assessment of the level of pollution generated in the production of food from animal origin derived from the use of the selected drugs has been carried out by analyzing water samples collected from rural areas with high and low density of farming activities. Urban areas located close to the discharge of WWTPs, were included in the study for comparison and based on the common presence of some of the drugs in these types of sampling points. Therefore, the method was validated in house and has been applied to the analysis of 549 surface water samples collected in rural and urban areas of the Galician environment during two different sampling periods.
## Experimental section
## Chemicals, reagents and stock solutions
Brilliant green, diclofenac, difloxacin, enrofloxacin, malachite green, marbofloxacin, oxolinic acid, propanolol, sarafloxacin, sulfamethoxazole, tamoxifen, triamcinolone and trimethoprim (all purity > 98%) and the internal standards (ISs) sulfadoxine-d 3 and malachite green-d 5 picrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the therapeutic and chemical properties of the selected drugs are presented in [fig_ref] Table 1: Therapeutic class and chemical properties of the selected drugs [/fig_ref]. Methanol (HPLC-grade, ≥99.9%) was obtained from Scharlau Chemie (Barcelona, Spain), and formic acid (purity > 99% for analysis) was purchased from Acros Organics (Geel, Belgium). Hydrochloric acid solution (1 N HCl) was purchased from Merck (Darmstadt, Germany). Purified water was prepared in house with a Milli-Q water system from Millipore (Bedford, MA, USA), and nitrogen gas (purity > 99.98%) was generated by an in-house nitrogen generator from Peak Scientific Instruments Ltd. (Chicago, IL, USA). The drugs were accurately weighed (±0.0001 g) on an analytical balance from Ohaus ® GA200 (Nänikon, Switzerland) to prepare stock solutions of individual compounds at a concentration of 0.6 mg·mL −1 in methanol. These stock solutions were mixed with 0.1% of formic acid in methanol to obtain a stock solution of 1 µg·mL −1 , and this solution was further diluted with 0.1% of formic acid in methanol to obtain standard mixtures of drugs at 12.5, 25, 50, 75, 100 and 150 ng·mL −1 . The stock solutions of the ISs (sulfadoxine-d 3 and malachite green-d 5 picrate) at 0.6 mg·mL −1 were mixed to obtain a working stock solution of 1 µg·mL −1 with 0.1% formic acid in methanol. All of the standard solutions were stored in the dark at −18 °C for a maximum of six months.
## Equipment
Samples were analysed on an HPLC-MS/MS system consisting of an HPLC model 1200 G1312A from Agilent Technologies (Waldbronn, Germany) with a binary pump, a degasser and an autosampler. The MS was a model API 4000™ from Applied Biosystems MSD Sciex Instruments (Toronto, ON, Canada) with an integrated TurboIonSpray ® for molecule ionisation. The software Analyst 1.4.1, also from Applied Biosystems, MSD Sciex, was employed to acquire the data and control the system.
The chromatographic analyses were performed by injecting 10 µL of the extract into a Synergi 2.5 µm Polar-RP 100A column (50 × 2.0 mm) connected to a Polar-RP security-guard cartridge (4.0 × 2.0 mm), which were both obtained from Phenomenex (Macclesfield, UK). An MS2 Minishaker vortex mixer from IKA ® (Staufen, Germany) and a vacuum station manifold with Strata ® -X solid phase extraction (SPE) cartridges (60 mg, 3 mL), which were both obtained from Phenomenex (Macclesfield, UK), and a TurboVap ® II evaporator from Zyrmark (Hopkinton, MA, USA) were employed for sample preparation and extraction.
The physical and chemical parameters (nitrites, ammonium, conductivity, turbidity and pH) were measured for each collected sample. These analyses utilised the following equipment and kits: Visocolor ® ECO nitrite test (0.02-0.
## Hplc-ms/ms conditions
Separation of analytes was achieved using a gradient mixture of two components, A (0.1% formic acid in water) and B (0.1% formic acid in methanol). The gradient programme employed is shown in [fig_ref] Table 2: Gradient program of the HPLC-MS/MS method developed [/fig_ref] , and the flow rate was 0.2 mL·min −1 during the whole run. Mass spectrometric measurements were performed using positive electrospray ionisation (ESI + ), and drug identification was performed using two multiple reaction monitoring (MRM) transitions and their retention times (t R ). Standard solutions of the individual drugs in a concentration of 1 µg·mL −1 in 0.1% formic acid in methanol were infused directly into the mass spectrometer, using a 1 mL syringe pump (model Gastight ® 1001), which was purchased from Hamilton (Bonaduz, Switzerland), at a flow rate of 5 µL·min −1 . The optimum cone voltage and collision gas energy were selected when they gave the most intense signals. Then, the HPLC with a Synergy-Polar column was connected to the MS.
To monitor a specific transition between a precursor and a product ion, the following MS parameters needed to be set: declustering potential (DP), entrance potential (EP), collision energy (CE) and cell exit potential (CXP). These parameters varied for each compound and changed automatically during the run, as summarised in [fig_ref] Table 3: Retention time [/fig_ref]. The following MS parameters were kept constant during the run: a source temperature of 650 °C, a vacuum gauge of 4.1·10 −5 Torr, an ion spray voltage of 5,000 V and a curtain gas pressure of 12 psi. The first ion source was set at 45 psi, and the second ion source was set at 50 psi. For all monitored transitions, the dwell time was 10 ms. Notes: DP: Declustering potential; EP: Entrance potential; CE: collision energy; CXP: cell exit potential.
## Study site description
The geographical area investigated is located in the extreme northwest (NW) portion of the Iberian Peninsula, just north of Portugal. The Miño River is an international river with a length of approximately 340 km. The source of the Miño River lies in Pedregal of Irimia, which is 50 km north of Lugo in Galicia, Spain (Serra de Meira, Lugo, at 750 m elevation) and in the last 75 km, the river defines the border between Portugal and Spain. The Miño River is the most important drainage basin in NW Spain, extending over 17,757 km 2 with abundant areas dedicated to agriculture and livestock distributed along its path to the Atlantic Ocean. The landscape of Galicia is dominated by the valleys created by the Miño River and its tributaries. Samples were collected from the Miño River and one of its main tributaries, the Asma River. The study area encompassed the upper basin, which includes the metropolitan area of Lugo, with approximately 98,000 residents (INE 2011) and areas dedicated to agriculture and farm production.
## Sampling strategy, method and conservation
Our sampling strategy was designed based on previous work where veterinary drugs were investigated in river water from different areas with and without farming activity using a total of 14 sampling points. Six of the points were located in rural areas and the other eight were chosen at different points along the Miño River, Galicia's largest river, which goes across both rural and urban areas . . Locations of the sampling sites selected for the monitoring study.
The method was developed and validated with river water samples collected from the Miño River, and the monitoring study was conducted with Galician fresh water. A total of 267 river samples of 1-L were collected in 1-L polyethylene plastic vessels during two different sampling periods, the first sampling period over a nine-week period from October to December 2010 and the second sampling period over a seven-month period between November 2011 and May 2012. The sampling sites were distributed along the Miño River between the villages of Rábade and Chantada in the province of Lugo. Of a total of 14 points, eight were located at the Miño River, one in its tributary the Asma River and five in streams and brooks .
All the samples for method validation and monitoring studies were filtered upon arrival at the laboratory and divided into replicate samples of 500 mL and were stored at 4 °C until analysis was carried out, which was always performed the day after sampling. The extracts were stored at −18 °C until their HPLC-MS/MS analysis was performed which took place within no more than seven days after the extraction. To quantify the analytes correctly all the river water samples were spiked with 50 µL of the IS solution and a blank and two control samples (Milli-Q water and Milli-Q water samples spiked at 75 and 150 ng/L, respectively) were filtered, stored and processed in the same way as our natural water samples for identification of contamination risk during sample processing.
## Analytical procedure
Analytical determinations were carried out using a modification of a previously reported method [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] based on solid-phase extraction (SPE) and determination by high performance liquid chromatography coupled to tandem mass spectrometry. Filtered samples were acidified with 1 N HCl solution to a pH of 3 and loaded in to a SPE cartridge (Strata ® -X, Phenomenex, Torrance, CA, USA) activated with 4 mL of methanol and 4 mL of water at pH 3. The analytes were eluted with 8 mL of methanol and evaporated to dryness under a nitrogen stream at 45 °C. The extracts were reconstituted with 200 µL of 0.1% formic acid in methanol and stored at −18 °C until further analysis by HPLC-MS/MS. The extracts were analysed within one week following extraction.
The samples employed for the determination of physical and chemical parameters (nitrites, ammonium, conductivity, turbidity and pH) were stored at 4 °C in the laboratory until analysis, which was carried out within one day of sampling. The physical and chemical parameters were determined following the manufacturers' instructions for the respective kits and equipment.
## Validation procedure
As the method employed is a modification of a previously reported method, validation had to be performed.
The linear response of the instrument to standard solutions containing all the selected drugs at six different concentrations (1, 5, 10, 50, 100 and 200 ng·mL −1 ) was investigated. These standard solutions were employed to obtain the instrument calibration curves (ICCs). The instrument detection limit (IDL) was defined as the concentration that gave a signal-to-noise (S/N) ratio above 3, and the instrument quantification limit (IQL) was defined as the concentration that gave an S/N above 10. The linearity of the whole method, extraction and HPLC-MS/MS detection, was evaluated using matrix-matched samples with river water samples spiked with all the selected drugs at the following concentrations: 1, 5, 10, 20, 50, 75, 100, 150 and 200 ng·L −1 . These samples were employed to build sample calibration curves (SCCs) and to determine the limit of detection (LOD) for the method, which is defined as the lowest concentration that gave an S/N above 3 and a method limit of quantification (LOQ) at an S/N above 10. Once the LOD was established, the method detection limit (MDL) was also calculated following the Environmental Protection Agency (EPA) procedure from CFR 136, Appendix B criteria. The MDL is defined as the minimum concentration of a substance that can be measured and reported with a 99% confidence that the analyte concentration is greater than zero. The MDL is determined from the replicate analysis of a sample in a given matrix containing the analyte.
The analytical method was validated in terms of selectivity, linearity, LOD, LOQ, MDL repeatability, precision and accuracy.
# Statistical analysis
Once the results from the identification and quantification of the selected analytes and the results from the determination of the physicochemical parameters were obtained from the monitoring study, they were analysed using the software PASW Statistics 18 (SPSS Inc., Chicago, IL, USA) to identify statistically significant trends in the drug concentrations. The effects of the weather conditions, sampling site characteristics (rural and urban areas), sampling date and physical and chemical parameters (nitrites, ammonium, conductivity, turbidity and pH) of the water samples collected were tested using one-way ANOVA (p < 0.05).
# Results and discussion
## Optimisation of the hplc-ms/ms and extraction protocols
Initially, the method employed by Nebot et al. [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] was selected for the analysis of the collected samples. However, based on the capability of a Synergy-Polar column in the laboratory, this column was selected to perform the analysis instead of a Luna column. This change of the chromatographic columns could explain the results obtained for the paracetamol peak, which did not have a Gaussian shape. The results of the analysis of this compound consequently had to be discarded. More compounds were also included in the method (brilliant green, cefalexin, difloxacin, enrofloxacin, gentian violet, leucomalachite green, malachite green, marbofloxacin, norfloxacin, oxolinic acid, sarafloxacin and triamcinolone). The mobile phase employed was different: methanol and water acidified with formic acid instead of ammonium acetate was employed because the best peak resolution and peak shape were achieved with a combination of these solvents. Formic acid aided in the prevention of peak tailing and provided sufficient ionisation [bib_ref] Quantification of human and veterinary antibiotics in water and sediment using SPE/LC/MS/MS, Kim [/bib_ref] [bib_ref] Simultaneous determination of sulfonamides, fluoroquinolones, macrolides and trimethoprim in wastewater and river..., Senta [/bib_ref]. The reproducibility of the t R of the selected drugs was evaluated through the RSDs, and their values were in the range between 0.44% and 5.40%.
The SPE protocol described by Nebot et al. [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] was initially employed but for the final method some modification were applied. The cartridges were conditioned with 1 mL less of methanol and water, the sample flow was the same but the elution of the drugs was conducted differently. Methanol alone was employed instead of acetone and methanol because a larger number of analytes were recovered. Instead of 2 L of sample, a volume of 500 mL was employed to spend as little time as possible during extraction and to avoid product degradation due to the laboratory conditions. The acidification of the water samples to pH 3 with 1 N HCl solution was performed to improve the interaction between the elution solvent and the analytes in the SPE [bib_ref] Efficient approach for the reliable quantification and confirmation of antibiotics in water..., Pozo [/bib_ref]. From 19 pharmaceuticals (brilliant green, cefalexin, diclofenac, difloxacin, enrofloxacin, erythromycin, gentian violet, leucomalachite green, malachite green, marbofloxacin, mefenamic acid, norfloxacin, oxolinic acid, propanolol, sarafloxacin, sulfamethoxazole, tamoxifen, triamcinolone and trimethoprim), a total of 13 analytes were extracted satisfactorily. Recoveries of cefalexin, erythromycin, gentian violet, leucomalachite green, mefenamic acid and norfloxacin could not be taken into account because of their low recoveries. Some steps during the extraction of the fortified samples led to degradation and low recoveries of the analytes. In particular, the pH adjustment inhibited the extraction of erythromycin and mefenamic acid. Recoveries of trimethoprim, sulfamethoxazole, propanolol, diclofenac and tamoxifen from fortified river water were higher in this study compared to Nebot et al. [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] most likely due to a different extraction protocol, but the LOD and LOQ were lower certainly due to lower sample volume. Another factor that could have improved recoveries would be the use of an internal standard, contrasting with the procedure of Nebot et al. [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] , who quantified 12 human drugs without the employment of any IS. In this research, the IS sulfadoxine-d 3 was employed to quantify the selected drugs in accordance with Hao et al., Kasprzyk-Hordern et al. [bib_ref] Determination of pharmaceuticals in environmental waters by liquid chromatography/electrospray ionization/tandem mass spectrometry, Hao [/bib_ref] [bib_ref] Determination of selected human pharmaceutical compounds in effluent and surface water samples..., Hilton [/bib_ref] [bib_ref] Multi-residue method for the determination of basic/neutral pharmaceuticals and illicit drugs in..., Kasprzyk-Hordern [/bib_ref] , who employed one or two ISs to quantify 13 drugs from different therapeutic classes in water samples.
A test was conducted to determine the percent of compound lost during the extraction protocol, in which a battery of water samples was fortified at a concentration of 25 ng·L −1 after the evaporation step. The results showed a loss between 40%-50% of diclofenac, MG, oxolinic acid, propanolol, sulfamethoxazole and tamoxifen during the extraction step.
# Method validation
The selectivity of the method was investigated by comparing the two MRM transitions of the selected analytes obtained from a standard solution, non-fortified river samples and fortified river samples. The method selectivity was demonstrated by noting the absence of interference peaks at the t R of the analytes and IS for both selected transitions.
When a standard solution containing a mixture of the analytes was injected at different concentrations, the IDL obtained ranged from 0.2 to 6.4 ng·mL −1 , and the IQL ranged from 0.3 to 6.7 ng·mL −1 . A linear response was also observed for concentrations between 1-200 ng·mL −1 , with a mean R 2 higher than 0.991. The linearity of the whole procedure, samples spiked and extracted as described previously, was observed to have a mean R 2 above 0.979 for a concentration range between 1 and 200 ng·L −1 [fig_ref] Table 5: Regression [/fig_ref].
For fortified samples, the LOD was in the range of 1-3.43 ng·L −1 , and the LOQ was in the range of 3.0-5.15 ng·L −1 . Although there are reports of lower LODs and LOQs, for example, for tamoxifen (0.03 and 0.08 g·L −1 ) [bib_ref] Quantification of human pharmaceuticals in water samples by high performance liquid chromatography-tandem..., Nebot [/bib_ref] , triamcinolone (0.5 and 1.67 ng·L −1 ) [bib_ref] Simultaneous determination of corticosteroids, androgens, and progesterone in river water by liquid..., Tölgyesi [/bib_ref] , and marbofloxacin (LOD = 0.8 ng·L −1 ) [bib_ref] Efficient approach for the reliable quantification and confirmation of antibiotics in water..., Pozo [/bib_ref] , other studies present LOD and LOQ values for enrofloxacin (LOD = 34 ng·L −1 , LOQ = 120 ng·L −1 ) and trimethoprim (LOD = 91 ng·L −1 , LOQ = 310 ng·L −1 ) higher than those reported in this work [bib_ref] Simultaneous analysis of multiple classes of antibiotics by ion trap LC/MS/MS for..., Batt [/bib_ref] where ion trap MS was used by Dinh et al. [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref] for enrofloxacin (LOD = 3. [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref]. Data from Ashton et al. [bib_ref] Investigating the environmental transport of human pharmaceuticals to streams in the United..., Ashton [/bib_ref] showed much higher values than those obtained in this work for sulfamethoxazole, trimethoprim, propanolol, diclofenac and tamoxifen. To the authors' knowledge, no LOD and LOQ values are available for malachite green and brilliant green in surface water to compare with those obtained in this study.
The MDL was calculated for each analyte using river samples fortified with the pharmaceuticals at a concentration of 25 ng·L −1 . MDL values for each analyte are summarised in , and the range was between 2 and 15.1 ng·L −1 . The MDL values for diclofenac and sulfamethoxazol achieved during this research were lower than those published by Cahill et al. and Yang et al. [bib_ref] Determination of pharmaceutical compounds in surface-and ground-water samples by solid-phase extraction and..., Cahill [/bib_ref] [bib_ref] Quantitative determination of trace concentrations of tetracycline and sulfonamide antibiotics in surface..., Yang [/bib_ref] ; Muñóz et al.
reported the same MDL for propanolol (2 ng·L −1 ). However, data obtained for trimethoprim and enrofloxacin were higher than those reported by Senta et al., who achieved MDLs for these compounds between 1-8.5 ng·L −1 [bib_ref] Simultaneous determination of sulfonamides, fluoroquinolones, macrolides and trimethoprim in wastewater and river..., Senta [/bib_ref]. Batt et al. also report MDLs for sulfamethoxazole and propanolol (5.5 and 1.3 ng·L −1 ) [bib_ref] Analysis of ecologically relevant pharmaceuticals in wastewater and surface water using selective..., Batt [/bib_ref] , and Kasprzyk-Hordern et al. report an MDL for diclofenac (0.05 ng·L −1 ) [bib_ref] The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs..., Kasprzyk-Hordern [/bib_ref]. Once again, no data for malachite green and brilliant green in surface water samples were found. . Instrument detection limit (IDL), instrument quantification limit (IQL), limit of detection (LOD), limit of quantification (LOQ) and method detection limit (MDL). Mean recoveries of the selected drugs are summarised in [fig_ref] Table 5: Regression [/fig_ref] , where recoveries higher than 60% can be observed. The highest recoveries were obtained for brilliant green, propanolol, sulfamethoxazole and trimethoprim with values above 80% and RSDs lower than 25%.
Diclofenac, malachite green, sarafloxacin and tamoxifen showed recoveries between 72% and 79%, and the RSDs were not higher than 25%. The lower recoveries (60%-68%) corresponded to the quinolone drugs (difloxacin, enrofloxacin, marbofloxacin and oxolinic acid) and triamcinolone with the RSD between 22% and 27%.
Dinh et al. reported results for recoveries of trimethoprim and sulfamethoxazole (90% and 94%) when the spiked level was 200 ng·L −1 [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref]. Recoveries for enrofloxacin (83%) and oxolinic acid (75%) were higher and for sarafloxacin were similar (79%). Tamtam et al. obtained, in general, lower recoveries with a spiked level of 100 ng·L −1 in river water samples using UPLC-MS/MS for trimethoprim, sulfamethoxazole, sarafloxacin and difloxacin (80%, 68%, 44% and 55%) with high RSDs (11%-29%) [bib_ref] Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of..., Tamtam [/bib_ref]. Recoveries for enrofloxacin were approximately 60% as in the present study. Other publications reported a range of mean recoveries between 38% to 88% for the selected drugs investigated in this study [bib_ref] Simultaneous determination of sulfonamides, fluoroquinolones, macrolides and trimethoprim in wastewater and river..., Senta [/bib_ref] [bib_ref] Investigating the environmental transport of human pharmaceuticals to streams in the United..., Ashton [/bib_ref] [bib_ref] Solid-phase extraction of fluoroquinolones from aqueous samples using a water-compatible stochiometrically imprinted..., Benito-Peña [/bib_ref] , lower than the values obtained in this research.
## Monitoring the presence of drugs in spanish rivers
Of 549 water samples collected during the two sampling periods, 100 samples (18%) were positive for the presence of one or more of the selected drugs. The 13 analytes investigated were detected in at least one sample. The mean percentage of positive samples according to the type of area was 17% in urban areas, 4% in rural areas with farming activity and 2% in rural areas without farming activity. Overall, the percentage of positive samples obtained for each sampling period was similar: 19% and 17% for the first and the second, even if the number of samples collected was different, 235 (first sampling period) and 314 (second sampling period). When results for each sampling area were compared by sampling period, results were similar, particularly for those samples collected from urban areas. Sampling points located in these areas gave a higher percentage of positive samples: 16% during the first sampling period and 18% during the second sampling period. However, we noticed that even if the total percentage of positive samples detected in rural areas did not exceed 6%, during the first sampling period the percent of positive samples was double the percentage of positive samples in the second sampling period. Of all the sampling points located in urban areas, those situated after the discharge point of a WWTP (sites 3 and 13) gave the higher number of positive samples, with a range of detections between 24% and 33% of the positive samples and with a percent of detection at each site of 60% and 80% for sites 3 and 13. These results for the area selected from the Galician environment correlated with the results reported by many other authors who detected the presence of pharmaceuticals in surface water samples collected after the discharge of a WWTP [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref] [bib_ref] Multi-residue method for the determination of basic/neutral pharmaceuticals and illicit drugs in..., Kasprzyk-Hordern [/bib_ref] [bib_ref] Determination of antibiotics in aquatic environment by solid-phase extraction followed by liquid..., Rao [/bib_ref]. Sites classified as rural areas with low farming activity were the sites with the lowest number of detections but with the highest variation between the different sampling periods. These results support the idea that contamination of the samples analysed comes from human activity.
It should be highlighted that the number of analytes detected was different in each sampling period. In the second sampling period, all the analytes selected for the study were detected in at least one sample. In the first sampling period, only four drugs were measured (diclofenac, marbofloxacin, sulfamethoxazole and trimethoprim). These results could certainly be due to a longer sampling time, as the second period was conducted over seven months while the first was conducted over only three months. Consequently, during the second sampling period, 60 more samples were collected.
The fact that the second sampling period took longer may have highlighted the influence of livestock in terms of health, feeding or environmental conditions. Weather conditions were also different during the two sampling periods. Mean solar radiation was 70% higher during the second sampling period, and mean precipitation was 73% higher during the first sampling period.
Analytes detected and their concentrations are summarised in [fig_ref] Table 6: Maximum, minimum and mean concentrations of the selected drugs detected in Spanish... [/fig_ref] and chromatograms of a surface water sample are represented in [fig_ref] Figure 2: MRM 64 [/fig_ref]. If the results are assessed by the type of drugs and their frequency of detection, diclofenac was the drug with more detections in both sampling periods, with 29 (in the first) and 40 (in the second), followed by sulfamethoxazole with nine (in the first) and 33 (in the second) and by trimethoprim with seven (in the first) and 39 (in the second) also the number of detections of sulfamethoxazole and trimethoprim was in the same range for each sampling period, possibly because these drugs are usually administered together in both veterinary and human medicine as they have a synergistic effect. Their high frequency of detection could be due to their frequent use because of their low cost and their broad spectrum of activity to treat bacterial infections [bib_ref] Review on immunoanalytical determination of tetracycline and sulfonamide residues in edible products, Pastor-Navarro [/bib_ref]. Sulfonamides such as sulfamethoxazole and trimethoprim have been detected in surface waters worldwide [bib_ref] Detection of pharmaceuticals and personal care products (PPCPs) in near-shore habitats of..., Ferguson [/bib_ref] [bib_ref] Fast and comprehensive multi-residue analysis of a broad range of human and..., Gros [/bib_ref] [bib_ref] Spatial and seasonal distribution of selected antibiotics in surface waters of the..., Yang [/bib_ref]. These results correlated with data reported by Huang et al. who suggested that sulfonamides are the most common water pollutants among various antibiotics based on information concerning their environmental fate and predicted concentrations [bib_ref] Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis, Huang [/bib_ref]. Huang et al. predicted that, for example, sulfamethoxazole was most likely to be present in municipal effluent and sulfamethazine in agricultural runoff [bib_ref] Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis, Huang [/bib_ref]. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), commonly used to treat inflammatory rheumatic diseases in humans, so its detection at sites located after the discharge of a WWTP is quite expected and, in fact, this drug was detected in 76% of the samples (n = 21). When the data were treated statistically, significant differences (p < 0.05) were found only for sulfamethoxazole and diclofenac according to sampling period. These two analytes were more frequently detected during the second sampling period when their detection was double. When a similar analysis was conducted by sampling month, no significant differences were observed for diclofenac or sulfamethoxazole, but significant differences were observed for trimethoprim. Concentration and frequency of detection of these analytes also showed significant differences (p < 0.05) when they were analysed according to sampling points. Site 13 was the site with the highest number of detections: 29 for diclofenac, 24 for trimethoprim, 19 for sulfamethoxazole and 10 for propanolol.
The concentrations of the drugs measured in this research were between 2.8 to 171.4 ng·L −1 [fig_ref] Table 6: Maximum, minimum and mean concentrations of the selected drugs detected in Spanish... [/fig_ref]. Based on the therapeutic class of the drugs, antimicrobial is the group most frequently detected, with 104 detections and a mean concentration of 53.4 ng·L −1 . The second group most frequently detected was the anti-inflammatory agent diclofenac, with 69 detections and a mean concentration of 13.6 ng·L −1 , followed by the anti-hypertensive propanolol. The highest concentration of drugs measured during this study corresponded to sarafloxacin but this compound was detected only once, in the sampling site located downstream from the discharge of one WWTP. The detection of sarafloxacin could be due to the entrance of some animal residue into the WWTP because this fluoroquinolone is specially employed in poultry to prevent and treat infections. The drug measured at the second highest concentration (164.5 ng·L −1 ) was also a fluoroquinolone, enrofloxacin. This sample was collected at one of the sites located in a rural area with farming activity (point 7). This site is surrounded by greenery where cow droppings were found on the bank of the river, most likely due to the easy access for grazing livestock to drink water. Like sarafloxacin, the frequency of detection of enrofloxacin was low: enrofloxacin was measured in only three samples collected from three different sites , each site classified differently. Trimethoprim was the analyte measured at the third highest concentration (110.4 ng·L −1 ). As mentioned earlier, this drug was frequently detected with a mean concentration of 13.8 ng·L −1 in both sampling periods. The highest observed concentration of diclofenac, the compound most frequently detected, was 45.9 ng·L −1 , and its mean concentration was 16. Another active compound frequently detected during this research was propanolol, which is a beta blocker drug involved mainly in relaxing blood vessels and decreasing heart rate to improve blood flow and lower blood pressure. Propanolol was detected in 14 samples in the range of 4.2-62.6 ng·L −1 , similar to the values reported by Kasprzyk-Hordern et al. ng·L −1 ) in the rivers of the UK [bib_ref] The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs..., Kasprzyk-Hordern [/bib_ref] and higher than the values measured by Alder et al. in Switzerland (7-8 ng·L −1 ) [bib_ref] Fate of b-blocker human pharmaceuticals in surface water: Comparison of measured and..., Alder [/bib_ref]. The highest concentration of propanolol found during this work (37.4 ng·L −1 ) was measured in a sample collected at a site described as a rural area. Most of the detections of this compound occurred at the sites located after the discharge of the WWTPs, most likely due to low removal during treatment at the WWTP as reported by Gros et al. who observed a rate of removal for this compound below 20% [bib_ref] Removal of pharmaceuticals during wastewater treatment and environmental risk assessment using hazard..., Gros [/bib_ref]. These results are in accordance with data published by Martín et al., who detected propanolol in 100% of the samples collected downstream of discharge from four WWTPs [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref]. The range of concentrations reported for this compound was between 30 to 720 ng·L −1 , ten times higher than the concentrations measured in the River Miño. However, the area investigated was more or less than ten times less populated than the Guadalquivir area studied by Martín et al. [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref]. As these drugs are not allowed in food-producing animals, the presence of propanolol in a sample collected from a rural area could be due to its application to animals not dedicated to food production, or the presence of this compound could also be due to a leak from the septic tanks. The antimicrobial marbofloxacin was detected with a range in concentration from 3.6 to 20.1 ng·L −1 , where the highest value was found at a rural site (point 1) located in the Miño River flow. The second greatest concentration was detected at a site also located in the Miño River but on its way through the city of Lugo (point 12). The rest of the analytes selected in this study were detected at a percentage less than 10%. Like sarafloxacin, oxolinic acid was detected only once (39.1 ng·L −1 ) in a sample collected at a site located near the discharge of a WWTP (point 13). Oxolinic acid had also been found in the Seine River at a similar concentration (23 ng·L −1 ) by Dinh et al. [bib_ref] Measurement of trace levels of antibiotics in river water using on-line enrichment..., Dinh [/bib_ref].
The presence of the triphenylmethane dyes, brilliant green and malachite green, in the Galician water is a matter for further study, even if brilliant green was detected only twice and malachite green once in concentrations that do not exceed 9.0 ng·L −1 . As previously mentioned, these compounds are multi-organ toxics to mammals that show serious negative effects [bib_ref] The potential for human exposure, direct and indirect, to the suspected carcinogenic..., Oplatowska [/bib_ref]. The detections of these two compounds were at the same sampling site, with two of them observed on the same day.
The compounds were found at a site located near the discharge of the WWTP located at site number 13, flowing downstream of the city of Lugo. The hypothesis is that these residues could originate from a paper industry located on the upper side of the city and perhaps be swept away by the Miño River to be found in such low concentrations on the lower side or maybe due to the fish farms that are located near the city of Lugo, but this is only a hypothesis because the use of these drugs is forbidden for processing of all categories of edible fish, including fish eggs.
For each collected sample, physicochemical parameters such as nitrites, ammonium, conductivity, turbidity and pH were measured. Statistical analysis (ANOVA and a linearity test) was conducted for the concentration of the analytes and these parameters. However, no significant differences (p < 0.05) were found for ammonium, nitrites, conductivity, turbidity and pH. When statistical analysis was conducted with weather conditions (mean temperature (°C), mean precipitation (mm), mean humidity (%) and mean solar radiation (10 kJ/m 2 )), significant differences (p < 0.05) were observed for diclofenac and mean temperature. Samples that contained a measurable concentration of diclofenac were collected on days when the temperature was equal to or above 15 °C. Diclofenac was more frequently detected in samples collected in urban areas, especially after the discharge of a WWTP, but in the samples collected from rural areas, diclofenac was detected on days when the mean temperature was above 17 °C, most likely because treated animals were put out to pasture. Significant differences (p < 0.05) were also observed for propanolol concentration and precipitation. This analyte was generally detected on days when rainfall was not registered. The lack of detection on rainy days could have being due to a dilution effect, as most samples contained a concentration of propanolol below 10 ng·L −1 . No significant differences (p < 0.05) were found for any of the drugs and mean humidity, but significant differences were observed for diclofenac concentration and mean solar radiation. Similarly to mean temperature, those positive samples for diclofenac that were collected in rural areas were collected on days with low mean solar radiation. As demonstrated by Bartels et al. [bib_ref] Solar radiation influence on the decomposition process of diclofenac in surface waters, Bartels [/bib_ref] , diclofenac is sensitive to solar radiation, and its concentration decreased with solar radiation. Based on these findings, diclofenac could have been liberated into the Galician environment, and its frequency of detection in this study could have been higher but solar radiation helped to decrease its concentration to undetectable levels with the method employed.
This study has demonstrated that even if Galicia is an area of Spain with high rainfall and low population density compared with other areas of Spain, the presence of active compounds such as pharmaceuticals in the aquatic environment is common, and these results were similar to results published for other areas of Spain such as Catalonia, the Spanish Mediterranean area, Valencia, Madrid and Sevilla [bib_ref] Determination of 19 sulfonamides in environmental water samples by automated on-line solid-phase..., García-Galán [/bib_ref] [bib_ref] Screening of antibiotics in surface and wastewater samples by ultra-high-pressure liquid chromatography..., Ibáñez [/bib_ref] [bib_ref] Analysis and occurrence of pharmaceuticals, estrogens, progestogens and polar pesticides in sewage..., Kuster [/bib_ref] [bib_ref] Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): Occurrence..., Martín [/bib_ref] [bib_ref] Detection of pharmaceutically active compounds in the rivers and tap water of..., Valcárcel [/bib_ref] [bib_ref] Determination of drugs in surface water and wastewater samples by liquid chromatography-mass..., Farré [/bib_ref]. Although drug concentrations were below 200 ng·L −1 [fig_ref] Table 6: Maximum, minimum and mean concentrations of the selected drugs detected in Spanish... [/fig_ref] , toxic effects of these compounds in non-target organisms have previously been reported. Potential toxicity affected plants and soil organisms due to sulfamethoxazole [bib_ref] Environmental levels of ultraviolet light potentiate the toxicity of sulfonamide antibiotics in..., Jung [/bib_ref] [bib_ref] Toxic and genotoxic evaluation of six antibiotics on non-target organisms, Isidori [/bib_ref] and freshwater crustaceans and fish as a result of their exposure to propanolol and diclofenac [bib_ref] Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus..., Kim [/bib_ref] [bib_ref] Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part I: Histopathological alterations..., Schwaiger [/bib_ref]. Based on these findings reported by other authors and results obtained for samples collected at sites located next to the collection point of a drinking water treatment plant, more than 5% of the samples collected at this site were positive for the analysed active compounds, causing significant concern.
# Conclusions
The occurrence of drugs in the aquatic environment has recently become a matter of concern. This work presents a suitable HPLC-ESI-MS/MS method for the simultaneous extraction, detection and quantification of residues of 13 active substances (antimicrobials, glucocorticosteroids, non steroidal anti-inflammatories, anti-hypertensives, anti-cancer drugs and triphenylmethane dyes) in fresh water. In-house validation according to EPA guidelines makes this method suitable for government or private laboratories dedicated to investigating the presence of drugs in the aquatic environment. The method was also employed to conduct a monitoring study for the presence of the selected analytes in the Galician region with 549 water samples. All the analytes were detected and in a range of concentrations between 2.8 and 171.4 ng·L −1 . Even if the number of sampling sites located in rural areas (with and without farming activity) was more than double the number of sampling sites located in urban areas, more positive samples were obtained in samples collected from sites located in urban areas, in particular, sites located after the discharge of a WWTP. Rural areas with farming activities also had positive samples but always at a percentage below 15% of the samples collected. The relationship between concentrations and environmental conditions, physicochemical parameters, dates and sampling points were statistically tested, and significant differences were observed only for diclofenac and temperature and solar radiation and between propanolol and precipitation. Based on these results, active compounds are also present in the Galician environment and even if farming activities are conducted in an area, the WWTPs act as a concentration point.
[fig] 3: ng·L −1 , LOQ = 11 ng·L −1 ) and oxolinic acid (LOD = 1.7 ng·L −1 , LOQ = 5.7 ng·L −1 ) and sarafloxacin (LOD = 1.1 ng·L −1 , LOQ = 3.6 ng·L −1 ). Martín et al. and Batt et al. reported LOD and LOQ values for propanolol (LOD = 1 ng·L −1 , LOQ = 2 ng·L −1 and LOD = 0.6 ng·L −1 , LOQ = 2.1 ng·L −1 ) [32,46], lower than the present work. However, for the compounds diclofenac, sulfamethoxazole and trimethoprim (LOD = 15 ng·L −1 , LOQ = 49 ng·L −1 , LOD = 8 ng·L −1 , LOQ = 28 ng·L −1 , LOD = 6 ng·L −1 , LOQ = 20 ng·L −1 ), the values were considerably greater [/fig]
[fig] 6: ng·L −1 in both sampling periods. Jiang et al. published values of concentrations in the same range for sulfamethoxazole (4.86-53.24 ng·L −1 ) and trimethoprim (2.23-62.39 ng·L −1 ) in China [61]. Diclofenac was detected in the range of 2.8-46 ng·L −1 in 69% of the positive samples during this research. A similar frequency of detection was reported by Kasprzyk-Hordern et al. who detected diclofenac in rivers of the UK in the range of 1-261 ng·L −1 at a mean percentage frequency of 74% [51]. The greater concentration of trimethoprim (110.4 ng·L −1 ) was detected in a sample collected from a rural site surrounded by bovine livestock farms, specifically dairy cattle. Dinh et al. also reported concentrations for sulfamethoxazole (3.6-1,435 ng·L −1 ) and trimethoprim (8-254 ng·L −1 ) downstream of WWTPs [14]. However, if data obtained during this research are compared with concentrations of residues found in surface waters in Spain (up to 20 ng·L −1 ), the United Kingdom (up to 42 ng·L −1 ), South Korea (3.2-5.3 ng·L −1 ) and in Serbia (25 ng·L −1 ) [47,62-64], the concentration of the analytes measured in the Galician environment were higher. [/fig]
[fig] Figure 2: MRM 64.3 ng·L −1 ) and diclofenac (31.4 ng·L −1 ) detected in galician surface water. [/fig]
[table] Table 1: Therapeutic class and chemical properties of the selected drugs. [/table]
[table] Table 2: Gradient program of the HPLC-MS/MS method developed. [/table]
[table] Table 5: Regression [/table]
|
Institutional Racism and Health: a Framework for Conceptualization, Measurement, and Analysis
Despite growing interest in the health-related consequences of racially discriminatory institutional policies and practices, public health scholars have yet to reach a consensus on how to measure and analyze exposure to institutional racism. The purpose of this paper is to provide an overview of the conceptualization, measurement, and analysis of institutional racism in the context of quantitative research on minority health and health disparities in the United States. We begin by providing definitions of key concepts (e.g., racialization, racism, racial inequity) and describing linkages between these ideas. Next, we discuss the hypothesized mechanisms that link exposure to institutional racism with health. We then provide a framework to advance empirical research on institutional racism and health, informed by a literature review that summarizes measures and analytic approaches used in previous studies. The framework addresses six considerations: (1) policy identification, (2) population of interest, (3) exposure measurement, (4) outcome measurement, (5) study design, and (6) analytic approach. Research utilizing the proposed framework will help inform structural interventions to promote minority health and reduce racial and ethnic health disparities.Supplementary InformationThe online version contains supplementary material available at https:// doi.
to explore the ways in which institutional policies and practices-both historic and contemporary-contribute to minority health and health disparities [bib_ref] Racial inequalities in health: framing future research, Hicken [/bib_ref] [bib_ref] Structural racism and health inequities in the USA: evidence and interventions, Bailey [/bib_ref] [bib_ref] Structural racism and health inequities: old issues, new directions, Gee [/bib_ref] [bib_ref] Racism and health I: pathways and scientific evidence, Williams [/bib_ref] [bib_ref] Racism and health II, Williams [/bib_ref] [bib_ref] Racism and health: evidence and needed research, Williams [/bib_ref]. This trend in public health scholarship reflects a broader societal shift toward thinking about racism as a structural problem rather than solely as an interpersonal phenomenon. The purpose of this paper is to provide an overview of the conceptualization, measurement, and analysis of institutional racism in the context of quantitative research on minority health and health disparities in the United States. Building on recent papers calling for greater conceptual clarity, increased consideration of historical context, and enhanced methodologic rigor in this area of research [bib_ref] Systemic and structural racism: definitions, examples, health damages, and approaches to dismantling, Braveman [/bib_ref] [bib_ref] Improving the measurement of structural racism to achieve antiracist health policy, Hardeman [/bib_ref] [bib_ref] Measuring structural racism: a guide for epidemiologists and other health researchers, Adkins-Jackson [/bib_ref] [bib_ref] Looking back to leap forward: a framework for operationalizing the structural racism..., Dennis [/bib_ref] , we begin by providing definitions of key concepts, such as racialization, racism, and racial inequity, and explaining linkages between these ideas. Next, we discuss the hypothesized mechanisms that link exposure to institutional racism with health. Finally, we propose a framework to guide methodologic considerations for future studies, informed by a literature review of measures and analytic approaches used in prior research on institutional racism and health.
## Framing the problem
## Definitions and conceptual model
Although studies examining institutional racism as a determinant of health have become increasingly common in recent years for reviews, see 2, [bib_ref] Naming institutionalized racism in the public health literature: a systematic literature review, Hardeman [/bib_ref] [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref] [bib_ref] Public health's approach to systemic racism: a systematic literature review, Castle [/bib_ref] , conceptual ambiguity remains widespread. Thus, we begin by defining relevant concepts. First, racialization refers to the social construction of racial categories, such as White, Black, Latino, [bib_ref] Racial inequalities in health: framing future research, Hicken [/bib_ref] and Asian [bib_ref] Racial formation in theory and practice: the case of Mexicans in the..., Massey [/bib_ref] [bib_ref] The racialization of "illegality, Menjívar [/bib_ref]. While racial categories vary across place and time, they are typically based on phenotypic characteristics, such as skin color, eye shape, and hair texture, that reflect differences in continental ancestry [bib_ref] Race as biology is. fiction, racism as a social problem is real..., Smedley [/bib_ref]. Importantly, phenotypic differences are commonly believed to reflect other important differences between the so-called races, including differences in intelligence and morality [bib_ref] Race as biology is. fiction, racism as a social problem is real..., Smedley [/bib_ref]. Thus, in racialized social systems, the process of racial differentiation is inextricably intertwined with the process of racial stratification (i.e., the hierarchical ranking of people according to race), which results in differential access to power and other resources [bib_ref] Rethinking racism: toward a structural interpretation, Bonillasilva [/bib_ref] [bib_ref] Racism and health: evidence and needed research, Williams [/bib_ref].
We use the general term racism to refer to both the ideology of racial superiority/inferiority (i.e., ideological racism, including internalized racism 2 ), as well as the resulting inequitable treatment of individuals according to race (i.e., actualized racism). We distinguish between two forms of actualized racism: institutional and interpersonal. Institutional racism refers to racially discriminatory policies and practices 3 embedded in social institutions such as the government, the economy, the education system, the healthcare system, religious institutions, the family, and the media. Institutional racism is said to be systemic [bib_ref] The race discrimination system, Reskin [/bib_ref] or structural [bib_ref] Structural racism and health inequities in the USA: evidence and interventions, Bailey [/bib_ref] [bib_ref] Structural racism: the rules and relations of inequity, Gee [/bib_ref] when it operates as a system across multiple interconnected institutions. Interpersonal racism refers to discriminatory treatment by race among individual actors. Finally, racial inequity refers to inequitable, or unjust, outcomes by race, including inequities in education, economic mobility, and health outcomes. Relationships among these concepts are shown in .
## Racism and health: mechanisms
Racism may negatively impact health through both psychosocial and material pathways. For example, the internalization of ideological racism could affect mental health (e.g., depression, anxiety) by decreasing self-esteem among members of stigmatized minority groups [bib_ref] The lived experience of race and its health consequences, Smedley [/bib_ref] [bib_ref] A meta-analysis of the relationship between internalized racial oppression and health-related outcomes, Gale [/bib_ref] , while repeated exposure to acts or threats of interpersonal racism could affect mental and physical health by triggering chronic activation of physiologic stress response systems [bib_ref] Stress-related biosocial mechanisms of discrimination and African American health inequities, Goosby [/bib_ref]. Institutional racism is hypothesized to negatively affect health and well-being through material pathways by shaping access to health-promoting resources [bib_ref] Is racism a fundamental cause of inequalities in health?, Phelan [/bib_ref] [bib_ref] Levels of racism: a theoretic framework and a gardener's tale, Jones [/bib_ref]. For example, the institutional practice of redlining drove residential segregation and depressed home values and homeownership rates in minority neighborhoods. By restricting where Black families could live, redlining contributed to overcrowding, which was then used as a rationale for demolishing homes in Black communities to make way for interstate highways. Along with dividing minority communities, the highways contributed to environmental injustice by increasing air pollution [bib_ref] Historical redlining is associated with present-day air pollution disparities in U.S. cities, Lane [/bib_ref]. Additionally, while not explicitly racist, programs such as the GI Bill, which was designed to provide resources such as higher education and mortgage lending to those returning after World War II, had disparate impacts by race because the program relied upon racist institutional policies, including redlining and racially discriminatory college admissions processes. Though redlining is no longer legal, residential segregation is firmly entrenched in the United States and is a powerful determinant of access to Conceptual model linking racialization to racial inequity via ideological and actualized racism [bib_ref] Racial inequalities in health: framing future research, Hicken [/bib_ref] While the US government classifies Latino as an ethnic group rather than a racial group, we include it in this list to acknowledge that Latinos have been historically racialized as non-White through legislation, immigration enforcement practices, and media framing. [bib_ref] Structural racism and health inequities in the USA: evidence and interventions, Bailey [/bib_ref] Internalized racism is a specific type of ideological racism in which members of stigmatized racial or ethnic groups accept negative stereotypes about their own group. [bib_ref] Structural racism and health inequities: old issues, new directions, Gee [/bib_ref] In the post-Civil Rights era, institutional policies or practices are considered racially discriminatory if they result in inequitable outcomes by race, regardless of intent. a broad range of health-promoting resources, including education, employment, housing, and healthcare [bib_ref] Racial residential segregation: a fundamental cause of racial disparities in health, Williams [/bib_ref]. In addition to its effects on material pathways, restricted access to socially valued resources resulting from institutional racism may further harm health through psychosocial mechanisms, such as perceived injustice or feelings of hopelessness.
While redlining is the most often cited racially discriminatory policy in health research, it is not the only racist policy or set of policies that have affected non-White populations in the USA. Other examples include the seizure of American Indian lands, Reconstruction era Black Codes/vagrancy laws, Jim Crow/American Apartheid policies, failure of Congress to protect Black Americans from lynching, exclusion of specific occupations in the receipt of social security benefits [bib_ref] The echoes of slavery: recognizing the racist origins of the agricultural and..., Perea [/bib_ref] , internment of specific ethnic groups, the War on Drugs, removal of desegregation orders in public education [bib_ref] Brown fades: the end of court-ordered school desegregation and the resegregation of..., Reardon [/bib_ref] , and public charge rules, among many others. It is important to note that institutional policies, including some of those listed above, are often enacted by individual agents of institutions, such as judges, poll workers, and police officers, who may or may not also engage in acts of interpersonal racism when interacting with racial or ethnic minorities. Thus, while we can distinguish between ideological, interpersonal, and institutional racism conceptually, they operate simultaneously and interactively, making it difficult to disentangle the effects of various forms of racism empirically.
## Framework for advancing institutional racism and health research
In this section we discuss key considerations in advancing institutional racism and health research. As shown in , the framework outlines six conceptual and analytic considerations when empirically examining questions on institutional racism and health: (1) institutional racism policy identification, (2) population of interest, (3) institutional racism exposure measurement, (4) health outcome measurement, (5) study design, and (6) analytic approach. This framework aligns with research calling for a historical/contextual and theoretical link between historical racist policies and contemporary health outcomes [bib_ref] Systemic and structural racism: definitions, examples, health damages, and approaches to dismantling, Braveman [/bib_ref] [bib_ref] Improving the measurement of structural racism to achieve antiracist health policy, Hardeman [/bib_ref] [bib_ref] Measuring structural racism: a guide for epidemiologists and other health researchers, Adkins-Jackson [/bib_ref] [bib_ref] Looking back to leap forward: a framework for operationalizing the structural racism..., Dennis [/bib_ref] [bib_ref] Structural racism: the rules and relations of inequity, Gee [/bib_ref] , strong measurement of institutional racism [bib_ref] Improving the measurement of structural racism to achieve antiracist health policy, Hardeman [/bib_ref] [bib_ref] Measuring structural racism: a guide for epidemiologists and other health researchers, Adkins-Jackson [/bib_ref] , and methodological approaches that can strengthen the causal evidence base [bib_ref] Measuring structural racism: a guide for epidemiologists and other health researchers, Adkins-Jackson [/bib_ref]. However, this framework builds on previous work [bib_ref] Measuring structural racism: a guide for epidemiologists and other health researchers, Adkins-Jackson [/bib_ref] [bib_ref] Looking back to leap forward: a framework for operationalizing the structural racism..., Dennis [/bib_ref] by incorporating and focusing on research methods such as linking the network of policies that created a racially disparate impact in resources, the exposure and outcome measurement, matching the timing and geographic region of the policy and outcome, and the design of the research. This framework for the study of institutional racism allows for modern racist policies (e.g., Muslim bans, modern voting restrictions, attacks on "Critical Race Theory") and changing definitions of race (e.g., person of Hispanic/Latinx or Middle Eastern nationalities forming new racial groups), and is broad enough to incorporate research on any racial or ethnic group.
To inform our framework, we conducted a systematic literature review, building on a recent review by Groos and colleagues [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref] , to summarize and critically assess current approaches to the measurement and analysis of institutional racism in quantitative health research. The Groos et al. review [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref] includes articles published between January 1, 2007 and December 31, 2017. The authors searched PubMed and EMBASE databases to identify studies that included the terms "structural racism," "systemic racism," "institutional racism," and/or "institutionalized racism" in the title or abstract. [bib_ref] Racism and health I: pathways and scientific evidence, Williams [/bib_ref] conducted in the United States, explicitly measured an indicator of structural racism, included a health-related outcome, full-text available, and used quantitative methods. Given growing interest in research on institutional racism in recent years, we conducted an updated search using the methods described in Groos et al. [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref]. As shown in , we identified 36 additional papers published between January 1, 2018 and December 15, 2020 that met the authors' inclusion criteria. [fig_ref] Table 1: Summary of institutional racism measures used in quantitative health research published between... [/fig_ref] summarizes the 20 papers included in the Groos et al. [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref] systematic review, as well as the 36 papers from our updated search.
BN performed the initial review of each abstract to determine whether the paper met the Groos et al. [bib_ref] Measuring inequity: a systematic review of methods used to quantify structural racism, Groos [/bib_ref] inclusion criteria. Next, BN extracted information on the measures used, domains examined, levels of measurement for exposures and outcomes, racial and ethnic groups examined, analytic approaches, and links to policy. A second coauthor (T.A or K A) then reviewed each paper to verify the accuracy of the information. In the event of a disagreement, a third coauthor reviewed the paper, and all three coauthors involved in the literature review met to make a final determination about the information in [fig_ref] Table 1: Summary of institutional racism measures used in quantitative health research published between... [/fig_ref].
## Identifying institutional racism policies
Making the links between policy and exposure measurement explicit can help advance empirical research on institutional racism and health. First, the closer research is tied to the harm of specific policies, including the enactment and the enforcement of policies, the stronger the evidence base for eliminating racist policies. Second, even if the exposure measurement is not explicitly tied to a specific policy, researchers can provide the past and present policy context of an institutional racism measure. This is important because, historically, institutional racism was enacted through overtly racist policies that were later modified to be covertly racist "color-blind" policies. Recently, scholars have emphasized the importance of better understanding and articulating the policy context in the measurement of institutional racism. For example, Dennis et al. provide a framework that details major periods of structural discrimination in the USA, including specific policies and domains that have affected different racial/ethnic populations [bib_ref] Looking back to leap forward: a framework for operationalizing the structural racism..., Dennis [/bib_ref]. Hardeman et al. also highlight the importance of understanding the historical context to better understand how structural racism impacts health [bib_ref] Improving the measurement of structural racism to achieve antiracist health policy, Hardeman [/bib_ref].
When policies can be measured directly, researchers can consider both the impact of the enactment of a policy as well as the potential for differential enforcement. For example, the Reagan Administration did not adequately enforce all aspects of the Civil Rights Act, such as employment discrimination, which may be important to consider when examining the impact of the policy over different time periods. However, we also recognize that it is not always possible to make direct links between historic policies that have laid the foundation for racial inequities, especially in cases when the legacy of past policies is omnipresent and difficult to measure or disentangle from concurrent policies. Nonetheless, by strengthening the theoretical linkage between specific policies and institutional racism exposure measurement in our framing of the research and interpretation of results, we can improve the quality of research.
In our literature review, few articles mentioned specific policies that form their basis of exposure measurement, and even fewer explicitly incorporated policies in their analyses. For example, only 15 out of 39 papers that used arealevel indicators of institutional racism included more than a cursory discussion of discriminatory policies or practices related to the indicator(s), while 13 papers included a minimal or generic discussion of policy, and 11 papers did not discuss policy at all. Out of 18 papers that included at least one individual-or family-level indicator of institutional racism, only four included a detailed discussion of discriminatory policies or practices related to the indicator(s), while two included a minimal or generic discussion of policy, and 12 did not discuss policy at all. It is noteworthy that three of the four papers with detailed policy discussions were in the immigration domain, where overtly discriminatory policies remain legal. Only one paper that used individual-or family-level indicators of institutional racism directly measured exposure to a discriminatory policy [bib_ref] From undocumented to lawfully present: do changes to legal status impact psychological..., Patler [/bib_ref].
## Population of interest
The population of interest should be well defined. Researchers must decide whether to take a minority health approach, in which people who identify as a specific racial/ethnic group are the focus of the analysis [bib_ref] Novel approaches to advance minority health and health disparities research, Duran [/bib_ref] , or to compare differences in exposure to or effect of discriminatory policies for different racial/ethnic groups. Studies that include a single racial/ethnic group in the analytic sample are appropriate when the focus is on understanding the health effects of varied exposure to institutional racism for racial or ethnic minorities or when the focus is on identifying potential moderation in causal mechanisms between institutional racism and health. These kinds of analyses acknowledge that the experience of institutional racism can vary within the same racial/ethnic group, either in exposure or in moderating variables that mitigate its health effect. For instance, exposure to institutional racism may vary by geographic location, such as among Hispanic populations who reside in states that differ in their immigration enforcement policies. Researchers could also identify how coping strategies, such as social support through extended family or "fictive kin" networks, mitigate the health risks of institutional racism within Black Americans [bib_ref] Clashes of common sense: on the previous child care experience of teenage..., Geronimus [/bib_ref].
In contrast, studies on the extent to which differences in exposure to or effect of discriminatory policies or practices contribute to racial/ethnic health disparities require analytic samples with more than one racial/ethnic group. For example, a multi-racial/ethnic population would be needed to determine the differential impact of a specific criminal justice policy on health outcomes across racial/ethnic groups. Multi-racial/ethnic populations are also needed to test hypotheses on whether institutional racism can actually benefit Whites while being detrimental to racial/ethnic minorities [bib_ref] Structural racism and myocardial infarction in the United States, Lukachko [/bib_ref] [bib_ref] Stress during pregnancy: the role of institutional racism, Mendez [/bib_ref] [bib_ref] Separate and unequal: structural racism and infant mortality in the US, Wallace [/bib_ref] [bib_ref] Methods for the scientific study of discrimination and health: an ecosocial approach, Krieger [/bib_ref].
In our literature review, we found that just over one third of papers took a minority health approach, examining a single racial/ethnic group, while approximately two thirds of papers took a health disparities approach, examining two or more racial/ethnic groups. Of the 21 papers that took a minority health approach, 17 included Black respondents only, three included Hispanic respondents only, and one included Chinese American respondents only. Of the 35 papers that took a health disparities approach, 27 included Black and White respondents, one included Black and Hispanic respondents, and seven included multiple racial/ ethnic groups. Just 23 of the 35 papers that included more than one racial/ethnic group examined effect modification by race, which is important for understanding group differences in vulnerability to institutional racism. While neither the minority health approach nor the health disparities approach is inherently superior, we recommend that researchers employing a health disparities approach include as many racial/ethnic groups as possible and examine effect modification by race if the study is adequately powered to test interactions.
Finally, whether studies focus on a single racial/ethnic group or multiple groups, to best define their population of interest, researchers should also consider the timing and historic context of the research question (related to the policy discussion above, and Timing of Exposure Measurement, below). For example, policy impacts may compound across an individual's life course, resulting in greater health impacts for older individuals, and may also compound across generations, resulting in the intergenerational transmission of poor health, beginning at birth. In addition, for specific racial/ethnic groups, immigration wave and immigration generation may be important considerations when defining the population of interest for a given study.
## Institutional racism exposure measurement
Improving exposure measurement for institutional racism is a key component of our framework. Below we consider five elements to specify for measures: (1) single vs. multiple domains, (2) area vs. individual level, (3) direct vs. proxy, and (4) timing of measurement.
## Single vs. multiple domains
Institutional racism is considered structural or systemic when multiple institutions work together to produce and sustain a racist system [bib_ref] Structural racism and health inequities in the USA: evidence and interventions, Bailey [/bib_ref] [bib_ref] Looking back to leap forward: a framework for operationalizing the structural racism..., Dennis [/bib_ref] [bib_ref] The race discrimination system, Reskin [/bib_ref] [bib_ref] Structural racism: the rules and relations of inequity, Gee [/bib_ref]. As such, researchers must first determine whether to examine the relationship between one institutional domain, or multiple domains, and health. To capture the systemic, largely latent (i.e., covert and not directly observable) nature of structural racism, researchers should consider ways to incorporate multiple domains into their work.
The 56 papers from the literature review included measures of institutional racism in seven domains: criminal justice, economics and labor, education, healthcare, housing/ residential segregation, immigration, and political participation/representation. Just over half of the papers measured institutional racism in a single domain, while slightly less than half included measures across multiple domains-an approach that is more consistent with the conceptualization of institutional racism as a "race discrimination system" that operates across many domains [bib_ref] The race discrimination system, Reskin [/bib_ref]. Housing-related measures, including residential segregation, were the most commonly used indicators of institutional racism (37 papers), followed by measures in economics and labor (22 papers), criminal justice (15 papers), education (13 papers), healthcare (4), political participation/representation (4), and immigration (3). We recommend that researchers focusing on a single domain identify causal mechanisms within that domain that impact health, while researchers examining measures from multiple domains incorporate theoretical and analytic approaches (e.g., latent models) that treat the measures as part of a connected system as opposed to discrete systems.
Complementary to focusing on a single or multiple domains is determining whether a single or multiple indicators will be used to represent a particular domain. Measurement approaches that combine multiple indicators across multiple domains have the potential to capture the systemic nature of institutional racism [bib_ref] Structural racism and health inequities in the USA: evidence and interventions, Bailey [/bib_ref] [bib_ref] The race discrimination system, Reskin [/bib_ref] [bib_ref] Structural racism: the rules and relations of inequity, Gee [/bib_ref] but may obscure the effects of individual policies or practices that greatly influence health. In our literature review, we found that 17 papers included a single indicator of institutional racism, while 11 included multi-item scales assessing experiences of discrimination within institutional settings, and 28 included multiple indicators of institutional racism, either within a single domain or across multiple domains. We recommend using multiple indicators when trying to capture a more comprehensive measure of institutional racism that may be related to multiple historical policies, and thus difficult to isolate as a single policy measure (e.g., arrests, encounters, and probations to measure institutional racism in the criminal justice system). A single indicator may be appropriate when trying to isolate the effect of a specific policy or practice (e.g., presence/absence of a drug policy or drug-related arrests after implementation of a drug policy).
## Area-level vs. individual-level measures
The level of exposure measurement is also critical. Researchers may consider measurement at the area level (e.g., state or Census tract) or individual level. In most cases, to capture the structural nature of institutional racism, we recommend using area-level exposure measures rather than individuallevel measures, such as experiences of discrimination in institutional settings. There may be exceptions where the individual-level exposure represents a structural policy, such as immigration status for individuals from different countries, but these exceptions should consider the policies that led to the individual-level status.
When considering area-level measures, the geographic level of enactment or enforcement of the racially discriminatory policy should ideally match the geographic level of the exposure to minimize exposure misclassification. For example, if exposure is operationalized as racial disparities in police use of force, and policing policies regarding use of force are primarily made at the district or city level, then exposure should be measured at the district or city level rather than the county or state level. Other specific policies, such as voting rights restrictions, are often at the state level.
In our literature review, we found that 39 of the 56 papers we reviewed included at least one area-level measure of exposure to institutional racism, which is consistent with the conceptualization of institutional racism as a macro-level phenomenon. Area-level indicators in the housing/residential segregation domain included contemporary measures of residential segregation, such as the index of dissimilarity, the isolation index, and the index of concentration at the extremes [bib_ref] The impact of residential segregation on pancreatic cancer diagnosis, treatment, and mortality, Blanco [/bib_ref] [bib_ref] An examination of preterm birth and residential social context among Black immigrant..., Blebu [/bib_ref] [bib_ref] Testing the association between traditional and novel indicators of county-level structural racism..., Chambers [/bib_ref] [bib_ref] Using index of concentration at the extremes as indicators of structural racism..., Chambers [/bib_ref] [bib_ref] Triple jeopardy: the joint impact of racial segregation and neighborhood poverty on..., Do [/bib_ref] [bib_ref] Measuring structural racism and its association with BMI, Dougherty [/bib_ref] [bib_ref] A multilevel analysis of the relationship between institutional and individual racial discrimination..., Gee [/bib_ref] [bib_ref] The role of racial residential segregation in black-white disparities in firearm homicide..., Knopov [/bib_ref] [bib_ref] Segregation, racial structure, and neighborhood violent crime, Krivo [/bib_ref] [bib_ref] Institutional racism and pregnancy health: using Home Mortgage Disclosure act data to..., Mendez [/bib_ref] [bib_ref] Stress during pregnancy: the role of institutional racism, Mendez [/bib_ref] [bib_ref] Institutional racism, neighborhood factors, stress, and preterm birth, Mendez [/bib_ref] [bib_ref] The relationship between structural racism and black-white disparities in fatal police shootings..., Mesic [/bib_ref] [bib_ref] Racial residential segregation, socioeconomic disparities, and the White-Black survival ga, Popescu [/bib_ref] [bib_ref] The impact of racial residential segregation on colorectal cancer outcomes and treatment, Poulson [/bib_ref] [bib_ref] Residential racial segregation and disparities in breast cancer presentation, treatment, and survival, Poulson [/bib_ref] [bib_ref] The impact of racial residential segregation on prostate cancer diagnosis and treatment, Poulson [/bib_ref] [bib_ref] The relationship between racial residential segregation and black-white disparities in fatal police..., Siegel [/bib_ref] [bib_ref] Structural racism and its influence on the severity of atopic dermatitis in..., Tackett [/bib_ref] [bib_ref] Differences in racial disparities in firearm homicide across cities: the role of..., Wong [/bib_ref] [bib_ref] Housing discrimination, residential racial segregation, and colorectal cancer survival in southeastern Wisconsin, Zhou [/bib_ref] ; historical measures of residential segregation based on Home Owner's Loan Corporation (HOLC) redlining maps [bib_ref] The impact of historical racism on modern gun violence: redlining in the..., Benns [/bib_ref] [bib_ref] The enduring impact of historical and structural racism on urban violence in..., Jacoby [/bib_ref] [bib_ref] Structural racism, historical redlining, and risk of preterm birth, Krieger [/bib_ref] ; and contemporary measures of mortgage discrimination and redlining [bib_ref] Neighborhood-level redlining and lending bias are associated with breast cancer mortality in..., Collin [/bib_ref] [bib_ref] A multilevel analysis of the relationship between institutional and individual racial discrimination..., Gee [/bib_ref] [bib_ref] Mortgage discrimination and preterm birth among African American women: an exploratory study, Matoba [/bib_ref] [bib_ref] Institutional racism and pregnancy health: using Home Mortgage Disclosure act data to..., Mendez [/bib_ref] [bib_ref] Stress during pregnancy: the role of institutional racism, Mendez [/bib_ref] [bib_ref] Institutional racism, neighborhood factors, stress, and preterm birth, Mendez [/bib_ref] [bib_ref] Housing discrimination, residential racial segregation, and colorectal cancer survival in southeastern Wisconsin, Zhou [/bib_ref]. These measures were assessed at a variety of geographic levels, including HOLC-defined neighborhoods, Census tracts, zip codes, metropolitan areas, counties, and states. Other area-level measures of institutional racism included Black-White inequities in the domains of economics and labor, criminal justice, education, and political participation/representation [bib_ref] Associations between obesity, obesogenic environments, and structural racism vary by countylevel racial..., Bell [/bib_ref] [bib_ref] Self-rated health and structural racism indicated by county-level racial inequalities in socioeconomic..., Bell [/bib_ref] [bib_ref] An examination of preterm birth and residential social context among Black immigrant..., Blebu [/bib_ref] [bib_ref] Testing the association between traditional and novel indicators of county-level structural racism..., Chambers [/bib_ref] [bib_ref] Measuring structural racism and its association with BMI, Dougherty [/bib_ref] [bib_ref] Access to social determinants of health and determinant inequity for the black..., Hahn [/bib_ref] [bib_ref] Segregation, racial structure, and neighborhood violent crime, Krivo [/bib_ref] [bib_ref] The relationship between structural racism and black-white disparities in fatal police shootings..., Mesic [/bib_ref] [bib_ref] Structural racial inequities in socioeconomic status, urban-rural classification, and infant mortality in..., Owens-Young [/bib_ref] [bib_ref] Structural racism and odds for infant mortality among infants born in the..., Pabayo [/bib_ref] [bib_ref] The effects of social adversity, discrimination, and health risk behaviors on the..., Simons [/bib_ref] [bib_ref] Separate and unequal: structural racism and infant mortality in the US, Wallace [/bib_ref] [bib_ref] Differences in racial disparities in firearm homicide across cities: the role of..., Wong [/bib_ref] [bib_ref] Joint effects of structural racism and income inequality on small-for-gestational-age birth, Wallace [/bib_ref]. These measures were assessed at the city, county, and state levels. In the criminal justice domain, two recent studies used the number of police killings of Black people in the county [bib_ref] Emergency Department visits for depression following police killings of unarmed African Americans, Das [/bib_ref] and metropolitan statistical area [bib_ref] Police killings of Black people and rates of sexually transmitted infections: a..., Ibragimov [/bib_ref] as an indicator of institutional racism. The measurement of area-level racism at different geographic scales makes it challenging to compare results across studies but may be justified based on theoretical or policy considerations. Thus, we recommend that researchers using area-level measures provide an explicit rationale for the geographic level examined.
Seventeen of the papers we reviewed included at least one individual-level measure of exposure to institutional racism. Most of these studies asked respondents to report experiences of discrimination in multiple institutional settings, such as schools, workplaces, and the criminal justice system [bib_ref] Perceptions of race/ethnic discrimination in relation to mortality among Black women: results..., Albert [/bib_ref] [bib_ref] Experiences of racism and subjective cognitive function in African American women, Coogan [/bib_ref] [bib_ref] Racial/ethnic discrimination and alcohol use disorder severity among United States adults, Glass [/bib_ref] [bib_ref] Systemic racism moderates effects of provider racial biases on adherence to hypertension..., Greer [/bib_ref] [bib_ref] The role of age in understanding the psychological effects of racism for..., Greer [/bib_ref] [bib_ref] Perceived racism in relation to telomere length among African American women in..., Lu [/bib_ref] [bib_ref] Structural racism in the workplace: does perception matter for health inequalities?, Mccluney [/bib_ref] [bib_ref] Peer social support is associated with recent HIV testing among young black..., Scott [/bib_ref] [bib_ref] The influence of cognitive development and perceived racial discrimination on the psychological..., Seaton [/bib_ref] [bib_ref] Racial discrimination and telomere length in midlife African American women: interactions of..., Thomas [/bib_ref] [bib_ref] Workplace stress and discrimination effects on the physical and depressive symptoms of..., Zambrana [/bib_ref] [bib_ref] Differential associations between everyday versus institution-specific racial discrimination, self-reported health, and allostatic..., Thomas [/bib_ref]. One study asked parents to report concerns about their children's exposure to racism in institutional settings [bib_ref] Stress of caring for children: the role of perceived racism, Vines [/bib_ref] , while two studies used individual-level characteristics like income and access to healthcare as indicators of exposure to institutional racism [bib_ref] Asthma disparities during the COVID-19 pandemic: a survey of patients and physicians, Baptist [/bib_ref] [bib_ref] The effects of social adversity, discrimination, and health risk behaviors on the..., Simons [/bib_ref]. Studies in the immigration domain included individual-level measures of immigration status [bib_ref] From undocumented to lawfully present: do changes to legal status impact psychological..., Patler [/bib_ref] and reports of sightings and interactions with immigration officials [bib_ref] Everyday violence, structural racism and mistreatment at the US-Mexico border, Sabo [/bib_ref] as indicators of exposure to institutional racism, while one study measured fear of deportation at the family level.
We note that it is not possible at the individual level to distinguish between experiences of institutional racism and experiences of interpersonal racism that occur within an institutional setting. For example, if an individual responds "yes" to the question, "Were you ever treated unfairly during the job hiring process because of your race?" it is hard to determine to what extent this unfair treatment was due to institutional policies/practices (e.g., institutional practice of asking job candidates about their conviction historybecause non-Whites are disproportionately more likely to have a conviction history compared to Whites, this practice precludes non-Whites from having a fair chance at employment) versus interpersonal racism (e.g., an employer choosing not to call back non-White applicants for a job interview because of their subconscious bias that non-White persons are less competent or less professional than White persons, despite laws in place that make it illegal for an employer to discriminate based on someone's race/ethnicity). The distinction between experiences of institutional racism and experiences of interpersonal racism that occur within an institutional setting is important to make in order to sufficiently address racism, which requires a multi-level approach. For instance, within employment, efforts to address interpersonal racism (e.g., anti-racism training for employers) may prevent individual employers from discriminating against non-White employees. However, without institutional policies in place (e.g., an amendment to the Civil Rights Act that prevents employers from discriminating against people with criminal records or requiring employers to delay a criminal background check until after an offer is made or addressing racism within the criminal justice system), non-White persons will not have a fair chance at employment and will continue to face institutional racism within employment and its subsequent health impacts. It is also not possible to determine the extent to which an individual's education or income is the result of exposure to discriminatory policies or practices. Thus, we recommend against using such measures to assess exposure to institutional racism.
## Direct vs. proxy measures
Another consideration is the use of direct or proxy measures of exposure to discriminatory policies or practices. In some cases, direct measures of exposure to specific policies may be preferable to strengthen the connection to a health outcome. For proxy measures, researchers must clearly articulate the conceptual link between institutional policies, whether historic or contemporary, and the exposure measurements. For example, a direct measure of a policy exposure would be living in a state with restrictive voter ID laws, whereas a proxy measure in the political participation/representation domain could be the Black:White ratio of the population proportion who voted in the last presidential election. In the immigration domain, a direct measure may be living in a county where local law enforcement work closely with immigration enforcement (e.g., Secure Communities [bib_ref] Policing immigration, Cox [/bib_ref] , whereas a proxy measure could be fear of deportation.
In our literature review, we found that, with the exception of three papers that used HOLC maps to measure exposure to a specific racially discriminatory policy [bib_ref] The impact of historical racism on modern gun violence: redlining in the..., Benns [/bib_ref] [bib_ref] Structural racism, historical redlining, and risk of preterm birth, Krieger [/bib_ref] [bib_ref] The enduring impact of historical and structural racism on urban violence in..., Jacoby [/bib_ref] , the studies in [fig_ref] Table 1: Summary of institutional racism measures used in quantitative health research published between... [/fig_ref] that used area-level indicators of institutional racism relied on proxy measures, rather than direct measures of exposure to discriminatory policies or practices. Given the challenges inherent in measuring institutional racism in an era of color-blind policies, researchers have increasingly come to rely on measures of racial inequality in domains, such as education or political participation/representation, as proxies for exposure to institutional racism. Though rarely stated explicitly, the rationale for this approach is that contemporary racial inequalities are the result of discriminatory policies and practices and, therefore, are a reasonable proxy for exposure to the policies and practices themselves. Explicitly linking policies to proxy measures is crucial to support the claim that racial/ethnic inequalities in specific domains are due to institutional factors rather than individual choices. Recent work by Agenor et al. provides a template for considering specific state-level policies in different institutional domains, which can be examined in future health studies [bib_ref] Developing a database of structural racismrelated state laws for health equity research..., Agénor [/bib_ref].
## Timing of exposure measurement
Lastly, researchers should consider the timing of their exposure measures within the context of the disease processes for their health measures. Historic timing is critical in linking exposure to health outcomes. As researchers, in order to provide the strongest evidence for causal inference, we must ensure that the timing of the policy precedes specific health outcomes in ways that allow for any latency period in disease progression [bib_ref] Measures of racism, sexism, heterosexism, and gender binarism for health equity research:..., Krieger [/bib_ref]. For example, prior empirical studies have incorporated administrative data on school quality from the Jim Crow South and linked it with later life cognitive functioning [bib_ref] Indicators of childhood quality of education in relation to cognitive function in..., Crowe [/bib_ref] [bib_ref] Race inequity in school attendance across the Jim Crow South and its..., Walsemann [/bib_ref]. The historic timing of policies has further implications for measurement, as more proximal events will be easier to measure directly. For example, it may be easier to link recent immigration policies that vary across states in their design and implementation [bib_ref] Included, but deportable: a new public health approach to policies that criminalize..., De Trinidad Young [/bib_ref] to acute health outcomes (e.g., birth outcomes, heart attacks) than to link older policies like the 1984 Immigration Reform and Control Act to chronic health outcomes. It is useful to consider a few specific questions when thinking about the timing of exposure measurement: are contemporary exposure measure(s) appropriate to adequately capture the link between policy and the health outcome? Are historic measures during a particular period of the life course more appropriate? And, accordingly, are intergenerational measures needed to capture the relevant exposures for specific health outcomes? For example, sensitive periods, such as gestation, may require a particular timing for specific birth outcomes, as in research that examines the timing of immigrant raids during different trimesters of pregnancy on low birth weight [bib_ref] Change in birth outcomes among infants born to Latina mothers after a..., Novak [/bib_ref].
## Health outcome measurement
As in other areas of epidemiologic research, health outcomes are best measured at the individual level. Area-level health outcome data, analyzed in an ecologic framework, can provide initial evidence on the extent to which institutional racism measures affect health, but cannot be used to identify causal relationships. Area-level health outcome measurement may mask the influence of the exposure on the health outcome for different racial/ethnic minority populations, depending on the distribution of race/ethnicity in a particular Census tract, state, or other geographic area. Moreover, measuring health outcomes at the individual level allows for an examination of effect modification of the institutional racism-health relationship by race/ethnicity, to help elucidate differences in the relationships across racial/ethnic populations.
## Study design
The study design will be determined by the type of data a researcher has, as well as the level of exposure and outcome measurement. Ecologic studies examine associations between area-level exposures and area-level outcome measures. Multi-level study designs can be used when the exposure measurement is at the area level, and the outcome measurement is at the individual level. Individual-level study designs are needed when both the exposure and outcome are measured at the individual level. Because institutional racism is a contextual phenomenon, the strongest study designs will be multi-level in nature to enable researchers to estimate the contextual effects of area-level institutional racism on individual-level health.
Study designs may be purely observational or quasiexperimental. Observational studies examine the relationship between existing exposures and outcomes, based on observed patterns within or between populations. When longitudinal observational data are available, researchers should take a counterfactual approach to isolate the impact of structurally racist policies over time and as they operate within and across domains to produce racial disparities in health. For instance, causal mediation approaches that model time-varying relationships between variables allow researchers to treat "race" as part of the time-varying reciprocal or mutually reinforcing processes of racialization and racial discrimination within and across various socioeconomic, political, and cultural systems [bib_ref] Structural racism and quantitative causal inference: a life course mediation framework for..., Graetz [/bib_ref]. These approaches also allow researchers to decompose racial health disparities into different types of cumulative life course effects of institutional racism including unobserved racism (i.e., operating through unmeasured pathways), racial discrimination (i.e., the effect of an underlying system that first racialized individuals and then discriminated against them based on those racial categories), and emergent discrimination (i.e., system-wide race discrimination arising from pervasive racial disparities collectively across multiple domains). For a detailed discussion of these approaches, see Graetz et al. [bib_ref] Structural racism and quantitative causal inference: a life course mediation framework for..., Graetz [/bib_ref]. Quasi-experimental designs take advantage of an intervention, such as a policy change, that disrupts the ongoing pattern of health. In quasi-experimental designs, in contrast to experimental designs, the researcher does not define the intervention. However, quasi-experimental designs can enhance causal inference if adequate comparison groups can be identified to represent a counterfactual comparison of what would have happened if the intervention had not occurred. Recent examples include evaluating the impact of immigration raids or the 2017 Executive Order of the travel ban targeting individuals from Muslim majority countries [bib_ref] Change in birth outcomes among infants born to Latina mothers after a..., Novak [/bib_ref] [bib_ref] The Muslim Ban and preterm birth: analysis of U.S. vital statistics data..., Samari [/bib_ref].
In our literature review, we found examples of multi-level, ecologic, and individual-level study designs. Among papers that included at least one area-level measure of institutional racism, 22 were multi-level studies, while 17 were ecologic studies. In both the multi-level and ecologic studies, institutional racism was measured at the group level (e.g., the state-level Black:White ratio of felony disenfranchisement). This is appropriate given that discriminatory policies and practices are a property of institutions, not individuals. In the ecologic studies, health outcomes were also measured at the group level, despite being a property of individuals. The lack of individual-level data necessary to control for confounding can introduce bias when estimating the contextual effect of institutional racism on health in ecologic studies [bib_ref] Ecologic versus individual-level sources of bias in ecologic estimates of contextual health..., Greenland [/bib_ref]. Thus, we recommend that researchers use multi-level study designs when possible. We also found 16 papers that measured exposures and outcomes at the individual level, and one paper that measured exposure and outcome at the family level. As discussed in detail above, we recommend against measuring institutional racism at the individual level due to challenges inherent in measuring a macro-level phenomenon at the individual level.
## Analytic approaches
Finally, analytic approaches must incorporate the specific aspects of exposure and outcome measurement and study design. An important decision point will be whether to model indicators of institutional racism separately or together; the former allows researchers to better identify specific policies that matter for health, while the latter better captures the systemic nature of multiple interconnected policies. If the latter approach is chosen, researchers must decide if they want to pre-specify institutional racism domains of interest (e.g., criminal justice, economics and labor) or use data reduction techniques to generate profiles of institutional racism. One approach for pre-specifying indicators within given domains is confirmatory factor analysis (CFA), where each domain is treated as a latent construct. CFA is used to understand how each individual indicator of institutional racism loads onto the latent construct of the specified domain. CFA can also be used to generate single summary measures of institutional racism, as in a recent paper by Dougherty et al. [bib_ref] Measuring structural racism and its association with BMI, Dougherty [/bib_ref]. Other data reduction techniques, such as latent class analysis, may be useful to generate profiles of institutional racism without pre-specifying domains of interest.
Importantly, if examining institutional racism and health through a health disparities lens, effect modification of the relationship by race/ethnicity should be incorporated. For example, if researchers are examining the relationship between redlining and cardiovascular disease, testing for effect modification by race/ethnicity will help the research community understand if redlining affected the health of Black individuals only and/or to a greater extent than White individuals, as we would hypothesize given the targeting of the policy. As highlighted in our review, testing for effect modification of the relationship between institutional racism and health by race/ethnicity has not always been incorporated into recent research. Testing these types of hypotheses are essential to provide evidence for differential effects of policy.
# Conclusion
In this paper we defined key concepts relevant to the study of institutional racism and proposed a framework for advancing institutional racism and health research, supported by a review of recent literature examining the relationship between institutional racism and health. We hope this framework will inform future examination of the impacts of institutional racism on health, and that it will help promote the consideration of structural interventions to improve minority health and to reduce and eliminate health disparities [bib_ref] Structural interventions to reduce and eliminate health disparities, Brown [/bib_ref].
[table] Table 1: Summary of institutional racism measures used in quantitative health research published between January 1, 2007 and December 31, 2020 [/table]
|
Minimum Vertex-type Sequence Indexing for Clusters on Square Lattice
An effective indexing scheme for clusters that enables fast structure comparison and congruence check is desperately desirable in the field of mathematics, artificial intelligence, materials science, etc. Here we introduce the concept of minimum vertex-type sequence for the indexing of clusters on square lattice, which contains a series of integers each labeling the vertex type of an atom. The minimum vertex-type sequence is orientation independent, and it builds a one-to-one correspondence with the cluster. By using minimum vertex-type sequence for structural comparison and congruence check, only one type of data is involved, and the largest amount of data to be compared is n pairs, n is the cluster size. In comparison with traditional coordinate-based methods and distance-matrix methods, the minimum vertex-type sequence indexing scheme has many other remarkable advantages. Furthermore, this indexing scheme can be easily generalized to clusters on other high-symmetry lattices. Our work can facilitate cluster indexing and searching in various situations, it may inspire the search of other practical indexing schemes for handling clusters of large sizes.Clusters containing a few to several thousands of atoms serve to bridge the gap between an isolated atom and the bulk counterpart 1-3 . They may exhibit peculiar physical and chemical properties that are probably not to be observed in the bulk materials. Small clusters are anticipated to exhibit strongly size-dependent features, while those intermediate-and large-sized clusters might exhibit a smoothly varying behavior which approaches the bulk limit 3-5 . As the number of atoms increases, the crystal fragments were found to be more stable 5, 6 . Thus, the congruence-check of the crystal fragments is important, while there is a lack of a high-efficient method for distinguishing the clusters containing bonds of same lengths.The size and structure are the fundamental factors in determining the properties of a cluster, they are thus of great concern in the study and application of clusters. The conventional experimental methods for structural determination are hardly able to directly obtain the atomic configuration of clusters 7-11 , as collaborating with theoretical computation is usually a necessity 1-3, 12 , yet without a guarantee of success. This is of no surprise since finding the global minimum on a given potential-energy-surface, a key criterion for predicting ground state structure of a cluster, is essentially a formidable task. The number of local minima increases exponentially with the increasing cluster size 3, 6, 13 , as it is obviously an NP-hard problem that can be mapped to the traveling-salesman problem 13, 14 . In the past two decades, several global optimization methods for structure prediction have been devised 15 , including simulated annealing 16 , basin/minima hopping 17-19 , genetic algorithm 19-27 , particle swarm optimization algorithm 28-33 , etc. To investigate the structural evolution by global optimization, a large number of trial structures are to be generated at each generation. Consequently, it is highly desirable to develop some techniques to label the transient structures and to judge the similarity or congruency among the structures available at the intermediate stages, which serve to avoid futile, repetitious computation so as to effectively accelerate the search process 33 . A complete sampling of all the minima on a potential-energy-surface is simply impossible, but a high-throughput screening under restricted conditions can be still very helpful 6, 34 , for which the congruence check of clusters is a crucial prerequisite. In order to congruence check two distinct clusters, it needs to establish a one-to-one mapping, or correspondence, between their structural elements. The most straightforward way would be to compare the coordinates of the corresponding atoms in those two clusters 35 . This coordinate-based method is very complicated because both clusters are subjected to a translation operation so that their centroids are at the Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 7: 392 |
origin of the coordinate system, and one of them needs an additional rotation to minimize the deviation in the corresponding coordinates. For two 2D clusters each containing n atoms, the largest amount of comparing data this way is significantly greater than 2n pairs (for square lattice, without distinguishing the enantiomers, it is usually 16n pairs). An alternative strategy is to compare the corresponding interatomic distances in the two clusters, i.e., the distance-matrix method [bib_ref] Global minimum structure searches via particle swarm optimization, Call [/bib_ref] [bib_ref] Significance of root-mean-square deviation in comparing three-dimensional structures of globular proteins, Maiorov [/bib_ref]. However, as Lv et al. [bib_ref] Particle-swarm structure prediction on clusters, Lv [/bib_ref] once pointed out, "the distance metric requires ordering of atoms in a structure, and thus is not able to unambiguously fingerprint structures". For two clusters of size n, the largest amount of comparing data in this way is much more than n(n − 1)/2 pairs. Per-atom distance sets fingerprint and diffraction-like fingerprint recently proposed by Oganov and Valle [bib_ref] How to quantify energy landscapes of solids, Oganov [/bib_ref] , from which a reasonable measure of structure of similarity was derived, can be used to discriminate structures of a majority of clusters, but they cannot be used to label distinct structures since there are still some clusters cannot be distinguished through the two fingerprints, even combining both of them (an example is illustrated in the supplementary materials). Other methods such as those involving Bond-Characterization-Matrix 32, 33 , Zernike descriptors 38 , spherical harmonic descriptors [bib_ref] A search engine for 3D models, Funkhouser [/bib_ref] , can provide an approximate, quantitative measure for structural similarity between two clusters, but cannot label the distinct structures. The aforementioned methods are all suitable for both amorphous and crystalline clusters. For 2D and 3D high-symmetry crystal-fragment clusters, where the interatomic distances among the nearest neighboring atoms are fixed, there is also another recognition technique based on the relative orientations of the bonds, which employs codes obtained by using the Balaban and von Schleyer's technique [bib_ref] Systematic classification and nomenclature of diamond hydrocarbons-I: Graph-theoretical enumeration of polymantanes, Balaban [/bib_ref]. In this approach the largest amount of comparing data is reduced to (n − 1) pairs, it is, however, not a fast structure retrieval method since finding the main and side chains of a given structure is very time-consuming.
In the current work we introduce a new indexing scheme, the minimum vertex-type sequence, to label and characterize the clusters on square lattice (below often simply referred to as cluster when no ambiguity may arise), which might meet some requirements for fast and effective decision making of artificial intelligence as in the game of Go 42 . This indexing scheme employs only the vertex type, or precisely the nearest-neighbor configuration, for each atom in a cluster. The atoms in a cluster are ordered and labeled following a special rule that the cluster can be indexed with a unique digit sequence, being independent from either the choice of the reference frame or the orientation of a cluster in given configuration. A one-to-one correspondence between the minimum vertex-type sequence and the cluster can be established. For clusters of size n, the largest amount of comparing data for congruency check is only n pairs.
# Results
Below we will demonstrate that for crystal-fragment clusters, the minimum vertex type sequence, which characterizes the nearest-neighbor configuration of each atom in a cluster, provides a practicable algorithm for fast structure comparison. The minimum vertex-type sequence method will be demonstrated in detail by treating clusters on square lattice.
Firstly, we classify the vertexes for atoms in a cluster on square lattice based on their nearest-neighbor configurations (the orientations of the bonds with the nearest-neighbors are discriminated). There are only fifteen different vertex types for clusters on square lattice, which are labeled accordingly with the integers 1-15 in [fig_ref] Figure 1: The fifteen possible vertex types appearing in a cluster on square lattice,... [/fig_ref].
Secondly, atoms in the cluster are labeled with their vertex types in the order of "left-to-right-and-bottom-up", i.e., the atom at the bottom left corner is set to be the first atom, it is then the turn of atoms in the same horizontal line from left to right till the end of that line, which is to be continued from the leftmost atom of the next upper The black stones denote the atoms of concern, while the white stones indicate the existence of possible nearest neighbors.
Scientific RepoRts | 7: 392 | DOI:10.1038/s41598-017-00398-z line (if there is any). This process will proceed to the atom at the upper right corner of the cluster, as shown in [fig_ref] Figure 2: The different orientations of a given structure for 4-atom cluster under the... [/fig_ref].
By assigning to all the atoms a corresponding vertex type in the aforementioned order, we obtain an n-digit vertex-type sequence, where n is the size of the cluster. Since the vertex types are discriminated with regard to the orientations of bonds to the nearest-neighbors, the same cluster in different orientations may have distinct vertex-type sequences. As the space group for square lattice is group D 4 with eight elements, a cluster here concerned may have at most eight different vertex-type sequences. In [fig_ref] Figure 2: The different orientations of a given structure for 4-atom cluster under the... [/fig_ref] displayed are the different configurations of a cluster of 4 atoms under the action of group D 4 , the eight vertex-type sequences are [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Understanding the stable boron clusters: A bond model and first-principles calculations based..., Xu [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref] , [bib_ref] The physics of simple metal clusters: self-consistent jellium model and semiclassical approaches, Brack [/bib_ref] [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Crystal structure prediction from first principles, Woodley [/bib_ref] , (2,6,10,3), (7,5,3,4), [bib_ref] Structural properties of nanoclusters: Energetic, thermodynamic, and kinetic effects, Baletto [/bib_ref] [bib_ref] Understanding the stable boron clusters: A bond model and first-principles calculations based..., Xu [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref] , [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref] , [bib_ref] Understanding the stable boron clusters: A bond model and first-principles calculations based..., Xu [/bib_ref] [bib_ref] The physics of simple metal clusters: self-consistent jellium model and semiclassical approaches, Brack [/bib_ref] [bib_ref] Crystal structure prediction from first principles, Woodley [/bib_ref] and (2,10,5,3), respectively. Among those eight (or less if the clusters possess rotation and/or reflection symmetries) vertex-type sequences, we adopt the one which has the minimum preceding numbers as the character of the cluster, and denote it as the minimum vertex-type sequence. For the cluster of 4 atoms shown in [fig_ref] Figure 2: The different orientations of a given structure for 4-atom cluster under the... [/fig_ref] , the minimum number for the first digit is 1, and in the two sequences beginning with 1, i.e., [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Understanding the stable boron clusters: A bond model and first-principles calculations based..., Xu [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref] and [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref] , the minimum number for the second digit is 5, thus the minimum vertex-type sequence for this cluster is [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref].
It can be strictly demonstrated that there is a one-to-one mapping between the minimum vertex-type sequence and the cluster (see the supplementary information for details). This is to say that the minimum vertex-type sequence can be used to characterize the individual clusters on square lattice, which can serve the identification and comparison of the clusters. With the minimum vertex-type sequence, the largest amount of data involved in the comparison of two clusters of n atoms can be reduced to only n pairs.
For the comparison of two clusters, this minimum vertex-type sequence scheme has two distinct advantages over the conventional coordinate-based methods. It uses only a few descriptive data and, more importantly, it is orientation independent. Unlike the distance-matrix method which suffers from ambiguous ordering of the atoms [bib_ref] Particle-swarm structure prediction on clusters, Lv [/bib_ref] , the labeling of atoms in the minimum vertex-type sequence is concise and determinate. In comparison with the Balaban and von Schleyer's technique [bib_ref] Systematic classification and nomenclature of diamond hydrocarbons-I: Graph-theoretical enumeration of polymantanes, Balaban [/bib_ref] , our minimum vertex-type sequence scheme employs only one single type of data, and it avoids the difficult process of finding out the main chains of a cluster, which is usually a formidable task. Roughly speaking, our minimum vertex-type sequence scheme uses only a series of integers labeling the vertex type of atoms, whereas the Balaban and von Schleyer's technique employs three types of data, i.e., digits denoting the orientation of the bonds, parentheses denoting the branches and commas denoting the different branches on the same level. An overview of the comparison of the minimum vertex-type sequence scheme to those conventional techniques is summarized in . Obviously, the minimum vertex-type sequence scheme can serve the fast congruence-check for clusters on high-symmetry lattices.
# Discussion
The minimum vertex-type sequences build a one-to-one correspondence with clusters on square lattice, thus a minimum vertex-type sequence can be regarded as the index tag of the corresponding cluster. That is to say that each cluster can be characterized by a unique minimum vertex-type sequence. For example, the clusters of size 2 can be indexed as [bib_ref] Structural properties of nanoclusters: Energetic, thermodynamic, and kinetic effects, Baletto [/bib_ref]. The cluster of size 4 displayed in [fig_ref] Figure 2: The different orientations of a given structure for 4-atom cluster under the... [/fig_ref] , which is one of the five possible configurations, can be indexed as [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Ground states of group-IV nanostructures: Magic structures of diamond and silicon nanocrystals, Yang [/bib_ref]. For clusters of size 7 in the two configurations in [fig_ref] Figure 3: The two configurations for cluster of 7 atoms corresponding to the minimum... [/fig_ref] (there are 108 different configurations in total), the minimum vertex-type sequences are [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] The physics of simple metal clusters: experimental aspects and simple models, De Heer [/bib_ref] [bib_ref] Modelling nano-clusters and nucleation, Catlow [/bib_ref] [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Crystal structure prediction from first principles, Woodley [/bib_ref] and [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Identifying structural patterns in disordered metal clusters, Doye [/bib_ref] [bib_ref] Understanding the stable boron clusters: A bond model and first-principles calculations based..., Xu [/bib_ref] [bib_ref] The physics of simple metal clusters: self-consistent jellium model and semiclassical approaches, Brack [/bib_ref] [bib_ref] Stacking Principle and Magic Sizes of Transition Metal Nanoclusters Based on Generalized..., Li [/bib_ref] [bib_ref] Crystal structure prediction from first principles, Woodley [/bib_ref] , respectively. Comparing with the Balaban and von Schleyer's indexing system [bib_ref] Systematic classification and nomenclature of diamond hydrocarbons-I: Graph-theoretical enumeration of polymantanes, Balaban [/bib_ref] , our minimum vertex-type sequence indexing system has at least two advantages. Firstly, the process of obtaining the minimum vertex-type sequence of a cluster is simpler than that to get the Balaban and von Schleyer's codes which usually require the determination of the main and the side chain(s) in a branched cluster. Secondly, the minimum vertex-type sequence indexing system is more economical for computer calculation, since the minimum vertex-type sequences use only one type of data, whereas the Balaban and von Schleyer's codes usually use three types of data.
In a practical implementation of congruence check for clusters of large sizes, approximate representation of single-number indexing can be used as a rapid primary filter, which is to be further confirmed by comparing the minimum vertex-type sequences. A good choice for the approximate representation of single-number indexing is to let
[formula] = ∑ ⋅ = f P S i ( ) i n i 1 6 [/formula]
, where S i is the i-th digit in the minimum vertex-type sequence of the cluster P. The final decision can be made by digit-by-digit comparing the corresponding minimum vertex-type sequences among the chosen candidates.
The minimum vertex-type sequence indexing scheme is orientation independent and employs only n integers to describe the configuration of a cluster on the square lattice. It is anticipated to provide a fast structure recognition method for large clusters. The excellent effectiveness of this indexing scheme can be verified in solving this typical problem: finding out all the isomers on square lattice for clusters as large as possible in a tolerable running time. This is a formidable task because the number of isomers increases exponentially with the cluster size 3, 6, 13 . When the cluster size reaches 13, as derived with the current method, there are 238591 types of isomers (with the enantiomers undistinguished). Each time when a new structure is generated, it needs to be compared with a great amount of existing isomers to judge whether a new type has occurred or not, which might immediately use up the computer resource. It is a difficulty for all structure recognition methods. And our minimum vertex-type sequence indexing scheme presented above provides a preferable fast structure retrieval method. Note that it is minimum vertex-type sequence. a m denotes the dimension of the system concerned. b If a structure is formed only from the main chain, the size of the descriptive data is (n − 1); if a structure is branched, the data size is then greater than (n − 1). not time-consuming for a computer to label a cluster on square lattice using our minimum vertex-type sequence indexing scheme (say 1000 atoms, about 0.4 second on a Lenovo_C560, a general type of personal computer). The minimum vertex-type sequence indexing also has many other merits. A minimum vertex-type sequence contains the information of vertex types of atoms, i.e., the nearest-neighbor configurations of atoms. It can be easily converted into the information of bonds. Therefore, the minimum vertex-type sequence indexing is preferable in the situation where the bond energy 4 is of concern.
Furthermore, the concept of minimum vertex-type sequence can be generalized to other high-symmetry lattices, such as the regular triangular lattice and the honeycomb lattice, following three typical steps: 1) classify the possible vertexes in clusters on a given lattice based on the nearest-neighbor configuration of an atom; 2) label the atoms in the cluster with their vertex types in the order of "left-to-right-and-bottom-up"; 3) carry out all the possible symmetry operations of the given lattice on the cluster to obtain several vertex-type sequences among which we adopt the one with the minimum preceding numbers as the character of the cluster. Note that in the case of square lattice there are 15 possible vertex types to be handled, while there are 63 and 14 for the regular triangular lattice and the honeycomb lattice respectively (more details will be presented in a forthcoming publication). By combining the minimum vertex-type sequence method with high-throughput screening procedure under restricted conditions, effective strategies can be devised to search new atomic cluster structures on those complicated lattices.
We devised the minimum vertex-type sequence indexing scheme to characterize clusters on square lattice. The minimum vertex-type sequence of a cluster comprises only one integer for each atom to label its vertex type, and the sequence is orientation independent. It turns out to be the most preferable scheme for fast recognition and strict comparison of clusters, as it employs fewer and simpler descriptive data. In particular, for fast preliminary filtering, the minimum vertex-type sequences can be at first converted into a single integer, which can further accelerate the procedure of structure comparison dramatically. Our work is anticipated to initiate the search of other practical indexing schemes for handling clusters of large sizes, which are desperately desired in many fields. It is true that clusters and crystals usually contain bonds of different lengths, but they are beyond the scope of the manuscript. Our MVTS indexing scheme cannot deal with this case. Note that the minimum vertex-type sequence indexing does not provide a metric and is not capable of computing degrees of similarity, although it provides a high-efficient way for no-fail detection of equivalence of clusters comprised of crystal fragments. This is exactly complementary to the Oganov-Valle fingerprints, which offer a good pragmatic measure of structural similarity but may fail in some cases. Recently, another promising method is also provided by Oganov [bib_ref] Machine learning scheme for fast extraction of chemically interpretable interatomic potentials, Dolgirev [/bib_ref] for improvement. The minimum vertex-type sequence indexing scheme would effectively speed up the ground state structure searching for the crystal-fragment clusters with large number of atoms.
[fig] Figure 1: The fifteen possible vertex types appearing in a cluster on square lattice, as labeled by integers 1-15. [/fig]
[fig] Figure 2: The different orientations of a given structure for 4-atom cluster under the action of group D 4 for square lattice and the corresponding vertex-type sequences. The minimum vertex-type sequence is(1,5,8,4). [/fig]
[fig] Figure 3: The two configurations for cluster of 7 atoms corresponding to the minimum vertex- [/fig]
|
Assessment of the quantity of microorganisms associated with bronchiectasis in saliva, sputum and nasal lavage after periodontal treatment: a study protocol of a randomised controlled trial
# Introduction
Periodontal disease (PD) has an infectious origin and affects dental supporting tissue, impacting 19% of the adult Brazilian population.It has been associated with various systemic diseases, such as diabetes and cardiovascular disease. In recent years, an association has been suggested between PD and pulmonary diseases, such as pneumonia, asthma and chronic obstructive pulmonary
## Strengths and limitations of this study
▪ Although some studies have correlated chronic obstructive pulmonary disease (COPD) with periodontal disease, to date, bronchiectasis has not been studied in the dentistry field. ▪ Other periodontopathogens, such as Tannerella forsythia and Treponema denticola, as well as other pathogens found in the oral biofilm, such as Haemophilus influenzae and Streptococcus pneumoniae, could be quantified, because they have been identified in tracheal aspirate of patients with COPD. However, since this is the first study, only the most representative microorganisms were chosen. Additional samples of this material will be collected for later analyses of these pathogens. ▪ Despite the reduction in bacteria in periodontal pockets being followed by their decrease in saliva, it would be interesting to perform microbiological evaluation in this niche, because it could be a potential reservoir for lung bacteria. This material will also be collected for later analyses.
disease (COPD), although the causal relationship between them is not clear. [bib_ref] Associations between periodontal disease and risk for nosocomial bacterial pneumonia and chronic..., Scannapieco [/bib_ref] The most accepted causal mechanism is aspiration of resident bacteria from the periodontal pocket, which could cause or exacerbate lung infections, since there is association between COPD and the plaque index, [bib_ref] Association between periodontitis and chronic obstructive pulmonary disease in a Chinese population, Si [/bib_ref] and PD and COPD have pathophysiological processes in common: connective tissue destruction and exaggerated inflammatory reaction. [bib_ref] The role of proteolytic enzymes in the development of pulmonary emphysema and..., Travis [/bib_ref] Systemic inflammatory markers are amplified for both, especially tumour necrosis factor (TNF)α, interleukin (IL)-6 and IL-8. [bib_ref] Association between chronic obstructive pulmonary disease and systemic inflammation: a systematic review..., Gan [/bib_ref] However, the levels of inflammatory markers are not similar among studies [bib_ref] Systematic review of the association between respiratory diseases and oral health, Azarpazhooh [/bib_ref] suggesting that more studies are necessary. [bib_ref] Association between chronic obstructive pulmonary disease and systemic inflammation: a systematic review..., Gan [/bib_ref] Although some studies have correlated COPD and PD, bronchiectasis has not been studied in the dentistry field to date. It is a condition with irreversible and permanent abnormal dilation of the bronchi and bronchioles, high morbidity and decreased quality of life. 14 Bronchiectasis is often misdiagnosed as asthma or COPD because of the similarities of the clinical manifestations, such as cough and sputum production. [bib_ref] Assessment and investigation of adults with bronchiectasis. Assessment and investigation, Drain [/bib_ref] The clinical treatment for this condition is based on control and prevention of recurrent acute exacerbations, which, in turn, reduce public health spending. The aetiology of bronchiectasis can vary widely, including congenital genetic defect and idiopathic causes.The most common extrinsic factor of bronchiectasis is aspiration of infected mucus, 6 which could be exacerbated by the presence of PD, but there are no previous studies investigating oral health in these patients. Inflammatory markers, such as IL-1, IL-6, IL-8 and TNFα are increased in bronchiectasis and in PD. [bib_ref] Inhaled fluticasone reduces sputum inflammatory indices in severe bronchiectasis, Tsang [/bib_ref] The most commonly isolated microorganism in infections is Pseudomonas aeruginosa, whereas Staphylococcus aureus, despite not being as common, is related to more complex infections. The presence of Ps. aeruginosa is associated with a higher number of hospital admissions. [bib_ref] A influência de bactérias patogênicas na transportabilidade do escarro e na qualidade..., Zanchet [/bib_ref] Periodontal treatment (PT) is not able to eliminate all subgingival pathogens because of inaccessible colonisation sites, and, furthermore, periodontopathogens such as Porphyromonas gingivalis can invade periodontal tissues, and it has been found in infected lung. Also, Ps. aeruginosa and S. aureus have been found in periodontal pockets of patients with COPD. [bib_ref] 16S rDNA-based metagenomic analysis of dental plaque and lung bacteria in patients..., Tan [/bib_ref] Antibiotics are relegated to adjuvants in PT, since the risks outweigh the benefits. [bib_ref] Associations between periodontal disease and risk for nosocomial bacterial pneumonia and chronic..., Scannapieco [/bib_ref] On the other hand, they are routinely administered in bronchiectasis, selecting resistant bacterial strains 23 in the whole body, including, possibly, the oral microbiome. Macrolides, fluoroquinolones and azalides are long-term antibiotics used for bronchiectasis, and they are effective against oral bacteria. Photodynamic therapy (PDT) is an antimicrobial, atoxic alternative for reducing the quantity of microorganisms in inaccessible sites after PT, [bib_ref] The potential for photodynamic therapy in the treatment of localized infections, O'riordan [/bib_ref] with no side effects and no bacterial resistance reported. [bib_ref] Photodynamic therapy in dentistry, Konopka [/bib_ref] PDT is the association of a photosensitiser with light in the presence of oxygen, [bib_ref] Short-term clinical effects of adjunctive antimicrobial photodynamic therapy in periodontal treatment: a..., Braun [/bib_ref] producing reactive oxygen species, such as cytotoxic superoxide and hydroxyl radicals. This reaction generates energy, which is transferred to the bacterial oxygen molecules, leading to cell death. [bib_ref] Influence of pseudomonas aeruginosa on exacerbation in patients with bronchiectasis, Chawla [/bib_ref] A common observation in patients with bronchiectasis is the presence of halitosis. It is expected that PT could partly solve this. To date it has not been tested if PT alone can eliminate halitosis in these patients or whether the pulmonary disease induces halitosis by itself.
Although bronchiectasis and COPD share very similar systemic clinical and immunological findings, there are no studies on the periodontal status of patients with bronchiectasis, which leaves a knowledge gap in this area. Therefore, the hypothesis to be tested is whether PT will decrease the number of microorganism species associated with bronchiectasis exacerbation in the saliva environment, sputum and nasal lavage. The primary objective of this study is to assess the decrease in the number of microorganisms associated with bronchiectasis exacerbation, as well as reduction of P. gingivalis (currently deemed a cornerstone in PD) in saliva, sputum and nasal lavage before PT and 3 months thereafter. The secondary objectives are to evaluate periodontal clinical parameters, halitosis and proinflammatory cytokines (IL-1, IL-6 and IL8, TNFα) in serum and saliva before PT and 3 months thereafter with and without PDT.
# Methods
A randomised, controlled, 3-month, single-centre, parallel-group clinical trial was designed according to the CONSORT Statement and is registered at http://www. clinicaltrial.gov. After verbal and written explanation of the study, patients will sign the informed consent form approved by the Research Ethics Committee (1.057.901) of Nove de Julho University (UNINOVE), and a researcher involved in the study (EHP) will enrol the patients. The study will be performed in accordance with the Declaration of Helsinki (revised in Fortaleza, Brazil, 2013.) A total of 182 patients under medical care at InCor-FMUSP with generalised chronic periodontitis [bib_ref] Development of a classification system for periodontal diseases and conditions, Armitage [/bib_ref] will receive PT at UNINOVE Dental Clinic, from September 2015 to September 2017. Patients with clinical bronchiectasis will be characterised by chronic sputum production and dilated bronchi confirmed by high-resolution thorax CT. The severity of bronchiectasis will be assessed using the FACED score. [bib_ref] Multidimensional approach to non-cystic fibrosis bronchiectasis: the FACED score, Martínez-García [/bib_ref] Calculation of sample size Sample size calculation was performed for the microbiota (P. gingivalis) as the main outcome. A sample size of 91 participants per group was found to be required to detect a between-group difference of 0.7 [bib_ref] Photodynamic therapy during supportive periodontal care: clinical, microbiologic, immunoinflammatory, and patient-centered performance..., Kolbe [/bib_ref] in the amount of bacteria from baseline to 3-month follow-up ( power=0.8, α=0.05). This predicted difference equates to an effect size of 0.42, considering an SD of 1.9 for group 2 (experimental group; PDT) and 1.4 for the root-planing group. [bib_ref] Photodynamic therapy during supportive periodontal care: clinical, microbiologic, immunoinflammatory, and patient-centered performance..., Kolbe [/bib_ref] We have also calculated the sample size for the secondary outcomes. A sample size of 42 participants per group was found to be required to detect a between-group difference of 0.84 41 in TNFα from baseline to the 3-month follow-up ( power=0.8, α=0.05). This predicted difference equates to an effect size of 0.62, considering an SD of 1.85 for group 2 and 0.49 for group 1 ( positive control group; gold standard). A sample size of 22 participants per group was found to be required to detect a between-group difference of 4.95 [bib_ref] Photodynamic therapy during supportive periodontal care: clinical, microbiologic, immunoinflammatory, and patient-centered performance..., Kolbe [/bib_ref] in IL-1β from baseline to the 3-month follow-up ( power=0.8, α=0.05). This predicted difference equates to an effect size of 0.87, considering an SD of 5.37 for group 2 and 5.98 for group 1. A sample size of 61 participants per group was found to be required to detect a between-group difference of 2.1 41 in IL-6 from baseline to the 3-month follow-up ( power=0.8, α=0.05). This predicted difference equates to an effect size of 0.51, considering an SD of 4.62 for group 2 and 3.48 for group 1. A sample size of 32 participants per group was found to be required to detect a between-group difference of 77.48 41 in IL-8 from baseline to the 3-month follow-up ( power=0.8, α=0.05). This predicted difference equates to an effect size of 0.72, considering an SD of 94.06 for group 2 and 120.33 for group 1. For halitosis, there is no study comparing scaling and root planing (SRP) +PDT with conventional treatment. However, a previous study comparing baseline with post-non-surgical PT (including oral hygiene (OH) instruction) [bib_ref] The levels of volatile sulfur compounds in mouth air from patients with..., Tsai [/bib_ref] demonstrated a high intragroup difference, determining an effect size of 0.77. Therefore, we believe that the sample size for the mean outcome (n=91) will cover the sample size required to detect intergroup difference for halitosis. The sample size calculation was performed on G*Power software (V.3.1.9). [bib_ref] A flexible statistical power analysis program for the social, behavioral, and biomedical..., Faul [/bib_ref] Calibration of clinical examiner One researcher (gold standard) will perform periodontal clinical examinations to achieve maximum reproducibility of the measurements. Ten patients with PD will be evaluated at six sites per tooth (mesiovestibular, vestibular, distobuccal, mesial lingual, lingual and distal-lingual) in the whole mouth, except third molars, for probing depth and clinical attachment loss. Patients who undergo these clinical examinations will not be included in the study groups. The intraclass correlation coefficient (ICC) will be calculated to evaluate intraexaminer concordance, which must be higher than 0.9. Periodontal probes from University of North Carolina (UNC-15) by Hu-Friedy, Chicago, Illinois, USA will be used. Data will be analysed by ICC test.The results will be expressed in tables, and compliance with the standard examiner must be higher than 0.8.
## Inclusion and exclusion criteria
Patients must be >35 years of age, of either gender, and have >10 teeth with chronic moderate PD-that is, more than 15% of teeth examined with ≥4 mm probing depth (PD). Patients with clinical bronchiectasis must be characterised by chronic sputum production and dilated bronchi confirmed by high-resolution thorax CT. All patients should be in a stable condition (no exacerbations requiring antibiotic use in the 3 months before and 3 months after PT). Exclusion criteria include smokers or former smokers who have quit less than 5 years previously, pregnant women, phenytoin or cyclosporin users (because they affect periodontal status), those with decompensated systemic diseases requiring prophylactic antibiotic therapy for PT, those who have used oral antiseptics or anti-inflammatory drugs in the past 3 months, or those who have had PT performed in the past 6 months. Patients with other pulmonary (eg, pulmonary cystic fibrosis, asthma) and nose or throat inflammatory diseases will also be excluded.
## Randomisation of patients and allocation concealment
After SRP, a researcher not involved in the study will divide the patients into two groups by randomised order (Microsoft Excel, 2013 version). Then, opaque envelopes randomly containing information about application of PDT or not will be labelled with sequential numbers. The researcher responsible for PT will open the first envelope and perform the procedure written therein. The internal contents will be revealed only after statistical analysis.
Experimental groups and study design Patients will be divided into two groups and they will be subjected to different treatments as follows: group 1, positive control group (gold standard; n=91; OH+SRP +simulation of PDT); group 2, experimental group (n=91; OH+SRP+PDT with methylene blue and the red laser diode (λ=660 nm)). All participants will receive PT from an experienced specialist with universal curettes (Hu-Friedy) and ultrasound (Ultra Sound Dabi Atlante-Profi Neo US, Ribeirão Preto, Brazil) in a full-mouth manner (figure 1).
No antibiotics or oral antiseptics will be prescribed. The periodontal supportive therapy will be performed 3 months after the end of PT. [bib_ref] Subgingival bacterial recolonization after scaling and root planing in smokers with chronic..., Feres [/bib_ref] After SRP, a researcher not involved in the study will divide the patients into two groups by randomised order (PDT use or not). In group 1, PT will be performed as described above with Therapy XT (see below) turned off as well. In group 2, the PT will be performed identically to that in group 1. PDT will be administered in periodontal pockets >4 mm. Methylene blue (Chimiolux 0.005%; DMC, São Carlos, São Paulo, Brazil) will be applied with a carpule syringe needle (with stop and no bisel) in the depth of periodontal pockets. After 5 min of application, the red laser diode (λ=660 nm) will be applied with an output power of 100 mW (Therapy XT; DMC) and 90 s of exposure-that is, 9 J in each point. The laser head will be placed in direct contact with the periodontal pocket. Applications will be held in six sites around the tooth in all teeth. To finalise, there will be 1 min of irradiation in scan around each tooth and rinsing with saline solution to remove the photosensitiser. All the proposed treatments in group 1 (OH+SRP+simulation of PDT) in each patient will be concluded within 1 week. The treatments for group 2 (OH+SRP+PDT) will be also concluded within 1 week. The data analysts who will assess outcome will be blinded during outcome analysis. All possible adverse effects will be noted and qualified during PT and during the maintenance period (3 months) through questionnaires developed for this protocol. The adverse events will be reported in the results section of the manuscript and will be discussed. The Research Ethics Committee of Nove de Julho University (UNINOVE) will be notified of any protocol alterations. In addition, the records at http://www. clinicaltrial.gov will be notified. [fig_ref] Figure 2: Schematic [/fig_ref] summarises the principal points of this protocol.
Sputum, saliva and nasal lavage collection for microbiological evaluation Fluid will be kept in the nasal cavities for 10 s and then washed out by allowing it to flow into a sterile container. The nasal wash will then be transferred to a graduated tube. The nasal lavage will be performed by asking the patient to breathe deeply and hold 10 mL sterile saline inserted through the nostrils. After collection, RNAlater (Qiagen, Valencia, California, USA) will be added to preserve the DNA.
The sputum will be collected in the morning before any food intake. Patients will be asked to breathe deeply several times and then cough deeply; the sample will be collected in a wide-mouth jar. In the laboratory, the sputum specimens will be decontaminated by N Acetyl-L-cysteine/NaOH centrifuged at 10 000×g for 5 min, and the pellet will be suspended in 50 μL buffer solution to extract DNA (1% Triton X-100, 0.5% Tween 20, 10 mM Tris/HC1, pH 8.0, and 1 mM EDTA). The material will be sent immediately to the laboratory.
The saliva will be collected and stored exactly as described above. The timeline in [fig_ref] Figure 3: Timeline showing the sequence of procedures and sample collection [/fig_ref] shows the sequence of procedures and sample collection.
## Microbiological evaluation
Samples will be defrosted and vortexed. DNA extraction will be performed with Master DNA Extraction Kit Pure Kit (Epicentre Technologies, Chicago, Illinois, USA) according to the manufacturer's instructions. Quantitative PCR of the total amount of Ps. aeruginosa, S. aureus and P. gingivalis will be performed with the SYBR Green System (Applied Biosystems, Foster City, California, USA) in a thermal cycler Step One Plus Real-Time PCR System (Applied Biosystems). This quantification will be performed before treatment and 3 months thereafter to verify the effectiveness of PT and to evaluate if it causes reduction of microbial content of buccal, nasal and lung niches. The outcomes of this protocol will determine the efficacy of PT in reducing the total amount of microorganisms in saliva, sputum and nasal lavage. These outcomes will be classified as (i) better, (ii) no change or (iii) worse than baseline. These results will be compared with a possible reduction of systemic and local inflammation and halitosis after 3 months.
## Determination of systemic and saliva inflammatory markers
Sample collections will be performed through venous puncture by a trained technician at baseline and after 3 months. Sample collections will be centrifuged and serum will be stored. Unstimulated saliva samples will be obtained in sterile collecting tubes and will be kept in a container with ice. All samples will be stored in dry tubes at −80°C until use.
Commercial kits (Peprotech, Rocky Hill, New Jersey, USA) for measuring inflammatory markers (TNFα factor, IL-1, IL-6 and IL-8) by ELISA will be used according to the manufacturer's instructions.
## Halitosis measurements
Air from inside the oral cavity will be collected with a syringe. With one movement it will be injected into an Oral Chroma (Abilit, Japan), a portable device connected to the computer which captures graph peaks of gas concentration, measuring the thresholds of volatile sulfur compounds (0-1000 parts per billion), after 8 min. To avoid bias, the examination will be conducted in the morning, and participants will be instructed just to brush with water during this day and not to eat food with garlic, onion and hot spices, not to consume alcohol and not to use antiseptic for 48 h before the oral assessment. They must not have eaten 2 h before the test and must not have had coffee, candies or chewing gum or used oral and personal care products with perfume (aftershave, deodorant, perfume and creams).
## Data collection and management
Microbiological collection will always be performed by the same researcher (CCBdC) supervised by a professor (ACRTH), as described under 'Sputum, saliva and nasal lavage collection for microbiological evaluation'. This material will be processed in the Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Brazil supervised by two professors (MPAM and PLL). The data will be analysed in an Excel worksheet developed for this purpose.
The collection of saliva to analyse inflammatory markers will be performed by CCBdC supervised by ACRTH, and systemic inflammatory markers will be collected through venous puncture by a single trained technician, as described under 'Determination of systemic and saliva inflammatory markers'. This material will be analysed by the same researcher (CCBdC) in the Laboratory of Biophotonics Applied to Health Sciences, Nove de Julho University, São Paulo, Brazil, supervised by two professors (KPSF and RAMF). The data will be analysed in an Excel worksheet developed for this purpose.
The collection of halitosis will be performed by the same researcher (CCBdC) supervised by SKB. The portable device, Oral Chroma, will be used for halitosis evaluation. Procedures will be performed as previously described. [bib_ref] Photodynamic therapy as a novel treatment for halitosis in adolescents: study protocol..., Lopes [/bib_ref] The air will be injected into the device with a single movement. The Oral Chroma will be connected to a computer (with specific software) allowing the capture of a chart with peaks of gas concentration after 8 min. The data will be analysed in an Excel worksheet developed for this purpose.
In order to improve adherence to the study, we will provide interested patients with information about the study results by mail.
Concerning data management, patients will be identified (ID) after randomisation. Every new datum or piece of material stored (records, documents, samples) will receive the same identifier (eg, ID-1A, related to patient 1 on baseline evaluation; ID-1B, related to patient 1 after 3 months of evaluation).
## Ct scan
A CT scan of patients' faces' sinuses for those who have not had a recent one (over 6 months) will be performed. Acquisition of the images will take about 15 min. The CT scan will be used to confirm the diagnosis of the underlying disease (bronchiectasis) and as one of the components for calculating severity criteria (FACED). [bib_ref] Multidimensional approach to non-cystic fibrosis bronchiectasis: the FACED score, Martínez-García [/bib_ref] Variables of the protocol The primary variable will be quantification of P. gingivalis, Ps. aeruginosa and S. aureus. The amount of microorganisms (measured as log) in saliva, sputum and nasal lavage at baseline will be compared with the amount of the same microorganisms after PT and between groups (group 1 and group 2). These outcomes will be classified as (i) better, (ii) no change or (iii) worse. All variables will be presented as mean±SD.
The secondary variables of the study will be quantification of inflammatory cytokines and halitosis. Cytokines will be quantified, ( pg/mL) in saliva and serum. Results will be compared between groups (group 1 and group 2) and with baseline. We expect the reduction in local cytokines to be followed by a decrease in systemic ones.
Halitosis will be quantified by measuring the thresholds of volatile sulfur compounds (hydrogen sulfide, methyl mercaptan and dimethyl sulfide). The gas concentrations will be given in standard units of ppd and ng/10 mL. Halitosis will be compared with baseline after PT and between groups (group 1 and group 2). We expect PT to reduce halitosis.
# Statistical analysis
The primary variable will be the quantification of periodontal pathogens and pulmonary microorganisms, and the secondary variables of the study will be quantification of inflammatory cytokines and halitosis. Dental clinical parameters will be assessed at baseline and 3 months after treatment. The Lilliefors test will be used to test the normality of the data. If the data behave normally, the Student t test (Bioestat 5.3, Pará, Brazil) will be used to compare continuous and dependent variables of group 1 and group 2 (quantification of microorganisms, inflammatory cytokines and halitosis). If the data do not behave normally, the Friedman test will be used. p<0.05 will be considered significant. The χ 2 test will be used to compare categorical variables between the groups (demographic data) in the comparison between baseline and 3 months. The results will be expressed as mean±SD.
Strategies for achieving adequate participant enrolment Participants will be recruited in InCor-FMUSP, the hospital where they undergo semi-annual medical care. A researcher (EHP) will perform the periodontal examination in an appropriate room (inside the hospital) and will invite them for PT (after 1 week) in the Dental Clinic of Nove de Julho University, right after periodontal diagnosis. They will be instructed about oral health and be given a set of instructions for performing daily OH.
# Discussion
Bronchiectasis is characterised as a serious lung disease with severe exacerbations, recurrent infections and frequent hospital admissions.The association between oral biofilm control and severity of lung diseases such as COPD has been demonstrated. [bib_ref] Association between periodontitis and chronic obstructive pulmonary disease in a Chinese population, Si [/bib_ref] Clinical trials are necessary to confirm whether this premise applies to bronchiectasis. In this study, the conventional PT (gold standard) will be compared with treatment with PDT. This alternative treatment was chosen because antibiotics are used more frequently in patients with bronchiectasis than in the general population. [bib_ref] Interventions for bronchiectasis: an overview of Cochrane systematic reviews, Welsh [/bib_ref] The PDT would provide an additional action, which does not select resistant microorganisms and does not have contraindications. [bib_ref] Photodynamic therapy in dentistry, Konopka [/bib_ref] Several microorganisms, such as T. forsythia, Tr. denticola, P. gingivalis, H. influenza, Ps. aeruginosa, S. aureus and Strep. pneumonia, have been identified in the tracheal aspirate. [bib_ref] 16S rDNA-based metagenomic analysis of dental plaque and lung bacteria in patients..., Tan [/bib_ref] Thus, any of them could have been selected as a microorganism indicator of bacterial contamination of saliva, nasal lavage and sputum. We chose to quantify three microorganisms of medical interest. S. aureus was chosen because of its high resistance to antimicrobials, [bib_ref] Microbiologic follow-up study in adult bronchiectasis, King [/bib_ref] and Ps. aeruginosa was chosen because of its biofilm-forming ability, high resistance to antimicrobials, and high prevalence in bronchiectasis exacerbation. The periodontal pathogen P. gingivalis was selected because of its well-established relationship with periodontitis and its association with extra oral infections. [bib_ref] Efficacy of antimicrobial photodynamic therapy in the management of chronic periodontitis: a..., Betsy [/bib_ref] The outcomes of this protocol will determine the efficacy of PT in reducing the total amount of microorganisms in saliva, sputum and nasal lavage. These outcomes will be classified as (i) better, (ii) no change or (iii) worse than baseline. These results will be confronted with a possible reduction in systemic and local inflammation and halitosis after 3 months. Greater improvement in all parameters measured over 3 months is expected for group 1 and group 2. It is expected that group 2 (PDT group) will have better results for periodontal parameters than PT alone after 3 months. [bib_ref] Adjunctive photodynamic therapy to non-surgical treatment of chronic periodontitis: a systematic review..., Sgolastra [/bib_ref] Regarding the other outcomes, such as local and systemic inflammatory cytokines, a slight decrease in local cytokines is expected after PT. Since these patients have a systemic inflammation per se, the reduction in local inflammation is expected to be followed by a slight systemic inflammation. In relation to the halitosis outcome, this is expected to be partly solved after PT, since PD is one of the most probable causes. As mentioned above, to date, it has not been tested if PT alone can eliminate halitosis in these patients or if the pulmonary disease itself induces halitosis.
Maintaining all these outcomes for a 1-2 year follow-up will probably improve lung disease, as demonstrated in patients with COPD. 51 These patients will be followed ( periodontal maintenance) for 2 years and future analyses will be possible. Contributors EHP: made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. PLL: made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. CCBdC: made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. SDC: involved in drafting the manuscript and revising it critically for important intellectual content. FDCL: involved in drafting the manuscript and revising it critically for important intellectual content. RS: gave final approval of the version to be published; RA: involved in drafting the manuscript and revising it critically for important intellectual content. KPSF: involved in drafting the manuscript and revising it critically for important intellectual content. MPAM: gave final approval of the version to be published. SKB: involved in drafting the manuscript and revising it critically for important intellectual content. RAMF: involved in drafting the manuscript and revising it critically for important intellectual content. ACRTH: gave final approval of the version to be published.
Competing interests None declared.
## Patient consent obtained.
Ethics approval Research Ethics Committee of Universidade Nove de Julho. The results of this study will be presented at international conferences and published in a peer-reviewed journal. Individual data of patients will be protected in order to protect their confidentiality. The identity of the patients will not be disclosed. All data related to the study will be saved in a computer at the Biophotonic Applied to Health Sciences laboratory. A backup will be copied in a cloud locked file with limited access. All laboratory specimens, data collection, reports and forms will be identified by an ID code to maintain confidentiality. All records containing personal identifiers (eg, informed consent forms) will be stored separately from study records and will be identified by code numbers. Only principal investigators (authors of this paper) will be given full access to the cleaned datasets. Project datasets will be saved on the computer of Biophotonics Applied to Health Sciences, Universidade Nove de Julho, UNINOVE, São Paulo, Brazil and all datasets will be protected by password.
[fig] Figure 1: Flow chart summarising the experimental design. [/fig]
[fig] Figure 2: Schematic [/fig]
[fig] Figure 3: Timeline showing the sequence of procedures and sample collection. Pinto EH, et al. BMJ Open 2016;6:e010564. doi:10.1136/bmjopen-2015-010564 [/fig]
|
A rare association of crossed fused renal ectopia
Background: Thrombocytopenia and absent radius syndrome (TAR) is a rare genetic disorder. It is an autosomal recessive disorder characterised by radial aplasia and thrombocytopenia that may have additional anomalies. We report a case of TAR syndrome with crossed fused renal ectopia. This anomaly has not been previously reported in association with TAR syndrome.Case presentation:A 24 years old female with Thrombocytopenia and absent radius syndrome admitted with pelvic fracture was investigated for recurrent urinary tract infections. Abdominal ultrasonography could not visualise the kidney on right side. Further extensive investigations in the form of intravenous urography (IVU), Magnetic resonance imaging (MRI) and renal isotope scans revealed a crossed fused renal ectopia.Conclusion:This report describes the new finding of a crossed fused renal ectopia associated with TAR syndrome that has not been reported before in the literature. Ectopic kidneys have increased susceptibility to develop complications like urinary infections, urolithiasis, and abdominal mass. There is a reported case of TAR syndrome with renal anomaly that developed Wilm's tumor. Finding of crossed fused renal ectopia warrants complete urologic investigation to rule out surgically correctable pathology in the urinary tract.
# Background
TAR syndrome is an autosomal recessive disorder with constant findings of thrombocytopenia and bilateral absence of radii with presence of thumbs . Many of the congenital anomalies have been described such as ulnar hypoplasia, malformed humeri, leucocytosis, tetralogy of fallot, atrial septal defect, ventricular septal defect and milk protein allergy.There are only three reports of renal anomalies associated with TAR syndrome.
Bradshaw et al. [bib_ref] Thrombocytopenia and absent radii syndrome associated with horseshoe kidney, Bradshaw [/bib_ref] reported a patient with TAR syndrome and horse shoe kidney, Chappel [bib_ref] Transposition of external genitalia in a case with Fanconi type deformity, Chappel [/bib_ref] reported TAR syndrome with penoscrotal transposition, i.e., insertion of penis below scrotum , Fivush et al. [bib_ref] Thrombocytopenia absent radii syndrome associated with renal insufficiency, Fivush [/bib_ref] reported TAR syn-drome with bilateral hypoplastic kidneys and poor renal function. Crossed fused renal ectopia is a very rare anomaly in which both kidneys are located on the same side and are fused. The autopsy incidence of renal ectopia is 5.9%.
We report the first patient of TAR syndrome associated with crossed fused renal ectopia and discuss the pathogenetic explanation for crossed fused renal ectopia.
## Case presentation
A 24 years old female with Thrombocytopenia absent radius syndrome (TAR) was admitted with fracture of pelvis in our department. Diagnosis of TAR syndrome had been made on the basis of radiographic findings of absent radii, radially deviated hands, presence of thumbs and a low platelet count. There was no family history of consanguinity or congenital malformations. USG was performed for recurrent urinary tract infections which revealed absent kidney on the right side. Further investigations including IVU, MRI scan and renal isotope scans revealed a crossed fused renal ectopia (fig 2, 3, 4).
# Conclusion
TAR syndrome was first described by Hauser in 1948. In 1956 Gross et al. [bib_ref] Congenital hypoplastic thrombopenia with aplasia of the radius, a syndrome of multiple..., Gross [/bib_ref] described it as a group of limb abnormalities including absent radii, ulnar hypoplasia and malformed humeri with hypo megakaryocytic thrombocytopenia. Genitourinary anomalies have been described in three cases of Tar Syndrome [ [bib_ref] Transposition of external genitalia in a case with Fanconi type deformity, Chappel [/bib_ref] and [bib_ref] Thrombocytopenia absent radii syndrome associated with renal insufficiency, Fivush [/bib_ref] ]. One infant had bilateral hypoplastic kidneys with poor renal function. The second child had transposition of external genitalia. The third child had TAR syndrome with a horse shoe kidney. Our patient represents fourth such case but with crossed fused renal ectopia.
Renal fusion anomalies can be categorized into 2 varieties: (1) horseshoe kidney and its variants and (2) crossed fused ectopia. Horseshoe kidney is probably the most common fusion anomaly. The term horseshoe kidney refers to the appearance of the fused kidney, which results from fusion at one pole. Horseshoe kidney is differentiated from crossed fused ectopia in which both fused kidneys lie on one side of the spine and the ureter of the crossed kidney crosses the midline to enter the bladder.
Crossed fused renal ectopia is a rare renal anomaly with incidence of 1:1300 -1: 7500 [bib_ref] WB Saunders: Philadelphia, Campbell [/bib_ref]. The formation of metanephros -the developing kidney depends on the presence of both the ureteric bud and metanephric blastema. The ureteric bud arises from the lower portion of wolffian duct and the metanephric blastema is a meso-derm tissue. Both these tissues migrate towards each other and merge to form the kidney and the urinary tract. Over bending and rotation of the caudal end of the embryo prevents the ureteric bud from merging with the ipsilateral metanephric blastema and thus is attracted towards the now more closer contralateral side [bib_ref] Fused kidneys: morphologic study and theory of Embryogenesis, Cook [/bib_ref].There is an increased prevalence of crossed renal ectopia in patients with scoliosis which supports the above theory [bib_ref] Anomalies of the genitourinary tract associated with congenital scoliosis and congenital kyphosis, Vitko [/bib_ref].
With growth the kidneys gradually ascend to be in the abdomen and away from the midline. Since the under ascent is more common than the over ascent, the ectopic kidneys are more commonly found in the pelvis or the lower abdomen. In most cases the fusion is between the lower pole of the orthotopic kidney and the upper pole of the ectopic kidney, it is usually the left side kidney which crosses to the right. In a recent clinical study of 34 patients with TAR syndrome Greenhalgh et al. found that renal anomalies occurred in seven cases (23%) [bib_ref] RA: Thrombocytopenia-absent radius syndrome: a clinical genetic study, Greenhalgh [/bib_ref].
## Intravenous urogram showing crossed fused renal ectopia
Most of the renal anomalies are incidental findings and the ectopic kidneys have a high incidence of stone formation. Although most of the patients with crossed fused renal ectopia are usually asymptomatic, they do present with increased susceptibility to develop complications like urinary infections, urolithiasis, and abdominal mass. There are reported cases of renal cell carcinoma and Wilm's tumor associated with crossed fused renal ectopia [bib_ref] Wilm's tumor in crossed fused renal ectopia, Redman [/bib_ref] [bib_ref] Ballestero Diego R: Renal cell carcinoma in crossed fused renal ectopia, Tubet [/bib_ref]. There is a reported case of TAR syndrome with renal anomaly that developed Wilm's tumor. [bib_ref] RA: Thrombocytopenia-absent radius syndrome: a clinical genetic study, Greenhalgh [/bib_ref] Finding of crossed fused renal ectopia warrants complete urologic investigation to rule out surgically correctable pathology in the urinary tract.
In summary, this report describes the new finding of a crossed fused renal ectopia associated with TAR syndrome. This association has not been reported before in the literature.
## Competing interests
The author(s) declare that they have no competing interests.
## Mri scan showing a crossed fused kidney on the left side
[fig] Figure 2: Intravenous Urogram showing crossed fused renal ectopia. AP radiograph of the upper limb showing absent radius with radially deviated hand Figure 1 AP radiograph of the upper limb showing absent radius with radially deviated hand. [/fig]
[fig] Figure 4: MRI Scan showing a crossed fused kidney on the left side. Isotope scan showing an ectopic kidney on the left side Figure 3 Isotope scan showing an ectopic kidney on the left side.Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours -you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv. [/fig]
|
Large Benefit from Simple Things: High-Dose Vitamin A Improves RBP4-Related Retinal Dystrophy
Citation: Smirnov, V.M.; Wilmet, B.; Nassisi, M.; Condroyer, C.; Antonio, A.; Andrieu, C.; Devisme, C.; Sancho, S.; Sahel, J.-A.; Zeitz, C.; et al. Large Benefit from Simple Things: High-Dose Vitamin A Improves RBP4-Related Retinal Dystrophy. Int. J. Mol. Sci. 2022, 23, 6590. https://
# Introduction
Inherited retinal diseases are a heterogeneous group of disorders, the most common of which-retinitis pigmentosa-progresses towards blindness with limited therapeutic options. Some rare forms of IRD, linked with inborn defects of small molecule metabolism can benefit from metabolic and/or dietary treatment [bib_ref] Treatable Forms of Retinitis Pigmentosa Associated with Systemic Neurological Disorders, Grant [/bib_ref] [bib_ref] Treatment of Retinal and Choroidal Degenerations and Dystrophies: Current Status and Prospects..., Weleber [/bib_ref]. For instance, vitamin A and E intake improves vision in patients with abetalipoproteinemia (OMIM# 200100), a hereditary defect of fat-soluble vitamin absorption and transport [bib_ref] Long-Term Assessment of Combined Vitamin A and E Treatment for the Prevention..., Chowers [/bib_ref]. Dietary arginine restriction and vitamin B6 intake can reduce blood ornithine level and slow retinal degeneration in patients with gyrate atrophy (OMIM#258870) [bib_ref] A Review of Treatment Modalities in Gyrate Atrophy of the Choroid and..., Balfoort [/bib_ref] [bib_ref] Resolution of Cystoid Macular Edema Following Arginine-Restricted Diet and Vitamin B6 Supplementation..., Casalino [/bib_ref]. Low phytanic acid diet may slow retinal degeneration progression rates in adult Refsum disease (OMIM#266500) [bib_ref] Adult Refsum Disease: A Form of Tapetoretinal Dystrophy Accessible to Therapy, Rüether [/bib_ref].
Retinol-binding protein 4 (RBP4, Uniprot#Q5VY30) is a major blood transporter of retinol from hepatocyte to target organs. Pathogenic variants in RBP4 are associated with both ocular developmental abnormalities and retinal degeneration (OMIM#615147). Ocular developmental abnormalities range from a mild iris coloboma to micro-and anophthalmia. Retinal degeneration consists of a rod-cone dystrophy, also known as retinitis pigmentosa. We report here that high-dose vitamin A is able to improve visual function in a new case of RBP4-associated retinopathy.
# Results
## Detailed case description
A male patient of Algerian ancestry was initially assessed at age 12 years. He had decreased visual acuity and progressive night blindness since early childhood. Best corrected visual acuity was 20/32 for both eyes with spectacle correction −0.50(−1.0)5 - in the right eye (RE) and −0.75(−0.25)170 - in the left eye .
Goldman kinetic visual field tested on V4e, V1e, and V1e targets was normal while the III1e and smaller targets were not perceived by the patient [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] in Supplementary Data). Automated static perimetry showed a pericentral ring scotoma. Dark-adapted responses (DA0.01) of ISCEV standard full-field electroretinogram (ffERG) were undetectable while DA3.0 and DA10.0 revealed severely reduced and delayed responses; light-adapted responses (LA3.0 and LA3.0 flicker) were severely reduced with implicit time shift in keeping with generalized rod-cone dysfunctionin Supplementary Data). Full Stimulus Threshold (FST) revealed severely reduced threshold for the white stimulus [fig_ref] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake [/fig_ref] in Supplementary Data). Fundus examination revealed numerous white dots scattered over the mid and far periphery with no pigmentary or atrophic retinal changes [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] and in Supplementary Data). On infrared reflectance (IRR) imaging, both maculae were granular [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref]. Increased image averaging on short-wave fundus autofluorescence (SWAF) revealed an ellipsoid-shaped ring of increased autofluorescence around the fovea on an overall hypoautofluorescent background [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref]. Near-infrared fundus autofluorescence (NIRAF) imaging also showed a reduced signal [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref]. Spectral domain optical coherence tomography (SD-OCT) centered on the fovea revealed an hyporeflective irregular ellipsoid zone (EZ) [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref]. The outer nuclear layer (ONL) thickness was preserved with an unusual hyper reflective band on both sides of the fovea [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] , yellow arrows). SD-OCT performed through the peripheral white dots revealed hyperreflective dots above the retinal pigment epithelium (RPE), interrupting the EZ [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] , white arrows).
A general examination was performed to detect any vitamin A deficiency-related alterations. The only skin issue was subtle acne vulgaris on the forehead. Past medical history was unremarkable and the patient declined any dietary restrictions.
## Genetic and functional studies
The family consisted of unaffected first-degree-cousin parents of Algerian descent and two unaffected sisters. Targeted NGS identified a novel homozygous nonsense variant c.255G >A, p.(Trp85*) in RBP4 which co-segregated with disease in this family. This variant was classified as pathogenic Ia (PVS1, PP1-S, PM2) in accordance with ACMG standards [bib_ref] Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus..., Richards [/bib_ref]. Western blot analysis revealed the absence of the RBP4 protein band in the serum of the patient. Blood retinol level was also undetectable. A general examination was performed to detect any vitamin A deficiency-related alterations. The only skin issue was subtle acne vulgaris on the forehead. Past medical history was unremarkable and the patient declined any dietary restrictions.
## Genetic and functional studies
The family consisted of unaffected first-degree-cousin parents of Algerian descent and two unaffected sisters. Targeted NGS identified a novel homozygous nonsense variant c.255G > A, p.(Trp85*) in RBP4 which co-segregated with disease in this family. This variant was classified as pathogenic Ia (PVS1, PP1-S, PM2) in accordance with ACMG standards [bib_ref] Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus..., Richards [/bib_ref]. Western blot analysis revealed the absence of the RBP4 protein band in the serum of the patient. Blood retinol level was also undetectable.
## Treatment and follow-up
After discussion with the patient and his parents and their informed consent, highdose oral vitamin A was initiated with retinol palmitate, 50,000 UI twice a week. Liver function was monitored. Five months after initiation of the treatment, the patient reported
## Treatment and follow-up
After discussion with the patient and his parents and their informed consent, highdose oral vitamin A was initiated with retinol palmitate, 50,000 UI twice a week. Liver function was monitored. Five months after initiation of the treatment, the patient reported a significant subjective night vision improvement and a better tolerance of light-to-dark transitions. There was an improvement in FST at this time point (+25% in RE and 40% in LE, respectively) sustained at 1-year follow-up [fig_ref] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake [/fig_ref] and [fig_ref] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake [/fig_ref] in Supplementary Data), while visual acuity, ERG, and retinal imaging (more specifically, white dots, ONL and EZ aspects) remained unchanged. We also observed a marked visual field improvement: III1e and II1e targets became perceived by the patient [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] in Supplementary Data). There were no signs of general retinol toxicity and liver function remained normal.
## Treatment and follow-up
After discussion with the patient and his parents and their informed consent, highdose oral vitamin A was initiated with retinol palmitate, 50,000 UI twice a week. Liver function was monitored. Five months after initiation of the treatment, the patient reported a significant subjective night vision improvement and a better tolerance of light-to-dark transitions. There was an improvement in FST at this time point (+25% in RE and 40% in LE, respectively) sustained at 1-year follow-up [fig_ref] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake [/fig_ref] and [fig_ref] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake [/fig_ref] in Supplementary Data), while visual acuity, ERG, and retinal imaging (more specifically, white dots, ONL and EZ aspects) remained unchanged. We also observed a marked visual field improvement: III1e and II1e targets became perceived by the patient [fig_ref] Figure 1: Multimodal retinal imaging [/fig_ref] in Supplementary Data). There were no signs of general retinol toxicity and liver function remained normal.
# Discussion
The visual system is highly dependent upon retinol supply. Retinoids are essential for normal ocular development as a whole and for the maintenance of a normal tissue structure. Abnormalities in nutritional supply, storage, transport, delivery, or metabolism of retinoids are linked with a broad spectrum of developmental and degenerative diseases of the eye [bib_ref] Phenotype in Retinol Deficiency Due to a Hereditary Defect in Retinol Binding..., Seeliger [/bib_ref]. . Full stimulus threshold before and after high-dose vitamin A intake. Mean FST ± SD at first assessment, at M + 5 and M + 12 follow-up. A significant improvement in FST was observed at M + 5 (25 and 40% decrease) and was sustained at M + 12 (123 et 215% in RE and LE, respectively). In green, normal FST range [bib_ref] Full-Field Stimulus Testing (FST) to Quantify Visual Perception in Severely Blind Candidates..., Roman [/bib_ref] [bib_ref] Psychophysical Assessment of Low Visual Function in Patients with Retinal Degenerative Diseases..., Klein [/bib_ref].
# Discussion
The visual system is highly dependent upon retinol supply. Retinoids are essential for normal ocular development as a whole and for the maintenance of a normal tissue structure. Abnormalities in nutritional supply, storage, transport, delivery, or metabolism of retinoids are linked with a broad spectrum of developmental and degenerative diseases of the eye [bib_ref] Retinoids and Retinal Diseases, Kiser [/bib_ref].
RBP4 is the major plasma carrier of retinol and retinoids. It forms a heterohexameric complex with transthyretin in the blood, a characteristic which protects small molecules of RBP4 (21k Da) from glomerular filtration. There are two different pathways for retinoids to reach their target tissues. The main pathway depends upon RBP4, which is complexed to retinol (holo-RBP4) and then recognized and bond to a membrane retinoid receptor STRA6, located at the basal side of the RPE. STRA6 allows the internalization of retinol [bib_ref] A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin..., Kawaguchi [/bib_ref]. The minor pathway is under the control of the scavenger class B type I receptor (Sr-BI) which enables the absorption of the protein-free fraction of retinoids transported within circulating lipoprotein complexes and chylomicrons. In the absence of RBP4, this second low-rate route could be sufficient to provide retinoids to all other tissues except for the RPE, leading to a retinal disease [bib_ref] Understanding the Physiological Role of Retinol-Binding Protein in Vitamin A Metabolism Using..., Quadro [/bib_ref] [bib_ref] Impaired Retinal Function and Vitamin A Availability in Mice Lacking Retinol-Binding Protein, Quadro [/bib_ref] [bib_ref] Transgenic Mice Over-Expressing RBP4 Have RBP4-Dependent and Light-Independent Retinal Degeneration, Du [/bib_ref].
Pathogenic variants in RBP4 are responsible for rod-cone dystrophy associated with various degrees of microphthalmia, coloboma, and comedogenic acne. Only few patients harboring RBP4 defects have been reported to date [bib_ref] Phenotype in Retinol Deficiency Due to a Hereditary Defect in Retinol Binding..., Seeliger [/bib_ref] [bib_ref] A Novel Homozygous c.67C>T Variant in Retinol Binding Protein 4 (RBP4) Associated..., Cehajic-Kapetanovic [/bib_ref] [bib_ref] Exome Analysis Identified a Novel Mutation in the RBP4 Gene in a..., Cukras [/bib_ref] [bib_ref] Vitamin A Deficiency Due to Bi-Allelic Mutation of RBP4: There's More to..., Khan [/bib_ref]. Our patient presents a mild phenotype unlike patients harboring RBP4 gene defects reported previously which could be explained in part by his young age (12 years) compared to the late adult cases with advanced retinal degeneration reported in the literature. The observed white dot retinopathy in our patient could be an early feature of progressive inherited retinal degeneration.
In contrast, our patient had a very unusual phenotype for RBP4 deficiency. His ocular findings were reminiscent of fundus albipunctatus (FA, OMIM#136880) due to the presence of night blindness, numerous peripheral white dots, and no signs of retinal degeneration. FA clinical phenotype is typically associated with pathogenic variants in RDH5 (OMIM#601617). However, retinal white dots in our patient were less numerous and clearly not organized in a network pattern as in RDH5-retinopathy. The ERG was also different showing a generalized severe rod-cone dysfunction distinct from the Riggs-type ERG [bib_ref] Electroretinography in Cases of Night Blindness, Riggs [/bib_ref] classically associated with RDH5-retinopathy including normal cone function and cone-dominated dark-adapted responses. Retinol-dehydrogenase 5 is a visual cycle enzyme oxidizing 11-cis-retinol into 11-cis-retinal. The lack of enzyme activity leads to 11-cis and 13-cis retinyl esters accumulation which are thought to be the origin of the white dots in FA [bib_ref] Disruption of the 11-Cis-Retinol Dehydrogenase Gene Leads to Accumulation of Cis-Retinols and..., Driessen [/bib_ref] [bib_ref] Characterization of a Dehydrogenase Activity Responsible for Oxidation of 11-Cis-Retinol in the..., Jang [/bib_ref].
Another retinal disease very close to our patient's presentation is retinitis punctata albescens (RPA, OMIM#136880 or Bothnia dystrophy, OMIM#607475). White dots are usually present in the early stages of the disease and are due to an accumulation of all-transretinyl esters in the retinal pigment epithelium secondary to an impairment of the cellular retinaldehyde-binding protein 1 (CRABP1, Uniprot#P29762), slowing down the isomerization of all-trans-retinyl esters in 11-cis-retinol. Pathogenic variants in RLBP1, encoding CRABP1, are responsible for this phenotype. As its name implies, RPA is a progressive retinal degeneration. Unlike in our patient, RPA is characterized by moderate narrowing of the retinal vasculature, optic disc pallor, pigmentary changes, and peripheral scalloped areas of chorioretinal atrophy [bib_ref] Genotype-Phenotype Correlations in Bothnia Dystrophy Caused by RLBP1 Gene Sequence Variations, Burstedt [/bib_ref] [bib_ref] Early-Onset Foveal Involvement in Retinitis Punctata Albescens With Mutations in RLBP1, Dessalces [/bib_ref]. ERG responses in RPA are, however, closer to those of our patient, with severely affected rod responses and more preserved cone responses. A limited recovery of ERG responses after prolonged dark adaptation is also reported in RPA [bib_ref] Effects of Prolonged Dark Adaptation in Patients with Retinitis Pigmentosa of Bothnia..., Burstedt [/bib_ref] as in RDH5-retinopathy. This was unfortunately not tested in our patient. White dots have also been described in RHO (OMIM#180380) [bib_ref] Retinitis Punctata Albescens Associated With the Arg135Trp Mutation in the Rhodopsin Gene, Souied [/bib_ref] , PRPH2 (OMIM#179605) [bib_ref] A Null Mutation in the Human Peripherin/RDS Gene in a Family with..., Kajiwara [/bib_ref] , LRAT (OMIM#604863) [bib_ref] A Homozygous Frameshift Mutation in LRAT Causes Retinitis Punctata Albescens, Littink [/bib_ref] [bib_ref] Long-Term Follow-Up of Retinal Degenerations Associated With LRAT Mutations and Their Comparability..., Talib [/bib_ref] , and RPE65 (OMIM#180069) [bib_ref] Fundus Albipunctatus Associated with Compound Heterozygous Mutations in RPE65, Schatz [/bib_ref] [bib_ref] RPE65-Mutation Associated Fundus Albipunctatus with Cone Dystrophy, Ramtohul [/bib_ref] gene defects, but the phenotype in these cases is more severe and progressive; thus, delineating it from our patient's clinical picture.
Functional and morphological retinal changes in our patient were close to those reported in vitamin A deficient retinopathy (VAD). Functionally, generalized photoreceptor dysfunction with rod responses being more altered than cone responses is also characteristic for VAD [bib_ref] Improvement in Visual Function and Fundus Findings for a Patient with Vitamin..., Apushkin [/bib_ref] [bib_ref] Fundus White Spots and Acquired Night Blindness Due to Vitamin A Deficiency, Genead [/bib_ref]. White retinal dots can be a feature in VAD [bib_ref] Improvement in Visual Function and Fundus Findings for a Patient with Vitamin..., Apushkin [/bib_ref] [bib_ref] Fundus White Spots and Acquired Night Blindness Due to Vitamin A Deficiency, Genead [/bib_ref] , but they are somewhat different in shape (indistinct borders) and hypoautofluorescent on SWAF [bib_ref] Retinal Structure in Vitamin A Deficiency as Explored with Multimodal Imaging, Aleman [/bib_ref]. Our case also presented intriguing finding with an additional hyperreflective band in the parafoveal region on OCT. We hypothesize that this alteration could be a partial duplication of the outer plexiform layer and may be related to the important role of retinoids in retinal development [bib_ref] Retinoic Acid Signaling in Mammalian Eye Development, Cvekl [/bib_ref].
Another inherited form of retinal degeneration linked with an altered absorption and biodistribution of vitamin A is abetalipoproteinemia (or Bassen-Kornzweig syndrome). Biallelic gene defects in MTTP (OMIM#157147), encoding Mitochondrial Triglyceride Transfer Protein (Uniprot#P55157) lead to impaired assembly and secretion of plasma lipoproteins that contain apolipoprotein B (very low-and low-density lipoproteins and chylomicrons). Lipoproteins facilitate absorption and carry a free fraction of fat-soluble vitamins (A, D, E, K). Upon reduced MTTP activity, there is a reduced retinol and tocopherol absorption, transport, and delivery to the target organs, including the eye, which results in retinal degeneration. Early treatment with high-dose vitamins A and E in patients with abetalipoproteinemia resulted in improvement in their retinal function and a slower retinal degeneration progression rate [bib_ref] Long-Term Assessment of Combined Vitamin A and E Treatment for the Prevention..., Chowers [/bib_ref] [bib_ref] Abetalipoproteinaemia in Adults: Role of Vitamin Therapy, Macgilchrist [/bib_ref] [bib_ref] Combined Vitamin A and E Therapy Prevents Retinal Electrophysiological Deterioration in Abetalipoproteinaemia, Bishara [/bib_ref].
Oral administration of high-dose retinol palmitate has already shown to raise the level of free plasma retinol and retinyl esters in RBP4-deficient patients [bib_ref] Biochemical but Not Clinical Vitamin A Deficiency Results from Mutations in the..., Biesalski [/bib_ref] but no visual outcome had been reported so far. Nevertheless, experimental studies report that RBP -/mice are able to use alternative RBP-independent pathways for retinol supply to the retina with phenotypic rescue provided by a retinol-sufficient diet [bib_ref] Impaired Retinal Function and Vitamin A Availability in Mice Lacking Retinol-Binding Protein, Quadro [/bib_ref]. In order to compensate for the lack of RBP4-related retinol transport and attempt to enhance the free fraction of retinoids delivered via the slow RBP4-independent pathway, we decided to prescribe high doses of retinol to our patient. After five months of high-dose oral vitamin A, the patient reported subjective improvement in dimly lighted environments which was supported by FST and kinetic perimetry changes from baseline. This effect further improved after one year follow-up.
Adverse and toxic effects of long-term high retinol intake are numerous including bone toxicity (hypercalcemia and osteoporosis), neurotoxicity (intracranial hypertension), and liver toxicity (liver enlargement, cirrhosis) [bib_ref] Evaluation of Vitamin A Toxicity, Hathcock [/bib_ref]. Saturation of the RBP4-independent pathway of retinol delivery could also be detrimental for xanthophyll pigment uptake by the retina, as this slow pathway is competitive between retinol and lutein/zeaxanthin transport [bib_ref] Xanthophylls Are Preferentially Taken up Compared with β-Carotene by Retinal Cells via..., During [/bib_ref]. However, toxicity did not occur in patients with abetalipoproteinemia on prolonged high-dose retinol treatment, as the overall blood levels remained low [bib_ref] Long-Term Assessment of Combined Vitamin A and E Treatment for the Prevention..., Chowers [/bib_ref]. Our patient did not experience any adverse effect after 1 year of retinol intake. Liver enzymes and blood calcium were normal. Long-term follow-up with close monitoring for vitamin A tolerability will determine whether high doses of retinol are able to prevent retinal degeneration.
# Materials and methods
## Clinical studies
The patient was clinically investigated at the national reference center for rare ocular diseases REFERET of the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts as previously described [bib_ref] An Unusual Retinal Phenotype Associated With a Novel Mutation in RHO, Audo [/bib_ref]. Briefly, best-corrected visual acuity (BCVA), refractive error, slit-lamp biomicroscopy, static and kinetic visual fields, full-field electroretinogram according to the standards of International Society for Clinical Electrophysiology of Vision(Espion, Diagnosys LLC, Lowell, MA), fundus photography, spectral domain optical coherence tomography (Spectralis OCT, Heidelberg Engineering, Inc., Heidelberg, Germany), infrared and short-wavelength autofluorescence (Heidelberg Retinal Tomograph, Heidelberg Engineering, Inc.) were performed. Full stimulus threshold was assessed using achromatic full-field stimuli (Diagnosys Espion system, Diagnosys LLC, Lowell, MA, USA) as previously described [bib_ref] Full-Field Stimulus Testing (FST) to Quantify Visual Perception in Severely Blind Candidates..., Roman [/bib_ref] [bib_ref] Psychophysical Assessment of Low Visual Function in Patients with Retinal Degenerative Diseases..., Klein [/bib_ref].
# Genetic analysis
Blood samples from the index case and from his parents were collected for genetic research and genomic DNA was extracted as previously reported [bib_ref] Novel C2orf71 Mutations Account for ∼1% of Cases in a Large French..., Audo [/bib_ref]. These DNA samples were stored and obtained from the NeuroSensCol DNA bank, for research in neuroscience (PI: JA Sahel, co-PI I Audo, partner with CHNO des Quinze-Vingts, Inserm and CNRS, certified NFS96-900). Targeted next generation sequencing (NGS) was performed in collaboration with an external company (IntegraGen, Evry, France) [bib_ref] Development and Application of a Next-Generation-Sequencing (NGS) Approach to Detect Known and..., Audo [/bib_ref]. The RBP4 (MIM#180250) variant selected after NGS was validated in the index case and relatives by Sanger sequencing (refseq: NM_001323517.1, primer sequences and conditions available on demand).
## Western blot
Western blot identification of RBP4 was performed as described in a previously published protocol [bib_ref] Shortcomings in Methodology Complicate Measurements of Serum Retinol Binding Protein (RBP4) in..., Graham [/bib_ref]. Loading control was performed using anti-transferrin monoclonal antibody (Abcam, Discovery Drive, Cambridge Biomedical Campus, Cambridge, UK, ab109503).
# Conclusions
We report here the first case of RBP4-related retinopathy manifesting as a whitedot retinopathy whose visual function improved after high-dose oral vitamin A intake. Such treatment should be considered in early stages of RBP4-related retinopathy. Future research will determine whether high level of vitamin A intake is able to reverse the phenotype sustainably.
[fig] Figure 1: Multimodal retinal imaging. (A) Fundus photo, multiple white dots scattered over the midperipheral retina. Note the absence of intraretinal pigment migration and the lack of retinal vessel attenuation. (B) Infrared reflectance image, macular granularity. (C) Short-wavelength fundus autofluorescence imaging obtained with significant averaging due to the generalized reduced autofluorescence, small peri-foveal hyperautofluorescent ring with indistinct borders. (D) Near infrared fundus autofluorescence, small hypoautofluorescent dots. (E) and (F) spectral domain optic coherence tomography (SD-OCT, top: horizontal scan; and bottom: vertical scan), hypo reflective and fragmented ellipsoid zone with no interdigitation zone; preserved outer nuclear layer thickness with an unusual hyper reflective band on both sides of the fovea (yellow arrows). (G) OCT (scan passing through white dots, white arrows), hyperreflective dots above the retinal pigment epithelium with a focal interruption of the ellipsoid zone. [/fig]
[fig] Figure 2: (A) Novel biallelic RBP4 variant co-segregating with the disease. (B) Western blot analysis showing the absence of RBP4 in peripheral blood of our patient (CIC08621) carrying the homozygous nonsense variant in RBP4, compared to an unaffected control and recombinant RPB4. Transferrin is used as serum loading control. (C) RBP4 gene and protein structure. SP-signal peptide domain. Disulfide bonds(22-178, 88-192, 138-147) are not shown. Previously reported and novel (in red) variants linked with inherited retinal degeneration[9,10]. [/fig]
[fig] Figure 3: Full stimulus threshold before and after high-dose vitamin A intake. Mean FST ± SD at first assessment, at M + 5 and M + 12 follow-up. A significant improvement in FST was observed at M + 5 (25 and 40% decrease) and was sustained at M + 12 (123 et 215% in RE and LE, respectively). In green, normal FST range[42,43]. [/fig]
[fig] Supplementary: Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23126590/s1. Author Contributions: Conceptualization, C.Z., J.-A.S. and I.A.; methodology, B.W., M.N., C.C., A.A., C.A., C.D. and S.S.; validation, C.Z., J.-A.S., I.A., B.W., M.N., C.C., A.A., C.A., C.D. and S.S.; formal analysis, V.M.S., C.Z., J.-A.S., I.A., B.W., M.N., C.C., A.A., C.A., C.D. and S.S.; investigation, V.M.S., C.Z., I.A., B.W., M.N., C.C., A.A., C.A., C.D. and S.S.; resources, C.Z., J.-A.S. and I.A.; data curation, V.M.S., C.Z., J.-A.S., I.A., B.W., M.N., C.C., A.A., C.A., C.D. and S.S.; writing-original draft preparation, V.M.S., writing-review and editing, V.M.S., C.Z. and I.A.; supervision, C.Z. and I.A.; funding acquisition, C.Z., J.-A.S. and I.A. All authors have read and agreed to the published version of the manuscript. Funding: LABEX LIFESENSES [reference ANR-10-LABX-65] supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir program [ANR-11-IDEX-0004-0]; IHU FOReSIGHT [ANR-18-IAHU-0001] supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir program; funding from RHU-Light4deaf [ANR-15-RHU-0001]; Foundation Fighting Blindness center grant [C-CMM-0907-0428-INSERM04], BR-GE-0619-0761-INSERM and fellowship award (MN) [CD-CL-0619-0759-INSERM]. Retina France, U UNADEV (Union Nationale des Aveugles et Déficients Visuels) in partnership with ITMO NNP/AVIESAN (alliance nationale pour les sciences de la vie et de la santé) for research on visual disorders. Institutional Review Board Statement: Research procedures adhered to the tenets of the Declaration of Helsinki and were approved by the local Ethics Committee (CPP, Ile de France V, Project number 06693, N•EUDRACT 2006-A00347-44, 11 December 2006). Informed Consent Statement: Prior to testing, written informed consent was obtained from the parents and participant who was under 18. No compensation or incentive was offered to the subject to participate in the study. Data Availability Statement: All data are contained within the article or Materials. [/fig]
|
Adolescent mothers' experiences of the transition to motherhood: An integrative review
a b s t r a c tA woman experiences heightened vulnerability and faces tremendous challenges when transitioning to motherhood. This is exacerbated for young mothers and studies have shown that adolescent mothers experience an increased burden of responsibility during the transition to motherhood. Recent research addressing the experiences of adolescent mothers has increased. However, the current literature on this topic is still fragmented. The aim of this study was to conduct an integrative review of the literature to explore adolescent mothers' experiences of transition to motherhood and identify associated factors. The literature was searched using electronic databases: Medline, Cumulative index to Nursing and Allied Health Literature (CINAHL), ProQuest, Scopus and PubMed. Relevant articles published in English from February 2005 to 2018 were included. Eighteen articles were included in the analysis. Based on this review, factors influencing a successful to transition to motherhood for adolescents included physical problems related to birth and breastfeeding, psychological well-being, ability to care for their baby, social support, education and economic strain and the provision of healthcare. The literature indicated a relationship between social supports and the development of positive maternal identity in the transition period for adolescent motherhood. Future healthcare interventions for adolescent mothers during the transitional period should aim to provide social support and the increase ability of adolescent mothers to manage the physical and psychological challenges of young motherhood, and enhance new mothers' knowledge about caring for babies.
# Introduction
In recent decades, adolescent pregnancy has become an important health issue in many countries. About 16 million girls aged 15e19 years and two million girls under the age of 15 years give birth every year. Having babies during adolescence can have serious consequences for the health of the girl and her infant [bib_ref] Adolescent mothers and older mothers: who is at higher risk for adverse..., Wang [/bib_ref] [bib_ref] Pregnancy and childbirth outcomes among adolescent mothers: a World Health Organization multicountry..., Ganchimeg [/bib_ref]. Adolescent bodies are not yet fully developed and too closely timed pregnancies, can result in girls who marry before the age of 18 years experiencing higher rates of life-threatening or debilitating conditions as a result of pregnancy. The prevalence of other complications among adolescent mothers with severe maternal outcomes was found in one study to be 75% among mothers aged under 15 years and 47.8% among mothers aged 16e19 years. A high risk of stillbirth was found among all adolescent age groups, but the risk was significant only among adolescent mothers aged 16e17 years (AOR ¼ 1.32; 95% CI, 1.11e1.57) [bib_ref] Pregnancy and childbirth outcomes among adolescent mothers: a World Health Organization multicountry..., Ganchimeg [/bib_ref] and there were increased rates of depressive symptoms in the postpartum period [bib_ref] Postpartum depression in adolescent mothers: an integrative review of the literature, Reid [/bib_ref].
The transition to motherhood is a major development in a woman's life [bib_ref] Becoming a mother versus maternal role attainment, Mercer [/bib_ref]. The transition begins during pregnancy and extends into the postpartum period. Settling-in process began as mothers began to feel competent and to develop confidence with their infants. Settling-in is usually achieved around four months following birth [bib_ref] Becoming a mother versus maternal role attainment, Mercer [/bib_ref]. Transition to motherhood for teenage girls has been associated with many additional challenges compared with older new mothers [bib_ref] Iranian pregnant teenage women tell the story of "fast development": a phenomenological..., Mohammadi [/bib_ref]. These girls must manage multiple significant life changes at the same time, the shift to adulthood, a possible marriage, pregnancy, and motherhood [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref].
A woman experiences heightened vulnerability and faces tremendous challenges when transitioning to motherhood [bib_ref] Nursing support of the process of becoming a mother, Mercer [/bib_ref]. This is exacerbated for young mothers and studies have shown that adolescent mothers experience an increased burden of responsibility during the transition to motherhood [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Motherhood as the vehicle for change in Australian adolescent women of preterm..., Sheeran [/bib_ref]. Adolescents often become mothers without the necessary knowledge, skills and resources to deal with early motherhood, which adds stress to their already challenged developmental level [bib_ref] Adolescents' lived experiences of their pregnancy and parenting in a semi-rural community..., Van Zyl [/bib_ref]. Thus, for effective adolescent motherhood transition support, an overview of the experiences of adolescent motherhood during pregnancy and childbirth and the factors associated with the transition to successful adolescent motherhood should be explored.
Recent research addressing the experiences of adolescent mothers has increased around the role of parental support [bib_ref] Experiences of adolescent mothers in Costa Rica and the role of parental..., Copeland [/bib_ref] , the influence of the father's involvement [bib_ref] The influence of father involvement and interparental relationship quality on adolescent mothers'..., Mallette [/bib_ref] , joys and challenges of adolescent mothers [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Joys and challenges of motherhood for Australian young women of preterm and..., Sheeran [/bib_ref] , and health care experiences of adolescent mothers [bib_ref] Treat me but don't judge me: a qualitative examination of health care..., Harrison [/bib_ref] [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. However, the current literature on this topic is still fragmented. The present review focused on identifying factors associated with the transition to motherhood for adolescent mothers. The knowledge gained from this study has importance in enabling healthcare professionals, as well as other professionals, dealing with pregnant teenagers to understand the key aspects of becoming and being a teenage mother, thus offering the teenage mothers they encounter optimum care. Thus, the aim of this paper was to identify associated factors during transition of adolescent mothers based on mothers' experiences.
# Method
# Literature search methods
An integrative approach is used to summarize previous research and to illustrate what is known about a particular phenomenon or health problem comprehensively and [bib_ref] The integrative review: updated methodology, Whittemore [/bib_ref] , in this case, the review focused on factors associated with a positive transition to adolescent motherhood. The literature was searched using electronic databases: Medline, Cumulative index to Nursing and Allied Health Literature (CINAHL), ProQuest, Scopus and PubMed. Keywords were: 'motherhood* OR adolescent mother* OR adolescent motherhood* OR teenage mother* OR teenage motherhood* OR becoming a mother* OR motherhood transition* OR maternal identity*. Inclusion criteria were as follows: (1) written in English language between 2005 and 2018, (2) reported on mothers aged younger than 21 years old, (3) specific to transition to adolescent motherhood, and (4) original research. The search included adolescent motherhood during the transition period, which was defined as the period of pregnancy up to six months postpartum. Exclusion criteria were: (1) Unpublished research due to lack of formal peer-review, (2) reviews of literature or (3) articles reporting on assessment tool development.
## Search outcomes
The search yielded 1288 titles related to adolescent motherhood (see details in [fig_ref] Figure 1: The Flow chart describing details of literature search and selection strategy [/fig_ref]. After duplicates were removed, 1175 manuscripts remained. Thirty-five papers remained after review of titles and abstracts, and these underwent full text review. Assessment of full texts excluded a further 17 papers leaving 18 papers for inclusion in this review.
## Data abstraction and quality assessment
The first author initially identified and reviewed citation on title and abstract. A second author independently reviewed and evaluated the papers selected for inclusion. We extracted the data related to author/s, year of publication, country, aim of the study, design and methods, sample and setting, key findings and evaluated the quality of the study [fig_ref] Table 1: Details of studies in this review [/fig_ref]. Research studies which met the inclusion criteria were assessed for quality using the standard quality assessment criteria of Joanna Briggs Institute dependent on study design. Quantitative studies were evaluated for the quality of: sample recruitment procedure, representativeness and sufficient sample size, inclusion criteria, connection between theoretical framework and hypothesis, instruments validity and reliability, ability to compare groups, suitable statistical data analysis, and generalizability of results. On the other hand, qualitative research studies were evaluated on: methodological congruency with the indicated philosophical view, study question, data collection procedures, data analysis methods, results interpretation, ethical consideration of study process and the basis of existing conclusion.
# Results
A total studies reviewed of 18 studies included 15 qualitative studies, two quantitative studies, and one mixed methods study were included in the review. All articles addressed adolescent motherhood transition. Studies were conducted in a range of countries around the world: four studies were from Australia, three studies were conducted in United States of America, two studies were conducted in Iran, and there was one study each from Canada, United Kingdom, Sweden, Ghana, Hong Kong, Vietnam, Costa Rica, Dominican Republic, and Jamaica. All populations studied were adolescent mothers (aged 14e20 years) during pregnancy and childbirth period with sample sizes from 4 to 126. The study setting varied from clinical to community sites with studies taking place in urban, suburban and rural areas.
Key factors reported in these studies reported by teenage mothers transitioning to motherhood related to physical problems associated with pregnancy, birth and breastfeeding, as well as psychological impacts with "mixed feelings", caring for the baby, heightened need for social support, education and economic strain and health care. A summary of these factors is presented in [fig_ref] Figure 2: Factors that influence the motherhood transition of adolescent women [/fig_ref].
## Physical problems
Transition to becoming a mother is an important event in the life of a woman. Most teenagers were not ready to become mothers and thus experienced multiple challenges [bib_ref] The experience of adolescent Motherhood : an exploratory mixed methods study, Kagawa [/bib_ref]. Three studies reported that adolescent mothers experienced physical problems during childbirth [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref] [bib_ref] Experiences of pregnancy and motherhood among teenage mothers in a suburb of..., Gyesaw [/bib_ref]. Teenagers stated that the pain associated with labor in the postpartum period become an obstacle to caring for the baby [bib_ref] Experiences of pregnancy and motherhood among teenage mothers in a suburb of..., Gyesaw [/bib_ref]. Many young mothers felt incapacitated because of tiredness and pain after giving birth [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. You mothers felt that this prevented them developing a deeper attachment with their newborn [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. Hunter et al. reported that teenage mothers felt tired and helpless after giving birth and thus needed additional assistance from health workers after giving birth [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref].
## Psychological aspects -"mixed feelings"
The majority of studies have shown the effect of psychological aspects during transition of adolescent mothers. Young mothers articulated complex emotions during pregnancy and giving birth and expressed mixed feelings, including joy and worry, about their new responsibility .
Negative feelings to becoming mothers were expressed as feelings of loss and regret related to their past lives and perceived future opportunities. The young women regretted the loss of their adolescence, and loss of continuing education [bib_ref] Experiences of adolescent mothers in Costa Rica and the role of parental..., Copeland [/bib_ref]. This feeling expressed by primiparous adolescent mothers. Two studies found that adolescent mothers felt isolated and alone [bib_ref] How adolescent mothers feel about becoming a parent, Devito [/bib_ref] [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref]. Feeling isolated was a significant experience of participants' lives early in pregnancy. They felt different to their peers and were outcasts at school [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref]. The same feeling was also reported by adolescent mothers after giving birth [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref]. Many adolescent said their peers did not understand their conditions and the demands of meeting the needs of their babies, and thus felt a lack of support and loss of friendships with their peers [bib_ref] How adolescent mothers feel about becoming a parent, Devito [/bib_ref].
Two studies reported psychological distress in adolescent primiparous mothers [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref]. The mothers felt distress because of their perceived unpreparedness for pregnancy [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref] and seven out of the 30 (23%) teenage mothers reportedly experienced depression, and 6,6% suicidal ideation or entertained thoughts of killing themselves [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref]. These feelings occurred with mothers who had an unwanted pregnancy and difficulty in accepting the responsibilities of motherhood [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref].
While several studies described young mothers negative feelings related to transition to motherhood, they also found most adolescent mothers revealed positive feelings associated with their transition [9,10,13,17, 30e33]. The majority of qualitative studies noted that adolescent mothers felt the transition as a life change into adulthood [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref] , they felt motherhood transition as a sense of maturity, and had positive self-perceptions when transforming to be good mothers [bib_ref] Iranian pregnant teenage women tell the story of "fast development": a phenomenological..., Mohammadi [/bib_ref] [bib_ref] Motherhood as the vehicle for change in Australian adolescent women of preterm..., Sheeran [/bib_ref] [bib_ref] One foot wet and one foot dry: transition into motherhood among married..., Klingberg-Allvin [/bib_ref] [bib_ref] Psychosocial factors and maternal wellbeing: an exploratory path analysis, Ngai [/bib_ref]. In this context, adolescent mothers were generally happy to have their babies and felt their lives had changed for the better since becoming mothers [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref]. Becoming a mother provided all the teenage mothers with the opportunity to develop. They described becoming more mature, less egocentric as they learned and grew to becoming good mothers [bib_ref] Motherhood as the vehicle for change in Australian adolescent women of preterm..., Sheeran [/bib_ref] [bib_ref] How adolescent mothers feel about becoming a parent, Devito [/bib_ref] [bib_ref] Psychosocial factors and maternal wellbeing: an exploratory path analysis, Ngai [/bib_ref]. To highlight the complexity, of new mothers feelings, one study found that despite the challenges, adolescent mothers predominantly felt unconditional love and positive feelings about their babies [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref].
## Caring for the baby
Many teenage mothers described that caring for their infants was the most challenging responsibility they had ever faced [bib_ref] Parenthood experiences during the child's first year: literature review, Nystrom [/bib_ref]. Three of the qualitative studies were concerned with mothers' care for their babies. The impeding factors maternal role competence included a lack of personal knowledge and experiences of infant care [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] and contradicting information from various sources [bib_ref] Chinese primiparous women's experiences of early motherhood: factors affecting maternal role competence, Ngai [/bib_ref]. However, Ngai et al. explored factors affecting maternal role competence, finding factors facilitating maternal role competence included positive experiences of infant care, well-being of the infant, success in breastfeeding and availability of social support. However, Similarly, Furthermore, one core essential characteristic of the competent mother is commitment towards caring for the emotional and physical wellbeing of the child [bib_ref] Chinese primiparous women's experiences of early motherhood: factors affecting maternal role competence, Ngai [/bib_ref].
## Social support
One important aspect for adolescent mothers is social support. The most important sources of social support were found to be from their parents, especially the new mother's mother. Social support from their partner was the second most important type of support [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] The influence of father involvement and interparental relationship quality on adolescent mothers'..., Mallette [/bib_ref] [bib_ref] The experience of adolescent Motherhood : an exploratory mixed methods study, Kagawa [/bib_ref] [bib_ref] Experiences of pregnancy and motherhood among teenage mothers in a suburb of..., Gyesaw [/bib_ref] [bib_ref] Young mothers' lived experiences prior to becoming pregnant in rural Victoria: a..., Roberts [/bib_ref]. One cross-sectional study showed that father involvement pre and post birth was positively associated with adolescent mothers' maternal identity [bib_ref] The influence of father involvement and interparental relationship quality on adolescent mothers'..., Mallette [/bib_ref]. In line with another descriptive correlational study, social support from her mother and the baby's father were factors that may contribute to the development of positive self-perception of adolescent parenting [bib_ref] Self-perceptions of parenting among adolescent mothers, Devito [/bib_ref] and they often needed assistance from their families to care for their babies [bib_ref] Experiences of pregnancy and motherhood among teenage mothers in a suburb of..., Gyesaw [/bib_ref].
Besides support from family, two qualitative studies reported support from friends and society was particularly needed by adolescent mothers [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref]. Wahn et al. found that received support was experienced in varied ways, both positive and negative. Support from families, friends and society was seen to contribute to successful motherhood, but the support from the professional network was not always adapted to the adolescents' sometimes uncommunicated needs [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref]. A study with African Australian adolescent mothers confirmed the need for some social support during the postpartum period to be available to assist them to meet challenges of early motherhood from family, friends, and the baby's father. However, none of the participants had received support for the baby from their community because of negative community perceptions of teen mothers that increased young women's risk of isolation [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref].
## Education and economic strain
Five studies described other challenges contributing to adolescent motherhood as economic strain and educational attainment [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Joys and challenges of motherhood for Australian young women of preterm and..., Sheeran [/bib_ref] [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref]. Practical difficulties with housing, transport and financial strain significantly impacted on experiences of some young mothers [bib_ref] Joys and challenges of motherhood for Australian young women of preterm and..., Sheeran [/bib_ref] [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref]. Some adolescent mothers felt regret having a baby while still at school, when they unable to finish their education, this then led to difficulty finding work compounding financial stressors. Meeting the needs of the baby, continuing their education and making friends were very difficult for teen mothers [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref]. One study revealed that having a newborn was seen my young mothers as their motivation to complete school, as they claimed they would continue their education in the future in order to provide the best future possible for their babies [bib_ref] Self-perceptions of parenting among adolescent mothers, Devito [/bib_ref]. Despite this, Mitchel et al. found that most of the teenage mothers discontinued their education and policies had not yet been implemented to support new mothers in school. Indeed, in Africa the discovery of pregnancy sometimes led to expulsion from school [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref].
## Health care providers
Four studies explored whether healthcare providers in clinical settings supported the transition process of adolescent mothers [bib_ref] Treat me but don't judge me: a qualitative examination of health care..., Harrison [/bib_ref] [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref] [bib_ref] One foot wet and one foot dry: transition into motherhood among married..., Klingberg-Allvin [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref]. One study found that young women often felt disempowered and vulnerable immediately after birth [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. When health professionals took control in providing care to the infant rather than supporting the new mother to do this herself, there was a compounded feeling of helplessness experienced by young mothers [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. Another study described variable experiences of adolescent mothers' healthcare. Negative experiences seemed more memorable and hurtful, demonstrating mothers' general sense of mistrust [bib_ref] Treat me but don't judge me: a qualitative examination of health care..., Harrison [/bib_ref] and lacking power with regard to decisions in relation to pregnancy, delivery, and contraceptive usage [bib_ref] One foot wet and one foot dry: transition into motherhood among married..., Klingberg-Allvin [/bib_ref]. However, positive attitudes and supportive open communication from midwives and social workers helped participants to feel more confident in becoming young mothers [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref].
# Discussion
This study reviewed existing literature on the transition experiences to adolescent motherhood to identified factors associated. We identified factors contributing to the transition to adolescent motherhood (both positive and negative) that included physical challenges associated with birth, pregnancy, breastfeeding and psychological aspects related to transiting to motherhood with "mixed feelings", concerns around caring for the baby, the need social support, and supportive rather than controlling healthcare.
The transition to becoming a mother requires extensive psychological, social, and physical adaptation. A woman experiences heightened vulnerability and faces tremendous challenges as she makes this transition [bib_ref] Nursing support of the process of becoming a mother, Mercer [/bib_ref]. Mothers faced several health problems related to pregnancy, childbirth, postpartum and breastfeeding [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref] [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref] [bib_ref] Experiences of pregnancy and motherhood among teenage mothers in a suburb of..., Gyesaw [/bib_ref]. Pain related to the birth process and feeling tired and experiencing pain after giving birth prevented new mothers from caring for their newborns [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref].
The adolescent mothers simultaneously faced multiple developmental challenges related to transition into adulthood, marriage, pregnancy and mothering responsibilities [bib_ref] Iranian pregnant teenage women tell the story of "fast development": a phenomenological..., Mohammadi [/bib_ref] [bib_ref] Exploring the challenges of adolescent mothers from their life experiences in the..., Mangeli [/bib_ref]. Meleis et al. states that good physical and psychological well-being of mothers would positively influence the process of maternal transition. Our review suggests that teenage mothers experienced some negative feelings associated with being a mother, mostly related to mourning for a life as it was with friends and school. However, feelings of love with their infants expressed by teenage mothers in studies in this review highlighted their enjoyment with the process of becoming mothers. When new mothers felt happiness as a mother they were more likely to demonstrate commitment to caring for their babies. However, this review did not compare primiarous and multipara mothers' related psychologic aspects.
The results of studies that explored maternal role during transition revealed caring for the baby was the most challenging mothering responsibility. The process of transition evaluated maternal competence in caring for their babies and comfort in going through the transition to becoming mothers and are important components and challenges in primary health care [bib_ref] Clinical assessment of mothering during infancy, Fowles [/bib_ref]. Adolescent mothers experienced lack of knowledge about infant care and contradictory information from various sources were obstacles to caring for their babies. However, essential characteristics of the competent mother included a commitment to care for the emotional and physical wellbeing of the infant, and availability of social support would facilitate competence in caring for the baby [bib_ref] Chinese primiparous women's experiences of early motherhood: factors affecting maternal role competence, Ngai [/bib_ref].
Social support is a well-meaning action that is given to someone with whom there is a personal relationship, and produces positive responses from the recipient [bib_ref] Clarifying the social support theory-research linkage, Hupcey [/bib_ref]. Brown et al. described social support received by adolescent mothers in the first six months postpartum, suggesting that social support in the form of emotional, informational, tangible support and problem-solving support showed a positive impact during the early postpartum period [bib_ref] Social support, parenting competence, and parenting satisfaction among adolescent, African American, mothers, Brown [/bib_ref]. Many studies indicated that social support positively influenced transition of adolescent mothers [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Experiences of adolescent mothers in Costa Rica and the role of parental..., Copeland [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref] [bib_ref] Self-perceptions of parenting among adolescent mothers, Devito [/bib_ref]. Young mothers believed social support increased their confidence in caring for their children [bib_ref] Maternal role attainment in adolescent mothers: foundations and implications, Sartore [/bib_ref]. Adolescent mothers not only needed to social support from their family, but also peers, the wider community and health services [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref].
Previous research have shown that adolescent mothers were faced with extreme economic and social marginalization [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref].
Some participants from studies within this review identified extreme financial hardship [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref]. The meaning and experience of mothering was also framed by the level of stability in their daily life, particularly in terms of housing, income, and appropriate support [bib_ref] Joys and challenges of motherhood for Australian young women of preterm and..., Sheeran [/bib_ref]. Compounding their poverty, another issue related to adolescent motherhood in these studies was educational attainment [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Self-perceptions of parenting among adolescent mothers, Devito [/bib_ref]. Some young mothers had regrets in relation to having babies while still at school, and verbalized a desire to return to school. In some contexts this was not supported by school policy, and the discovery of pregnancy was met with expulsion from school [bib_ref] Early motherhood: a qualitative study exploring the experiences of African Australian teenage..., Watts [/bib_ref] [bib_ref] Psychological health and life experiences of pregnant adolescent mothers in Jamaica, Wilson-Mitchell [/bib_ref]. This review found different policies across countries related to education for adolescent mothers.
Role transition theory explains that the transition process requires supplementation of roles through therapeutic nursing. Three studies explored whether health care providers in clinical setting contributed to transition process of adolescent mothers [bib_ref] Joys and challenges of motherhood for Australian young women of preterm and..., Sheeran [/bib_ref] [bib_ref] The meaning of motherhood: adolescent childbearing and its significance for poor Dominican..., Salusky [/bib_ref] [bib_ref] One foot wet and one foot dry: transition into motherhood among married..., Klingberg-Allvin [/bib_ref] [bib_ref] Becoming and being a teenage mother: how teenage girls in south western..., Wahn [/bib_ref]. All the studies reported on experiences of adolescent mothers about their health care during pregnancy, childbirth and postpartum. The adolescent mothers experienced positive as well as negative health care encounters, the negative was reportedly more memorable and hurtful [bib_ref] Treat me but don't judge me: a qualitative examination of health care..., Harrison [/bib_ref] [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref]. Health workers, and especially nurses, play an important role in each stage of achieving the mother's role. The trend towards shorter hospital stays during the postpartum period limits health care encounters for adolescent mothers. However, there is no standard for health care encounters for adolescent mothers related to maternal and babies' health. Therefore, during their short stay at the hospital, nurses and midwives must help new mothers and their partners to transition and to care for their babies before returning home. It is important to recognize specific adolescent needs [bib_ref] One foot wet and one foot dry: transition into motherhood among married..., Klingberg-Allvin [/bib_ref] offering encouragement and a provide a realistic appraisal of the challenges ahead [bib_ref] Disempowered, passive and isolated: how teenage mothers' postnatal inpatient experiences in the..., Hunter [/bib_ref].
This review found factors contributing to the transition to becoming a mother requiring extensive psychological, social, and physical adaptation. However, no studies evaluated cultural and policy related health care contributing to adaptation of adolescent mothers to improve their maternal roles. This is an area that could be explored further.
## Limitation and implication for future study
This integrative review has some theoretical and methodological limitations. Firstly, most reviewed studies used qualitative methods; hence it is difficult to determine if each factor had significant correlation to effective transition of adolescent mothers. Two studies using quantitative cross-sectional methods and one study using mixed methods showed social support and involvement of the baby's father was associated with maternal identity of adolescent mothers during the transition period. Furthermore, only studies conducted in English language were included so relevant studies published in other languages were not included.
Future studies should aim to build on previous work to clarify relationships between the factors outlined in this review and motherhood transition outcomes. Moreover, further studies are needed to determine the effectiveness of nursing and midwifery interventions in the transition period of young mothers.
# Conclusion
Our review has presented current knowledge of adolescents' experiences to the motherhood transition and identified influencing factors. Factors found to contribute included physical problems, psychological aspects, caring for the baby, social support, and healthcare provision. Social support was also associated with the development of maternal identity in the transition period of adolescent mothers.
This integrative review contributes to nursing and midwifery science in relation to further understanding the transition of adolescent mothers. Future interventions for adolescent mothers during the transitional period should aim to provide social support and increase the ability of adolescent mothers to deal with the challenges they experience when becoming young mothers.
## Conflicts of interest
No conflict of interest has been declared by the authors.
[fig] Figure 1: The Flow chart describing details of literature search and selection strategy. [/fig]
[fig] Figure 2: Factors that influence the motherhood transition of adolescent women. [/fig]
[table] Table 1: Details of studies in this review. [/table]
|
Trichomonas tenax: A Neglected Protozoan Infection in the Oral Cavities of Humans and Dogs—A Scoping Review
Trichomonas tenax is a flagellated protozoan parasite found in the oral cavities of humans and animals and has been associated with periodontal disease, the most prevalent inflammatory disease affecting them all. Studies have shown that T. tenax can cause damage to mammalian cells and secretes virulent proteins, such as cysteine. It is presently considered zoonotic. Despite the few studies that have been done, the pathogenicity of this oral protozoan is still not fully understood. A database search was performed in July 2022 using PubMed and Google Scholar to retrieve data eligible for this study. PRISMA-ScR guidelines were followed to conduct this scoping review. A total of 321 articles were found with 87 included in this review after applying the exclusion criteria. Due to its increasing prevalence worldwide in both humans and dogs, detecting and elucidating the pathogenicity of this parasite is paramount for effective global control and prevention of periodontal disease. However, there is a paucity in the literature on this neglected zoonotic trichomonad, which is in large contrast to the closely related human pathogen T. vaginalis. Here, we comprehensively review the history, morphology and reproduction, host, prevalence, diagnosis, pathogenicity, control, and prevention of T. tenax. Hopefully, this article will call attention to both medical and veterinary professionals as well as epidemiologists on this most neglected and zoonotic protozoan. More epidemiological and clinical studies need to be conducted on T. tenax to gain a better understanding of its pathogenicity, to increase the chances of developing effective drugs to aid in the control of this oral parasite, and reduce the spread of periodontal disease worldwide.
# Introduction
Trichomonas tenax is an anaerobic flagellated protozoan of an ancient eukaryotic lineage without mitochondria that lives in low-oxygen environments [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref] [bib_ref] Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax, Kucknoor [/bib_ref]. In contrast to its close relative, Trichomonas vaginalis found in the human urogenital tract, T. tenax is mostly found to inhabit the oral cavities of humans and animals, which may lead to periodontal disease [bib_ref] Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp.,..., Kellerová [/bib_ref]. The protozoan has also been found in other parts of the body, such as the lungs and bronchi, submaxillary glands, tonsils, and lymph nodes [bib_ref] Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques-In..., Dybicz [/bib_ref]. Despite its importance, relative to both human and animal health, very little attention has been paid to T. tenax. Therefore, this scoping review aims not only to identify future research gaps but also to call attention to both medical and veterinary professionals on the epidemiology, detection, and risk associated with contracting this greatly neglected oral protozoan.
# Materials and methods
## Data search strategy
This scoping review was executed to assess the following research questions: (1) Is T. tenax more prevalent in humans and dogs with periodontitis compared to those that are healthy? [bib_ref] Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax, Kucknoor [/bib_ref] Is there an association between T. tenax and periodontal disease? (3) What techniques are presently available to detect T. tenax? (4) Does T. tenax exosomes contains
## Study inclusion and exclusion criteria
The inclusion criteria included all peer-reviewed published articles in English that included any information on the history, morphology, reproduction, host preference, the prevalence in humans and dogs, diagnosis, pathogenicity, and control and prevention of T. tenax. All articles were required to have an abstract and/or full text available to be included in this study. The search was not restricted to the publication date; therefore, all articles up to 29 July 2022 were included once inclusion criteria were met. Studies were excluded if no abstracts were available, if the article was not in English, if the article was not relevant based on the title and abstract, and if it was a duplicate [fig_ref] Figure 1: Flow diagram of scoping review selection process [/fig_ref].
## Data search strategy
This scoping review was executed to assess the following research questions: (1) Is T tenax more prevalent in humans and dogs with periodontitis compared to those that are healthy? [bib_ref] Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax, Kucknoor [/bib_ref] Is there an association between T. tenax and periodontal disease? (3) What techniques are presently available to detect T. tenax? [bib_ref] Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques-In..., Dybicz [/bib_ref] Does T. tenax exosomes contains virulent proteins and RNA, such as its close relative T. vaginalis?Are there any treatments or measures available for the control and prevention of T. tenax? This scoping review was conducted by adapting the guidelines of the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (PRISMA-ScR) [bib_ref] PRISMA extension for scoping reviews (PRISMA-ScR): Checklist and explanation, Tricco [/bib_ref]. A literature search was conducted on 29 July 2022 with an open date using PubMed and Google Scholar databases for published articles. The databases were searched for the keywords Trichomonas tenax and Trichomonas elongata [fig_ref] Table 1: Results obtained from search strategies used in databases [/fig_ref]. The results were exported in .csv format and then opened and organized in Microsoft Excel.
## Study inclusion and exclusion criteria
The inclusion criteria included all peer-reviewed published articles in English that included any information on the history, morphology, reproduction, host preference, the prevalence in humans and dogs, diagnosis, pathogenicity, and control and prevention of T. tenax. All articles were required to have an abstract and/or full text available to be included in this study. The search was not restricted to the publication date; therefore, al articles up to 29 July 2022 were included once inclusion criteria were met. Studies were excluded if no abstracts were available, if the article was not in English, if the article was not relevant based on the title and abstract, and if it was a duplicate [fig_ref] Figure 1: Flow diagram of scoping review selection process [/fig_ref].
## Data extraction
For data extraction, two reviewers, (MM) and (CY), independently read the title and abstract of each article retrieved from the search to determine if the article was eligible. Full-text screening was conducted by (MM), (CY), and (JK), and the data were extracted independently. Any differences or disagreements on eligibility were discussed and resolved by the team. The data extracted included the author, publication date, title, history, morphology, reproduction, host preference, the prevalence in humans and dogs, diagnosis, pathogenicity, and control and prevention.
# Results
A total of 321 articles were retrieved after searching 2 databases (PubMed = 143; Google scholar = 178). Three hundred and twelve articles were screened after duplicates were removed. After inclusion and exclusion criteria were applied, 87 articles were eligible for this scoping review [fig_ref] Figure 1: Flow diagram of scoping review selection process [/fig_ref].
## History
The name Trichomonas tenax originated from the Greek words "trichos" (meaning tiny hair), "monas" (meaning simple creature), and the Latin word "tenere", which means to keep or to stick to. The trichomonad protozoan was originally discovered by Muller in 1773, in aqueous solutions of tartar derived from the oral cavities of human beings [bib_ref] Relation between Trichomonas tenax and pulmonary diseases, Hamadto [/bib_ref]. He named it Cercaria tenax [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref]. Up until the 1940s, different names were used, such as Trichomonas elongata, originating from Steinberg in 1862 [bib_ref] The common flagellate of the human mouth, Dobell [/bib_ref] , and Tetratrichomonas buccalis [bib_ref] Observations on Entamoeba gingivalis from the human mouth, with a note on..., Goodey [/bib_ref]. Although T. tenax was first observed by Muller in 1773, it was in 1917 that the first accepted name was given to the oral flagellate, which at that time was called Tetratrichomonas buccalis [bib_ref] Observations on Entamoeba gingivalis from the human mouth, with a note on..., Goodey [/bib_ref]. This was the first recorded report on the oral trichomonad in the 1900s. According to [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref] , it was [bib_ref] The common flagellate of the human mouth, Dobell [/bib_ref] and [bib_ref] Comparative morphology of the trichomonad flagellates of man, Wenrich [/bib_ref] who assumed that Trichomonas was the only flagellate in the buccal cavity and that its name should be tenax based on priority (hence, the name T. tenax) [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref].
## Morphology and reproduction
Trophozoites of T. tenax are either ellipsoidal or ovoid (pear-shaped) in shape and 5-16 × 2-15 µm in size. Each has five flagella-four free anterior flagella and one that extends posteriorly. An undulating membrane extends two-thirds of the body length and its accompanying costa typically lies next to the posterior flagellum [bib_ref] Comparative morphology of the trichomonad flagellates of man, Wenrich [/bib_ref] [bib_ref] Trichomonas tenax and periodontal diseases: A concise review, Marty [/bib_ref]. The four anterior flagella arise from a three-lobed blepharoplast, which gives rise to a chromatic basal rod and an axostyle. The nucleus is situated near the anterior end of the body and is generally ellipsoidal or ovoidal with an average size of 2.5 × 1.7 µm. It contains a single nucleolus, surrounded by clear halos [bib_ref] Animal parasites of the mouth and their relation to dental disease**from the..., Kofoid [/bib_ref] [bib_ref] Trichomonas tenax: Ultrastructure of giant forms, Ribaux [/bib_ref]. The anterior end of the capitulum runs continuously with the pelta and there is no cell mouth present [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref]. In addition to trophozoites, T. tenax has other forms. These forms include a round form that is usually larger than the trophozoite, an amoeboid cell form found swimming freely in axenic medium, and a pseudocyst form, which is found when the trichomonads are under stressful conditions. These forms can range in size from approximately 5 to 16 µm in length and 5-6 µm wide [bib_ref] Trichomonas tenax and periodontal diseases: A concise review, Marty [/bib_ref].
In the past, these different forms of the trichomonad were considered different lifecycle stages. However, more recently, it has been shown that these different forms are due to different environmental conditions of the trichomonad's habitat [bib_ref] Clinical and microbiological aspects of Trichomonas vaginalis, Petrin [/bib_ref].
Trichomonas tenax can be readily differentiated from other species of the Trichomonadidae family, except for two, T. vaginalis and T. gallinae. Honigberg and Lee (1959) stated that from a morphological standpoint, T. vaginalis is on average larger than the other two trichomonads and the Paracostal granules are larger and more numerous compared to the other two trichomonads. Paracostal granules are mostly absent or small in T. tenax whereas in T. gallinae the granules are found in the region of the axostyle [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref]. Furthermore, these trichomonads exhibit host and site predilection, with the T. vaginalis site of predilection being in the urogenital tract of humans, T. tenax in the oral cavities of humans and animals, and T. gallinae in the upper digestive tracts of avian hosts [bib_ref] Aberrant and accidental trichomonad flagellate infections: Rare or underdiagnosed?, Yao [/bib_ref].
Trichomonas tenax reproduces by asexual reproduction, followed by a process of mitosis involving six morphologically distinct chromosomes [bib_ref] The protozoa of the human mouth, Atwood Kofoid [/bib_ref]. [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref] reported that during division, the parental flagella are divided equally among the daughter mastigonts with full flagellar completion taking place right before cytokinesis. Additionally, each daughter cell receives one of the parabasal, one daughter cell retains the old undulating membrane and costa while the other develops new organelles. By the end of the division, the old axostyle and pelta are destroyed and a new supporting organelle develops in both daughter cells [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref].
## Host
Trichomonas tenax has been cited and recorded in different animal hosts over the years. It was first reported in the oral cavities of humans by Muller in 1773 [bib_ref] Relation between Trichomonas tenax and pulmonary diseases, Hamadto [/bib_ref]. This was confirmed by other studies that also found T. tenax in the oral cavities of humans [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref] [bib_ref] Investigation of the relationship between oral and dental health and presence of..., Özçelik [/bib_ref] [bib_ref] Trichomonas tenax in Basrah, Mahdi [/bib_ref]. It has also been observed in samples taken from the vaginas of monkeys [bib_ref] Trichomonads from the vagina of the monkey, from the mouth of the..., Hegner [/bib_ref]. Several studies reported that T. tenax can cause urogenital invasions in humans in addition to its close relative T. vaginalis [bib_ref] Protozoan genital invasions caused by the representatives of trichomonas and giardia, Fedorych [/bib_ref] [bib_ref] A false-positive Trichomonas vaginalis result due to Trichomonas tenax presence in clinical..., Brosh-Nissimov [/bib_ref] [bib_ref] Trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial..., Crucitti [/bib_ref]. detected T. tenax in the oral samples of small companion animals, such as dogs and cats, as well as large animals, such as horses [bib_ref] Molecular identification of trichomonas tenax in the oral environment of domesticated animals..., Dybicz [/bib_ref]. Other studies have reported on the occurrence of T. tenax in the oral cavities of dogs and cats [bib_ref] Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp.,..., Kellerová [/bib_ref] [bib_ref] The prevalence of canine oral protozoa and their association with periodontal disease, Patel [/bib_ref]. Recently, T. tenax was detected in the cloaca of birds [bib_ref] Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and..., Martínez-Herrero [/bib_ref] [bib_ref] Prevalence of trichomonads in the cloaca of wild wetland birds in the..., Landman [/bib_ref] [bib_ref] Prevalence of Trichomonas spp. in domestic pigeons in Shandong Province, China, and..., Jiang [/bib_ref] [bib_ref] Trichomonad parasite infection in four species of Columbidae in the UK, Lennon [/bib_ref] [bib_ref] Molecular analysis of clonal trichomonad isolates indicate the existence of heterogenic species..., Grabensteiner [/bib_ref] [bib_ref] Molecular characterization of the Trichomonas gallinae morphologic complex in the United States, Gerhold [/bib_ref]. With T. tenax being found in so many different hosts, it is important to determine whether T. tenax is a human parasite, which is the predominant current view, or if it is a parasite of animal origin with zoonotic potential. This question on host preference and specificity requires further investigation.
## Culture
Culturing T. tenax is important for studying the biochemistry, physiology, and metabolism of parasites, and to determine the nutrient requirements, morphological structure, pathophysiology, life cycle, and host-parasite relationship. Before 1917, T. tenax was of little or no interest to clinicians and biochemists since it could not be maintained in an axenic culture unlike its close relatives, T. vaginalis and T. gallinae [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref]. Although there were reports of T. tenax being cultured as early as 1915, these reports were not credible [bib_ref] Cultivation of the causative organism and experimental infection. (A Preliminary Communication), Lynch [/bib_ref]. In 1917, Ohira and Noguchi continued culture work on T. tenax, but this work was also brief with no final conclusions as to whether the culture was successful or not [bib_ref] The cultivation of trichomonas of the human mouth (Tetratrichomonas hominis), Ohira [/bib_ref]. Culture studies on T. tenax have increased over the years as they have (simultaneously) attracted the attention of researchers. Other culture work was followed by Hinshaw and Hogue in 1926, giving a better understanding of environmental conditions and nutrient requirements necessary to culture the oral trichomonad [bib_ref] Correlation of protozoan infections of human mouth with extent of certain lesions..., Hinshaw [/bib_ref] [bib_ref] Studies on Trichomonas buccalis, Hogue [/bib_ref]. [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref] tried without success to culture T. tenax axenically [bib_ref] Structure and division of Trichomonas tenax (O. F. Müller), Honigberg [/bib_ref]. Success was finally reached in 1962 when L. S. Diamond was able to induce axenic growth in a nutrient broth supplemented with serum and a cell-free extract of the chick embryo, which is now referred to as Diamond's medium [bib_ref] Axenic cultivation of Trichomonas tenax, the oral flagellate of man I. Establishment..., Diamond [/bib_ref]. Currently, Diamond's medium is still used by researchers worldwide to culture T. tenax. Although were successful in culturing T. tenax, other studies continued as the oral trichomonad gained interest. Work by [bib_ref] Collection, identification, and cultivation of oral protozoa, Wantland [/bib_ref] and [bib_ref] Growth of Trichomonas tenax in tissue culture medium containing complement and antiserum..., Asai [/bib_ref] was somewhat unsuccessful but still contributed to the overall understanding of the oral flagellate [bib_ref] Collection, identification, and cultivation of oral protozoa, Wantland [/bib_ref] [bib_ref] Growth of Trichomonas tenax in tissue culture medium containing complement and antiserum..., Asai [/bib_ref]. [bib_ref] Collection, identification, and cultivation of oral protozoa, Wantland [/bib_ref] used egg yolk, a non-synthetic serum, to culture T. tenax and Entamoeba gingivalis, which resulted in the near-axenic and mono-axenic culture of T. tenax and E. gingivalis. [bib_ref] Growth of Trichomonas tenax in tissue culture medium containing complement and antiserum..., Asai [/bib_ref] used RPMI 1640 and Eagle's minimum essential medium (MEM) to culture T. tenax but cultures failed to support the growth of T. tenax without bacterial growth. Additionally, researchers have shown that T. tenax isolated from humans can be successfully cultured if incubated at a temperature between 31 and 37 - C with a pH between 7.0 and 7.5 for 72 h [bib_ref] Collection, identification, and cultivation of oral protozoa, Wantland [/bib_ref] [bib_ref] Growth of Trichomonas tenax in tissue culture medium containing complement and antiserum..., Asai [/bib_ref]. All of these contributions to the culture of T. tenax were mainly from human strains with only one being successful, i.e., culturing T. tenax from dogs [bib_ref] Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp.,..., Kellerová [/bib_ref].
## Molecular diagnosis
Currently, microscopy and molecular methods are used in the diagnosis of T. tenax infection in humans and animals. Trichomonads of ellipsoidal or ovoid (pear-shape) shapes and 5-16 × 2-15 µm in size, as revealed by the microscopy along with the predilection site of the mouth, either directly or after being cultured, can be used as criteria for diagnosing T. tenax infection [bib_ref] Aberrant and accidental trichomonad flagellate infections: Rare or underdiagnosed?, Yao [/bib_ref] [fig_ref] Table 2: Morphological characteristics of trichomonad protozoa found in human infections a [/fig_ref]. However, molecular methods such as PCR are preferred for confirmation since they are considered more sensitive and specific than the conventional techniques of culture and microscopy [bib_ref] Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage..., Lin [/bib_ref]. PCR was first used to detect T. tenax in human oral samples in 1997 [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref] , where primers were designed for the 18S rRNA gene of T. tenax, which was aligned with T. vaginalis and T. foetus [fig_ref] Table 3: Molecular diagnosis of Trichomonas tenax [/fig_ref]. [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref] concluded that the method was specific to T. tenax and had a limit of detection of 100fg DNA or as low as 5 cells [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref]. In comparison with the gold standard microscopy in the detection of T. tenax (in ten healthy individuals and nine patients of periodontitis or gingivitis), the PCR only detected T. tenax in five patients, whereas microscopy was negative for all patients and healthy individuals [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref]. The data clearly show that PCR surpasses microscopy in sensitivity and yet maintains specificity. However, it was not until a case study by [bib_ref] Abd-Al-Hammza Abbass, Z. Canine Trichomonas tenax mandibular gland infestation, Szczepaniak [/bib_ref] that PCR was used to detect T. tenax in canine oral samples where primers for the ITS1-5.8S rRNA-ITS2 regions were used [bib_ref] Abd-Al-Hammza Abbass, Z. Canine Trichomonas tenax mandibular gland infestation, Szczepaniak [/bib_ref]. Over the last two decades, the loop-mediated isothermal amplification (LAMP), a nucleic acid amplification test, was developed to amplify the DNA of different pathogens for detection and diagnostic purposes [bib_ref] Loop-mediated isothermal amplification of DNA, Notomi [/bib_ref]. This technique has been proven to be more sensitive than traditional techniques of culture and microscopy and PCR that are presently used [bib_ref] The loop-mediated isothermal amplification technique in periodontal diagnostics: A systematic review, Lenkowski [/bib_ref] [bib_ref] Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in..., Kato [/bib_ref] [bib_ref] The development of a Loop-mediated isothermal amplification (LAMP) procedure for plague diagnostic, De Lira Nunes [/bib_ref]. It is rapid, does not require expensive equipment, and is ideal for future diagnoses, especially in developing countries [bib_ref] Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis, Reyes [/bib_ref] [bib_ref] Loop-mediated isothermal amplification for the detection of Xanthomonas arboricola pv. Pruni in..., Li [/bib_ref]. To date, only one LAMP assay has been developed to detect T. tenax in human and/or canine oral samples, which can be directly used for clinical samples without prior DNA extraction. The test has a limit of detection of 10fg DNA or 1 cell. A total of 8 out of 44 clinical canine samples were microscopically positive for T. tenax after culturing. They were also LAMP-positive when two cells were used in each reaction without prior DNA extraction. Therefore, this LAMP assay has the great advantage of being used in point of care in both developed and developing countries. No other new molecular techniques have been reported in the literature to specifically detect T. tenax in humans and/or animals.
## Prevalence
## Humans
The only reliable method available that could have identified or detected T. tenax in the 1900s and most of the 20th century was microscopy [bib_ref] Correlation of protozoan infections of human mouth with extent of certain lesions..., Hinshaw [/bib_ref]. [bib_ref] Correlation of protozoan infections of human mouth with extent of certain lesions..., Hinshaw [/bib_ref] found that 90% of the prisoners with advanced pyorrhea (periodontitis) were infected with T. tenax. Several prevalence studies in humans have followed since that time [fig_ref] Table 4: Prevalence of Trichomonas tenax in humans with or without periodontitis [/fig_ref] [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref] [bib_ref] Investigation of the relationship between oral and dental health and presence of..., Özçelik [/bib_ref] [bib_ref] Trichomonas tenax in Basrah, Mahdi [/bib_ref] [bib_ref] Trichomonas Tenax in human oral cavity, Potočki-Tukša [/bib_ref] [bib_ref] Investigation of Entamoeba gingivalis and Trichomonas tenax in periodontitis or gingivitis patients..., Yazar [/bib_ref] [bib_ref] Prevalence Trichomonas tenax in Karbala Governorate, Mohammed [/bib_ref] [bib_ref] Prevalence of oral Entamoeba gingivalis and Trichomonas tenax in patients with periodontal..., Ghabanchi [/bib_ref]. In Iraq, the prevalence of T. tenax was 8.4% in 143 mouth disease patients and 4.1% in 271 controls [bib_ref] Trichomonas tenax in Basrah, Mahdi [/bib_ref]. [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref] reported the prevalence of both E. gingivalis and T. tenax in patients with oral infections in Brazil with the prevalence of T. tenax being 51% (51 out of 100 patients). The study by [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref] also showed that the flagellate infection decreased with age in the control group and increased with age in those who were ill. Additionally, E. gingivalis and T. tenax infections increased in individuals with tooth loss, indicating a positive correlation between tooth loss and both infections [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref].
The difficulty in culturing and identifying the parasites made way for the development of molecular techniques, such as PCR and LAMP. [bib_ref] Prevalence of oral trichomoniasis in patients with periodontitis and gingivitis using PCR..., Athari [/bib_ref] reported on the prevalence of oral trichomoniasis in 160 patients with gingivitis and periodontitis using the PCR-amplifying 18S rRNA gene and microscopy. A total of 33 patients (20.6%) were PCR-positive whilst 28 (15.5%) were diagnosed microscopically. The study further found an association between the prevalence of T. tenax and the severity of periodontitis [bib_ref] Prevalence of oral trichomoniasis in patients with periodontitis and gingivitis using PCR..., Athari [/bib_ref]. [bib_ref] Prevalence of oral Trichomonas tenax in periodontal lesions of down syndrome in..., Mehr [/bib_ref] investigated the prevalence of T. tenax in the periodontal lesions of patients who also had Down's syndrome in Iran. Moreover, 52 patients were presented with periodontal disease and 52 with healthy gingiva; the prevalence was 14 (26.9%) and 5 (9.6%), respectively [bib_ref] Prevalence of oral Trichomonas tenax in periodontal lesions of down syndrome in..., Mehr [/bib_ref]. used PCR as a method of detection for T. tenax in patients with health issues, including diabetes, renal transplant, and rheumatoid arthritis. Healthy individuals were used as controls and the primers targeted the ITS1-5.8S rRNA-ITS2 region rather than the 18S rRNA, as mentioned earlier. The prevalence of T. tenax in the oral cavity of the control group was 10.2% (33 of 226), 14.1% (13 of 92) in diabetics, 12% (6 of 50) in renal transplant patients, and 14% (7 of 50) in rheumatoid arthritis patients. A higher prevalence of T. tenax was revealed in adults from all groups involved [bib_ref] Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques-In..., Dybicz [/bib_ref]. Two additional studies have been published, which used PCR to determine the prevalence of T. tenax in patients with periodontal disease. In one study, primers designed from the RNA polymerase II rpbI gene for T. tenax strain NIH4 [bib_ref] Specific clones of Trichomonas tenax are associated with periodontitis, Benabdelkader [/bib_ref] were used and the other study used primers designed from the β-tubulin gene of T. vaginalis [bib_ref] Observational cross-sectional study of Trichomonas tenax in patients with periodontal disease attending..., Bracamonte-Wolf [/bib_ref]. Benabdelkader et al. (2019) included 50 patients in the study-20 with gingivitis and 30 with periodontitis. The overall prevalence of T. tenax was 56% (28/50). Interestingly, it was found to be more prevalent in patients with periodontitis than in those with gingivitis, i.e., 70% (21/30) and 35% (7/20), respectively [bib_ref] Specific clones of Trichomonas tenax are associated with periodontitis, Benabdelkader [/bib_ref]. In a later study using PCR by Bracamonte-Wolf et al. (2019), out of 106 periodontitis patients and 85 healthy controls, the prevalence was 34% (36/106) in periodontitis patients and 28% (30/85) in the control group [bib_ref] Observational cross-sectional study of Trichomonas tenax in patients with periodontal disease attending..., Bracamonte-Wolf [/bib_ref]. Collectively, these data showed a strong correlation between T. tenax and periodontal disease in humans. Additionally, these data represent the evolution of techniques developed over the years to detect and assess T. tenax in humans.
The variability in the prevalence of T. tenax in humans is possibly due to the difference in the standards of oral hygiene in the different populations and due to the increased sensitivity of the detection methods. In populations with low oral hygiene and poor socioeconomic backgrounds, the prevalence of T. tenax is high compared to populations with average or good oral hygiene and socioeconomic backgrounds [bib_ref] Trichomonas tenax and periodontal diseases: A concise review, Marty [/bib_ref]. In a cross-sectional survey carried out in Iran, Azadbakht K et al. found that the odds ratio (OR) of individuals brushing their teeth was 0.43 (95% CI: 0.21-0.88) in comparison with those who did not brush. Further, the OR of people who resided in urban areas was 0.22 (95% CI: 0.1-0.47) compared with those in rural places [bib_ref] Prevalence and risk factors of oral cavity parasites in pregnant women in..., Azadbakht [/bib_ref].
## Domestic dogs
Trichomonas tenax was first reported in canines in 1927 with 22 out of 23 canines testing positive [bib_ref] Trichomonads from the mouth of the dog, Hegner [/bib_ref]. It was not until there was a PCR method designed to detect T. tenax that studies on canines started to increase. This was probably due to the difficulty in culturing and detecting the flagellate by microscopy in a canine host. Alternatively, it may be due to a lack of interest in this protozoan in veterinary medicine (until recently). To date, only three recorded studies have been reported on the prevalence of T. tenax in canines using PCR [fig_ref] Table 5: Prevalence of Trichomonas tenax in a domestic dog with or without periodontitis [/fig_ref] [bib_ref] Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp.,..., Kellerová [/bib_ref] [bib_ref] Molecular identification of trichomonas tenax in the oral environment of domesticated animals..., Dybicz [/bib_ref] [bib_ref] The prevalence of canine oral protozoa and their association with periodontal disease, Patel [/bib_ref]. The first study was reported by [bib_ref] The prevalence of canine oral protozoa and their association with periodontal disease, Patel [/bib_ref] in the United Kingdom. A total of 92 samples were collected from canine dental plaque and screened for the presence of T. tenax and Entamoeba spp.; the prevalence of T. tenax was 56.2% (52/92) and Entamoebae spp. was 4.34% (4/92). Furthermore, the next-generation sequencing of healthy, gingivitis, early-stage periodontitis, and severe periodontitis samples showed the prevalence of T. tenax at 3.51%, 2.84%, 6.07%, and 35.0%, respectively. These findings were the first conclusive evidence of the presence of T. tenax in canine oral plaque [bib_ref] The prevalence of canine oral protozoa and their association with periodontal disease, Patel [/bib_ref]. Kellerová and Tachezy (2017) also investigated the occurrence of oral trichomonads in 111 domestic dogs and 122 cats using cell culture, PCR, and sequencing of the ITS1-5.8S rRNA-ITS2 regions. The prevalence percentages of T. tenax in dogs and cats were 8.1% and 4.1%, whilst for the different Trichomonas spp., they were 30.6% and 6.6%, respectively. The study also identified T. brixi as a new species. It concluded that dogs 3 years or older, as well as crossbred dogs, showed an increased prevalence of T. tenax [bib_ref] Zoonotic Trichomonas tenax and a new trichomonad species, Trichomonas brixi n. sp.,..., Kellerová [/bib_ref]. used PCR to detect T. tenax in domesticated animals, such as horses, dogs, and cats. In the study, 142 dogs, 57 cats, and 102 horses were examined for the presence of T. tenax. The prevalence of T. tenax in canines was 4.92% (7 of 142). Additionally, 9 of 11 DNA sequences of trichomonad isolates showed 100% identity with T. tenax sequence obtained from the GenBank. The study concluded that oral trichomoniasis spreading between humans and domestic animals should be taken into consideration since the owners of three positive dogs also tested positive for T. tenax [bib_ref] Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques-In..., Dybicz [/bib_ref]. Studies on the prevalence of T. tenax in canines remain limited and more studies are needed worldwide.
## Pathogenesis and virulence factors
Two systematic reviews on T. tenax have concluded that there is an association between T. tenax and periodontal disease [bib_ref] The neglected role of Trichomonas tenax in oral diseases: A systematic review..., Eslahi [/bib_ref] [bib_ref] Assessment of the role of Trichomonas tenax in the etiopathogenesis of human..., Bisson [/bib_ref]. The increase in the prevalence and association of T. tenax with periodontal disease from previous studies leads to questions concerning the pathogenicity of the oral flagellate. Extensive studies have been conducted on the pathogenicity of its close relative of the urogenital tract, T. vaginalis, and bacteria associated with periodontal disease [bib_ref] Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax, Bózner [/bib_ref]. However, the pathogenicity of T. tenax with regard to periodontal disease is far less documented [bib_ref] Trichomonas tenax and periodontal diseases: A concise review, Marty [/bib_ref]. To date, only seven studies have focused on the pathogenicity of T. tenax, and the proteins secreted by the oral flagellate that exhibit virulent characteristics. The first reported study on the pathogenicity of T. tenax was by [bib_ref] Study of an oral protozoan Trichomonas tenax using scanning and transmission electron..., Ribaux [/bib_ref] , who investigated the proteolytic activity of T. tenax in whole cells. Unfortunately, only the abstract could be obtained for this scoping review [bib_ref] Study of an oral protozoan Trichomonas tenax using scanning and transmission electron..., Ribaux [/bib_ref]. [bib_ref] Immunohistochemical localization of fibronectin-like protein on the cell surface of the oral..., Ribaux [/bib_ref] reported another study on the immunohistochemical location of fibronectin-like proteins on the cell surface of T. tenax. Two strains of T. tenax were used to establish an immunofluorescence staining procedure. The cells gave a positive fluorescence stain with anti-fibronectin anti-serum and the controls remained negative. They concluded that T. tenax produces fibronectin-like proteins that could be responsible for tissue adhesion [bib_ref] Immunohistochemical localization of fibronectin-like protein on the cell surface of the oral..., Ribaux [/bib_ref]. These early works led other researchers to further investigate the proteolytic activity of T. tenax.
Bózner and Demeš (1991) carried out a study on the proteolytic activity in crude extracts and culture filtrates from T. tenax in SDS-polyacrylamide gels containing copolymerized gelatin. A total of seven distinct proteolytic bands were found, of which, three had molecular weights ranging from 35 to 56 kDa. These bands were SH-dependent, and their inhibitory sensitivities were characteristic of cysteine proteinases. The other four bands had molecular weights ranging from 76 to 270 kDa; these were SH-independent and were inhibited by a chelating agent, EDTA, suggesting they belong to the metalloproteinase family [bib_ref] Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax, Bózner [/bib_ref]. The authors continued studying the degradation of collagens I, III, IV, and V by extracellular proteinases of T. tenax. They concluded that the degradation of all four collagen types was temperature-dependent, with collagen IV being digested most effectively. They further stated that E-64 and the activation by reducing the agent dithiothreitol indicate that cysteine proteinases from T. tenax are responsible for the cleavage of collagen [bib_ref] Degradation of collagen types I, III, IV and V by extracellular proteinases..., Bózner [/bib_ref].
Another study that focused on the pathogenicity of T. tenax, carried out by [bib_ref] Two distinct hemolysins in Trichomonas tenax ATCC 30207, Nagao [/bib_ref] , investigated the ability of T. tenax to lyse the red blood cells of sheep, horses, and humans. To achieve this, five fractions derived from intact cells, culture supernatant, cultural filtrate, cell debris, and lipid enriched fractions were used to assess the hemolytic activities under various conditions; only the culture supernatant was negative for hemolytic activity, the other four samples were positive for hemolytic activity. The authors concluded that the hemolytic activities were due to two types of hemolysins, one which is protein-like and the other lipid-like. The protein-like hemolysin was heat-labile and inhibited by various cysteine-proteinase inhibitors [bib_ref] Two distinct hemolysins in Trichomonas tenax ATCC 30207, Nagao [/bib_ref]. A decade later, El Sibaei et al. (2012) investigated the proteinase activities of seven isolates of T. tenax obtained from clinical patients in Egypt. The study also concluded that proteinase bands were observed, and these bands were intensified with a cysteine proteinase activator and disappeared completely in the presence of the cysteine proteinase inhibitor, further suggesting that the proteinases found were also cysteine proteinases [bib_ref] Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates..., El Sibaei [/bib_ref]. These cysteine proteinases are the same virulent proteins that have been detected in T. vaginalis [bib_ref] Analysis of the proteinases of representative Trichomonas vaginalis isolates, Neale [/bib_ref]. Recently,performed a study where T. tenax fulfilled the requisites of a parasite, damaging different mammalian cells and behaving in a similar manner to T. vaginalis. In short, limited work has been done on the pathogenicity of T. tenax, although there is growing evidence that it contributes to periodontal disease. Therefore, more studies are needed to better understand the pathophysiological processes of T. tenax infections in humans and other mammalian hosts with reference to periodontal disease.
Regarding the proteomics of parasites, exosome release has been gaining attention. Research has shown that many parasites excrete proteins enclosed in exosomes, which are considered potential virulent factors [bib_ref] Research progress on the composition and function of parasite-derived exosomes, Nawaz [/bib_ref]. Exosomal studies performed on T. vaginalis have found that it secretes exosomes (such as those found in mammals) containing RNA and parasite-specific proteins [bib_ref] Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions, Twu [/bib_ref]. [bib_ref] Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions, Twu [/bib_ref] illustrated that T. vaginalis exosomes deliver their contents to the host cell, modulating the cell's immune response when fused. Additionally, the study was the first to show the potential role of exosomes in parasite-toparasite communication [bib_ref] Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions, Twu [/bib_ref]. Another study examined the major surface proteins (MSP) in the exosomes of the Leishmania spp. These proteins have been shown to digest extracellular matrix proteins. The study classified the MSP proteins released in L. infantum exosomes from promastigotes in avirulent procyclic (logarithmic), virulent stationary, and metacyclic stages, respectively, and found high levels of MSP in exosomes released from the stationary and metacyclic promastigotes than in the logarithmic promastigotes. Work on exosomes is the new path that scientists are taking to gain a better idea of the pathogenicity of parasites. Therefore, it is important to investigate the pathogenicity of T. tenax by isolating exosomes, finding virulent factors by proteomic analysis, and elucidating exosome interactions with epithelial cells. This would be a significant contribution and addition to the pathogenicity of T. tenax.
## Control and prevention
Limited studies have been reported on the control and prevention of T. tenax despite its high prevalence among the human and canine populations. This could be because the pathogenesis of the oral protozoa remains unclear. However, studies have been documented on the treatment and prevention of its close relative T. vaginalis [bib_ref] Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions, Twu [/bib_ref] [bib_ref] Prevention or treatment: The benefits of Trichomonas vaginalis vaccine, Cudmore [/bib_ref] [bib_ref] The effect of nonsurgical periodontal therapy on Trichomonas tenax and Entamoeba gingivalis..., Rashidi Maybodi [/bib_ref]. Trichomonas tenax can be transmitted between individuals by droplets from the mouth, kissing, or the use of contaminated dishes and drinking water [bib_ref] Trichomonas tenax in Basrah, Mahdi [/bib_ref]. Few studies have investigated treatment and prevention measures to control T. tenax infections. One study investigated the effects of non-surgical periodontal therapy (i.e., deep cleaning with scaling and root planning) on T. tenax and E. gingivalis in patients with chronic periodontitis. Rashidi Maybodi et al. reported that non-surgical treatment can reduce T. tenax and E. gingivalis in the oral environments of patients with chronic periodontitis [bib_ref] The effect of nonsurgical periodontal therapy on Trichomonas tenax and Entamoeba gingivalis..., Rashidi Maybodi [/bib_ref]. Another study investigated the in vitro activities of selected mouth rinses on the reference strains of T. tenax and E. gingivalis. In this study, two standard strains of T. tenax (ATCC 30207) and E. gingivalis (ATCC 30927) were used, and metronidazole was used along with fourteen mouth rinses. The activities of the preparations were evaluated based on the ratio of dead to living cells after incubation at (37 - C) for 1, 10, and 30 min. The death of protozoa was categorized by the lack of movement and changes in the shape and characteristics of cell disintegration. The study concluded that all mouth rinses tested were effective on both protozoa [bib_ref] The in vitro activity of selected mouthrinses on the reference strains of..., Moroz [/bib_ref]. There are no vaccines or drugs reported in the literature to effectively treat T. tenax infections in the oral cavity. However, metronidazole and tinidazole are the drugs approved by the U.S. FDA and EMA to treat vaginal trichomoniasis caused by T. vaginalis [bib_ref] Research progress on the composition and function of parasite-derived exosomes, Nawaz [/bib_ref] [bib_ref] Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions, Twu [/bib_ref] [bib_ref] Strategies for prevention and treament of Trichomonas vaginalis infections, Bouchemal [/bib_ref]. Since both protozoa are flagellates and closely related, there may be a possibility that metronidazole and tinidazole are effective at treating oral trichomoniasis caused by T. tenax as well [bib_ref] Trichomonas tenax empyema in an immunocompromised patient with advanced cancer, Shiota [/bib_ref]. Moreover, studies focusing on the treatment and prevention of T. tenax are needed since it is prevalent in both humans and animals that exhibit signs of periodontal disease [bib_ref] Microbiological basis of oral infections and sensitivity to antibiotics, Prieto-Prieto [/bib_ref]. It is worth noting that T. tenax has been found in the urogenital tracts of humans [bib_ref] Protozoan genital invasions caused by the representatives of trichomonas and giardia, Fedorych [/bib_ref] [bib_ref] A false-positive Trichomonas vaginalis result due to Trichomonas tenax presence in clinical..., Brosh-Nissimov [/bib_ref] and, hence, possibly contributes to human trichomoniasis (although how significant a contribution is to be determined).
# Discussion
Periodontal disease is a public health concern for humans and dogs worldwide. For years, the disease has been associated with the oral flagellate T. tenax, although the cause and effect have not been confirmed, and much is unknown about the pathology of the parasite. Trichomonas tenax was first seen in the oral cavities of humans, and site and host predilections were assumed. However, it is now found in the lungs [bib_ref] Pyopneumothorax from coinfection by Trichomonas tenax and Geotrichum capitatum in a child..., Wu [/bib_ref] [bib_ref] First report of Trichomonas tenax infections in the Philippines, Dimasuay [/bib_ref] [bib_ref] Pulmonary trichomoniasis: Improved diagnosis by using polymerase chain reaction targeting Trichomonas tenax..., Mahmoud [/bib_ref] [bib_ref] Pulmonary trichomoniasis and Trichomonas tenax, Hersh [/bib_ref] , lymph nodes [bib_ref] Lymph node infection by Trichomonas tenax: Report of a case with co-infection..., Duboucher [/bib_ref] , vaginal samples [bib_ref] Trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial..., Crucitti [/bib_ref] , and subhepatic abscesses [bib_ref] Trichomonas species in a subhepatic abscess, Jakobsen [/bib_ref]. When it comes to the host, T. tenax has been found in humans, dogs, birds, cats, horses, and monkeys. A parasite that has been found in so many different sites and hosts, with zoonotic potential, should not be neglected, especially since the transmission between these hosts is not yet understood. Studies have shown its parasitic and pathogenic capabilities when in contact with mammalian cells. Researchers are now questioning this protozoan zoonotic potential because of the wide range of hosts it has discovered [bib_ref] What is the importance of zoonotic trichomonads for human health?, Maritz [/bib_ref]. Its prevalence is increasing in humans and dogs, with the latter needing more research. The prevalence of T. tenax ranges between 1 and 90% in humans and 8-96% in dogs [fig_ref] Table 4: Prevalence of Trichomonas tenax in humans with or without periodontitis [/fig_ref]. This gradual increase in prevalence worldwide should not be taken lightly. The prevalence in humans is in accordance with results reported by [bib_ref] Abd-Al-Hammza Abbass, Z. Canine Trichomonas tenax mandibular gland infestation, Szczepaniak [/bib_ref] ; however, the prevalence in dogs was higher than what was reported by [bib_ref] Entamoeba Gingivalis (Gros, 1849) and Trichomonas Tenax (Muller, 1773) oral infections in..., Norberg [/bib_ref] , due to limited studies. Moreover, based on studies published so far, it is clear that there is a direct association between periodontal disease and T. tenax infection [bib_ref] Parasites in periodontal health and disease: A systematic review and meta-analysis, Martin-Garcia [/bib_ref] [bib_ref] Presence of Trichomonas tenax and Entamoeba gingivalis in peri-implantitis lesions, Arpag [/bib_ref] [bib_ref] Protozoans in subgingival biofilm: Clinical and bacterial associated factors and impact of..., Dubar [/bib_ref]. Presently, several methods can detect T. tenax, including culture, microscopy, PCR, and LAMP. Thus far, LAMP is the most sensitive and specific method used to detect T. tenax, with a limit of detection of one cell, followed by PCR, and then microscopy, which is considered a gold standard [bib_ref] Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction, Kikuta [/bib_ref]. Virulent proteins have been extracted from T. tenax similar to the ones found in its close relative, T. vaginalis [bib_ref] Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax, Bózner [/bib_ref] [bib_ref] Study of an oral protozoan Trichomonas tenax using scanning and transmission electron..., Ribaux [/bib_ref] [bib_ref] Immunohistochemical localization of fibronectin-like protein on the cell surface of the oral..., Ribaux [/bib_ref] [bib_ref] Degradation of collagen types I, III, IV and V by extracellular proteinases..., Bózner [/bib_ref] [bib_ref] Characterization of the cathepsin B-like proteinases of Trichomonas tenax ATCC 30207, Yamamoto [/bib_ref]. However, the pathogenicity is still largely unknown. The area of exosome studies has shown great potential in finding the pathogenicity of various eukaryotic cells. With this new area of proteomics and the recent publication of the T. tenax draft genome [bib_ref] Draft genome sequence of, Yang [/bib_ref] , we can possibly move closer to mapping the pathway of T. tenax and developing a possible drug therapy that can control and prevent the transmission of this oral flagellate.
# Conclusions
Periodontal disease is a major public health concern worldwide with prevalence ranging from 1 to 90%; it was ranked the 11th most prevalent disease condition in the world in 2016 [bib_ref] Global prevalence of periodontal disease and lack of its surveillance, Nazir [/bib_ref]. Presently, microscopy, PCR, and LAMP are the available techniques used to detect T. tenax, with the latter being the most sensitive. In this scoping review, gaps in the knowledge and areas of research on T. tenax are indicated. The evidence, however, suggests that not only does T. tenax play a role in periodontal disease but there is an association between both oral flagellate and periodontal disease patients. It is imperative that the mechanism in which T. tenax adheres (and causes damage) to gums be functionally elucidated. This knowledge could increase the ability to develop other effective drug therapies to control this parasite and potentially decrease the prevalence of periodontal disease.
[fig] Figure 1: Flow diagram of scoping review selection process. [/fig]
[fig] *: M-microscopy; N/A-not available. Author Contributions: Conceptualization, C.Y.; methodology, M.A.M., N.Y. and S.M.; investigation, M.A.M., J.K. and C.Y.; writing-original draft preparation, M.A.M.; writing-review and editing, N.Y., J.K., S.M. and C.Y.; supervision, C.Y.; project administration, C.Y.; funding acquisition, C.Y. All authors have read and agreed to the published version of the manuscript.Funding: This research was partially funded by intramural research grants from the Ross University School of Veterinary Medicine (RUSVM), grants 41002-2021 and 41004-2023. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
[table] Table 1: Results obtained from search strategies used in databases. [/table]
[table] Table 2: Morphological characteristics of trichomonad protozoa found in human infections a . [/table]
[table] Table 3: Molecular diagnosis of Trichomonas tenax. N/A-not available; LAMP-loop-mediated isothermal amplification. [/table]
[table] Table 4: Prevalence of Trichomonas tenax in humans with or without periodontitis. * M-microscopy; N/A-not available. [/table]
[table] Table 5: Prevalence of Trichomonas tenax in a domestic dog with or without periodontitis. [/table]
|
Subsets and Splits